Products for

Cell Biology

In-Cell Westerns
Phospho ELISAs
Protein Transfection
SUMOylation
Sample Preparation
Fluorescent Assays & Stains

More Tools to Analyze Nuclear Function

Products for Chromatin
Biology & Epigenetics

Products forTranscription Biology

Antibodies for the Studyof Nuclear Function

ChIP-IT™ Express Products
for Chromatin IP

Products for
DNA Methylation

Products for
Histone Analysis

TransAM™ Transcription
Factor ELISAs

FACE™ Cell-based
Phospho-specific ELISAs

Fluorescent Dyes, Conjugated
Secondaries & Labeling

Get any (or all) of our product information by mail or by download
In addition to this product area profile, Products for Cell Biology, Active Motif has createdproduct area profiles that describe our products in other areas of nuclear function, suchas Chromatin Biology & Epigenetics, Transcription Biology and Antibodies. These profiles,
more detailed brochures for most products and several informative posters can be downloaded or requested by mail at www.activemotif.com/info. Product manuals and technicaldata sheets for all products are also available.

Table of Contents
Products for Cell Biology
Phospho-Protein Detection
FACE™ Colorimetric & Chemiluminescent.................................2
FACE™ Maker..........................................................................................4
Suspension Cell FACE™.......................................................................4
Sandwich ELISAs
FunctionELISA™ IkBa...........................................................................5
FunctionELISA™ TRAIL.........................................................................5
Protein Transfection
Chariot™....................................................................................................6b-Galactosidase Staining Kit...........................................................7
Protein Labeling & Translocation
LigandLink™ Universal Labeling.......................................................8
Deubiquitination
DUB-Detector™................................................................................... 10
SUMOylation
SUMOlink™ SUMO-1........................................................................... 11
SUMOlink™ SUMO-2/3..................................................................... 11
DNA Repair
GTBP DNA Repair Kit....................................................................... 12
Ku70/86 DNA Repair Kit................................................................. 12
RPA DNA Repair Kit........................................................................... 12
GTPase Activation Assay
Ras GTPase Chemi ELISA.................................................................13
Nitric Oxide Quantitation
Nitric Oxide Quantitation Kit....................................................... 14

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Gene Silencing
gripNAs™................................................................................................. 15mRNA Isolation
mTRAP™ Maxi....................................................................................... 16mTRAP™ Midi....................................................................................... 16mTRAP™ Total...................................................................................... 16
Nuclear & Cytoplasmic Extraction
Nuclear Extract Kit............................................................................ 18
Mitochondrial Separation & Staining
Mitochondrial Fractionation Kit.................................................. 19
Chromeo™ Live Cell Mitochondrial Staining Kit.................... 19
Protein & Extract Quantification
ProStain™............................................................................................... 20
Albumin Quantification
Albumin Blue Fluorescent Assay Kit........................................... 21
Live Cell Membrane Staining
LavaCell™............................................................................................... 22
Cell Viability Assay
ToxCount™.............................................................................................23
Competent E. coli
RapidTrans™ TAM1.............................................................................. 24
Fluorescent Dyes, Antibodies & Labeling
Conjugated Secondary Antibodies........................................... 25
Chromeo™ Dyes.................................................................................. 25
Chromeo™ Py-Dyes........................................................................... 25

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1

Phospho-Protein Detection
FACE™
simple, efficient analysis of protein phosphorylation
Fast Activated Cell-based ELISA (FACE™)* Kits provide a simple,
efficient, cell-based method to monitor proteins activated byphosphorylation. FACE Kits enable modification-specific analysis
directly within the cell, without the need for cell extractions, gelelectrophoresis or membrane blotting. And, because the cells
are grown, stimulated and assayed in a single 96-well plate, FACE
Kits are easily automated.

Traditionally, protein phosphorylation has been studied using
radioactive kinase assays and Western blotting, which can betime-consuming, complicated and expensive. FACE assays are
non-radioactive and generate data that is more quantitative,
specific and reproducible than these other methods. This
makes FACE Kits the simplest, most cost-effective phosphospecific assays available.
* Developed in collaboration with Dr. M. Peppelenbosch and Dr. H. Versteeg.

The FACE advantage
FACE Kits are easy to use and require less than 2 hours of handson time. In the FACE method, cells are cultured in 96-well plates
and stimulated to induce the pathway of interest. Following
stimulation, the cells are rapidly fixed, which preserves activationspecific protein modifications. Each well is then incubated witha primary antibody specific for the activated protein of interest.
Subsequent incubation with secondary HRP-conjugated antibody
and developing solution provides a colorimetric or chemiluminescent readout that is quantitative and reproducible. The
number of cells in each well can be determined easily with theprovided Crystal Violet solution. FACE Kits also contain a

primary antibody specific for the total protein, so you canmonitor both total and activated protein levels in the same
experiment. FACE eliminates cellular extractions, radioactive
kinase assays, time-consuming Westerns and inefficient epitope
interactions that occur on membranes. FACE is a highly sensitive,
high-throughput assay designed for detecting activated proteins
within mammalian cells. FACE Kits are currently available formonitoring a large number of phosphorylated and total kinases,
including p38, JNK, AKT, ERK1/2 and EGFR to name a few; to see
a complete list of the over 20 targets available, please visit us
at www.activemotif.com/face.

Why use FACE?
• Simple, non-radioactive method that is easily quantified• Study any phospho-protein of interest using
FACE Maker (see page 4)• Fixing preserves activation-specific protein modifications• Chemiluminescent sensitivity to detect extremely smallchanges in phospho-protein levels• Cell-based assay eliminates cell extracts, gels and blotting• Minimal hands-on time

• Optimized module for working with suspension cells (page 4)• Antibodies and plates to compare both phospho andtotal protein levels• Compatible with high-throughput automation

Add anti-IgG
HRP conjugate

Add primary
antibody
3 hrs.

1 hr.

Cells arefixed following
stimulation

Flow chart of the FACE process.
Cells are grown, stimulated and fixed in the same
96-well plate. Addition of primary and secondary
antibodies detects phosphorylated protein.

2

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P

P

P

Add developing
and stop solution

Stimulatecells

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Cells cultured in
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Phospho-Protein Detection
Improved accuracy

1

Preparing cellular extracts for Western blotting is time-consuming, and additional protein modifications can occur during theextraction process that may alter the final results. To eliminate
these problems, FACE Kits use a special fixation step that “freezes” the cellular state of the cell and prevents further protein
modifications. This enables the detection of the exact proteinstate within the cell at a chosen time point, which provides you
with more accurate results.

1

Although sensitive, Western blots are more of a qualitative rather than quantitative tool. In contrast, FACE Kits provide data thatcan be easily quantified relative to the number of cells or to thetotal amount of target protein present. To illustrate, FACE p38
MAPK assays and Western blots were performed on anisomycintreated murine macrophage 4/4 cells. FACE Kits clearly yield
results that are more quantifiable than Western blot (Figure 1).

Reproducible results
FACE Kits are highly reproducible, which is extremely importantwhen measuring small changes in the amounts of a phosphorylated protein. To demonstrate, FACE assays were performed on
three different samples of macrophage 4/4 cells that had beentreated in an identical manner with PMA to induce ERK phosphorylation. Levels of phosphorylated ERK were highly consistent
between each sample (Figure 2). The high level of reproducibility
using FACE Kits makes it possible to accurately monitor subtle
differences in protein phosphorylation.

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4

A

2

3

4

B

total-p38

OD450 nm

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

Phospho p38
Total p38

C

0

0.1
1
10
Anisomycin induction (µg/ml)

FIGURE 1:
Phospho and total p38 MAPK assays.
Macrophage 4/4 cells were grown in 10 cm dishes to 80% confluency, serumstarved for 16 hours and stimulated with anisomycin for 15 minutes. Cell
lysates were made and Western blots performed using phospho- (A) andtotal-p38 antibodies (B). For FACE, 4/4 cells were grown in 96-well plates,
stimulated as above, fixed and then assayed in triplicate using the FACE p38
Kit (C). Data were corrected for cell number through use of the kit’s Crystal
Violet Dye. Western blot data provided courtesy of Dr. Henri H. Versteeg and
Dr. Maikel P. Peppelenbosch.

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More quantitative data3

phospho-p38

Unmatched specificity
To be certain that only the protein of interest is detected, all
FACE Kit antibodies are stringently tested for cross-reactivity byWestern blot analysis. In particular, the phospho-specific antibodies are assayed to verify that they detect only the activatedform of the target protein. The phospho and total antibodies areused in tandem to make sure that the phospho-antibody does
not interact with other phosphorylated proteins. This ensuresthat FACE Kits are highly specific and detect only the correct
protein of interest at the specific phosphorylated site.

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FIGURE 2:
Reproducible assay of phosphorylated ERK.
Murine macrophage 4/4 cells were cultured in 96-well plates and serum
starved for 16 hours. Cells were then stimulated with Phorbol 12-myristate
13-acetate (PMA) for 10 minutes and fixed. Levels of phospho ERK wereassayed in triplicate using the FACE ERK1/2 Chemi Kit. Data was plotted after
normalization for cell number (performed through use of the kit’s Crystal
Violet Dye).

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3

Phospho-Protein Detection
FACE Chemi – increased sensitivity

FACE NFkB p65 Profiler – study multiple residues

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All of Active Motif’s FACE Kits are available in a Chemi format
that is ideal for those researchers who require maximum sensitivity. These ultra-sensitive kits use chemiluminescent detection ona luminometer to accurately monitor even the smallest changes
in protein phosphorylation (Figure 3). Chemiluminescent detection provides more flexible measurement parameters than traditional colorimetric kits, enabling detection limits to be adjusted
to maintain linearity and ensure that the detection sensitivity isappropriate to the sample type being assayed. FACE Chemi Kits
require the use of a microplate luminometer or a CCD camera.

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Chemiluminescent detection of c-Jun phosphorylation using FACE Chemi.
The FACE c-Jun (S63) and c-Jun (S73) Chemi Kits were used to assay thelevels of total and phosphorylated c-Jun contained within uninduced andanisomycin-induced NIH/3T3 cells.

Many proteins, such as NFkB, are phosphorylated on multiple
residues and can be induced by a variety of stimuli. Being able todistinguish between different phosphorylation sites on a protein
can be difficult to determine using conventional Western blot
analysis. Active Motif’s FACE NFkB p65 Profiler Kits make it possible to rapidly profile the levels of different phosphorylationsites in one simple experiment. Plus, like all the FACE Kits, Profiler
Kits include an antibody against the total form of the protein,enabling comparisons of phosphorylated to native protein levels.
The FACE NFkB p65 Profiler Kit contains antibodies specific fortwo phosphorylated sites: serine 468 and serine 536 (Figure 4).

FACE Maker is an adaptable version of the FACE Kit, enabling you
to study any phosphorylated protein you choose in a simple, fastand sensitive cell-based assay. Using antibodies specific to yourdesired target protein, modification-state specific analysis is performed directly within the cell without the need for lysates andtime-consuming immunoblotting. FACE Maker Kits provide all
the optimized buffers of our target-specific FACE Kits, but FACE
Maker Kits do not include antibodies. So, you are able to study
any phosphorylated protein you want with your own antibodies while taking advantage of the effective FACE method andoptimized FACE reagents.

Suspension Cell FACE – measure more cells
The Suspension Cell FACE module was designed to be used withany colorimetric or chemiluminescent FACE Kit (Figure 5); it
provides you with 96-well filter plates that make it easier toperform washing & liquid handling steps through use of an
appropriate vacuum manifold. This helps eliminate the loss ofcells that can occur when performing wash and fixation steps
in 96-well plates. Because you’re able to measure more cells perwell, this makes the assay better able to discern small effects.

4

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80
Normalized Signal (Relative Percent)

FACE Maker – study any phospho-protein

FIGURE 4:
Monitoring phosphorylation of NFkB p65 at multiple sites.
The FACE NFkB p65 Profiler Kit was used to assay levels of total andphosphorylated NFkB p65 in uninduced and TNF-a + Calyculin A induced
HeLa cells. Data was plotted after correction for cell number (performed
through use of the kit’s Crystal Violet reagent).

70
60
50
40
30
20
10
0

Untreated

PMA treatedTotal ERK 1/2

Untreated

PMA treated

Phospho-ERK 1/2

FIGURE 5:
FACE ERK1/2 with the Suspension Cell FACE module.
The Suspension Cell FACE module was used with FACE ERK1/2 Chemi to assay
50,000 Jurkat cells per well. Jurkat cells were serum-starved and treated with
100 ng/ml PMA (Phorbol 12-myristate 13-acetate) for 15 minutes. A three-foldincrease in phospho-ERK1/2 was detected in the assay.

For an up-to-date list of available
FACE Kits, please visit us atwww.activemotif.com/face.
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Sandwich ELISAs
FunctionELISA™
accurate quantification of signal pathway proteins
FunctionELISA™ Kits offer a simple, rapid method to monitorchanges in signal pathway proteins. The FunctionELISA technique
is an improvement over other methods used to study proteins,
such as Western blotting, as it provides sensitive, quantitative

protein measurement in just hours. The 96-well format requires
only a small amount of extract, making it possible to perform
multiple-sample, high-throughput analysis.

How do the FunctionELISA Kits work?

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The FunctionELISA Kits utilize the Sandwich ELISA techniqueto capture and quantifiably measure the amount of a specificprotein present in a sample. Sandwich ELISAs use two antibodies that recognize different epitopes on the protein of interest.
The Capture Antibody is provided immobilized in the wells ofan ELISA plate. When cell lysate is added, the protein of interest
is bound by this antibody. The Detecting Antibody then binds
to the captured protein. FunctionELISA IkBa and TRAIL Kits
use a horseradish peroxidase (HRP) or alkaline phosphatase (AP)conjugated secondary antibody, respectively, that binds to the
Detecting Antibody to quantify the bound protein (Figure 1).

FunctionELISA IkBa
NFkB is regulated by the IkB family of inhibitory proteins.
Phosphorylation of IkBa leads, ultimately, to activation of
NFkB. Thus, analysis of the phosphorylation state of IkBa
provides insights about NFkB and the many genes it regulates.
FunctionELISA IkBa Kits can be used to study the phosphorylation state of IkBa, and to correlate this information with theactivation and translocation of NFkB (Figure 2). FunctionELISA
IkBa uses a Capture Antibody specific for phosphorylated
IkBa and highly sensitive and accurate chemiluminescent detection. Therefore, the kit requires the use of a luminometer.

0chemiluminescence

A

B

5
10
30
time (minutes)

120000
80000
40000
10

20

30
40
time (minutes)

50

60

70

FIGURE 2:
Induction of IkBa phosphorylation.
Jurkat cells were treated with 1 nM TNF-a and harvested at the indicatedtime points. Whole-cell extracts were assayed in Western blot analysis (A)using Phosphorylated-IkBa mAb (Cat. No. 40904) and the FunctionELISA
IkBa Kit (B).

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Sandwich ELISA schematics.
Capture and Detecting Antibodies are used for sensitive, accuratequantification of the antigen of interest (Ag).

Why use FunctionELISA?
• Eliminates the need to run, blot and develop gels
• Each kit includes a positive control protein to generatea standard curve for sample quantification• Ready-to-use format with Capture Antibody
precoated on the plate• Ability to assay both cell and tissue samples

Tumor Necrosis Factor-Related Apoptosis Inducing Ligand(TRAIL) is a member of the TNF ligand family. It induces apoptosis in many cancer cells but is not toxic to most normal cells.
FunctionELISA TRAIL can accurately measure picogram quantities of soluble TRAIL in lysates and fluid samples. It utilizes anAP-conjugated secondary antibody and detection is measured
through the use of a spectrophotometer.

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FunctionELISA TRAIL

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2000000;JAPAN +81 (0)3 5225 3638

Product

FormatCat. No.

FunctionELISA™ IkBa

1 x 96-well plate5 x 96-well plates

48005
48505

FunctionELISA™ TRAIL

1 x 96-well plate5 x 96-well plates

48010
48510

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5

Protein Transfection
Chariot™
simple, efficient protein delivery
Chariot™* is a revolutionary transfection reagent that efficiently
transports biologically active proteins, peptides and antibodies
directly into cultured mammalian cells in less than two hours1, 2.
The typical delivery efficiency is 65-95%. After delivery, living
cells can be assayed to determine the effects of the introduced
materials, without the need for fixation. This makes Chariot ideal
for a wide variety of functional studies, including delivery of
inhibitory proteins, organelle labeling, screening peptide libraries
and transient complementation studies.

While there are many techniques available for introducing DNA
into cells, including microinjection,3 calcium phosphate coprecipitation,4 cationic liposomes,5 viral vectors6 and electroporation,7 these methods have several limitations. After transfection,
the researcher must wait overnight to several days to detect
gene expression. A greater drawback is that the cytotoxicity ofthese methods can make it difficult to determine if any changes
observed are caused by the recombinant protein that was
introduced, or by the transfection method itself.
* Covered under U.S. Patent No. 6,841,535. Purchase includes the right to use forbasic research only. Other-use licenses available, please contact Tech Service.

Chariot advantages
Chariot is a peptide that forms a non-covalent complex withthe macromolecule to be delivered. This complex stabilizes theprotein, protecting it from degradation during delivery andpreserving its function. Upon addition to cells, the complex is
rapidly internalized, where it dissociates. The Chariot peptide
then localizes to the cell nucleus where it is degraded, leaving
the macromolecule free to proceed to its target organelle. By
directly delivering protein, peptide or antibody, Chariot completely bypasses the transcription-translation process associatedwith recombinant gene expression, saving hours to days of time.

Why use Chariot?
• Delivers active proteins, peptides and antibodies
directly into mammalian cells• Up to 95% efficiency in less than 2 hours• Facilitates functional studies in living cells• Non-cytotoxic and serum independent• Non-covalent binding preserves the function of delivered
proteins and eliminates the need to create fusion proteins• Works in a variety of cell lines, including hard-to-transfectcells and neurons, as well as in vivo with animal models

Applications of Chariot

Chariot delivers results

Direct delivery of active protein makes it easy to performstudies not even possible using classical methods. Common
applications for Chariot include:

Chariot is non-cytotoxic and efficiently delivers proteins intoa wide range of cell lines, including hard-to-transfect PC-12 andprimary cells. It has proven to be effective on both adherent andsuspension cells, as well as in vivo with animal models.8 As efficient delivery occurs at 4°C, the Chariot process is independentof the endosomal pathway,9 which can modify macromolecules
during internalization. The process is also serum-independent, sodelivery can occur in the presence or absence of serum.

• Delivery of inhibitory peptides and gripNAs™ (see page 15)• Transient complementation studies• Antibody studies• Screen peptide libraries• Protein half-life studies• Organelle labeling

Flow chart of the Chariot process.
Chariot is easy to use. Simply combine it with your macromolecule,wait 30 minutes, then add thecomplex to your cells. Activity canbe assayed in as little as 90 minutes
post-delivery.

6

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Protein Transfection
Proven Chariot performance
The ability of Chariot to effectively deliver biologically active
proteins, peptides and antibodies directly into a variety of different cell types has been well documented. Table 1 below lists
some of the results reported to Active Motif by Chariot users.
Please go to www.activemotif.com/chariot and select the tabfor User Data to view the complete table.

A

B

0

Cell Line or Model

Cell Type

Delivery Efficiency

3T3-L1

Mouse fibroblast

80%

A549

Human lung carcinoma

95%

Arabidopsis

Primary plant protoplasts

Not provided

COS-7

Monkey kidney

80%

Embryonic cells

Primary mouse

90%

HeLa

Human cervix carcinoma

95%

HMSC

Primary humanmesenchymal stem cells

80%

HS-68

Human foreskin fibroblast

95%

J774

Mouse macrophage

90%

JurkatHuman T-cell leukemia

75%

Mouse hepatocytes

Primary liver

95%

Mouse model (in vivo)

Alveolar wall tissue

88%

Neural retina cells

Primary chicken

80%

NIH/3T3

Mouse embryo

80%

PC-12

Rat pheochromocytoma

80%Primary chick and primary rat

80%

Primary human

90%

Human lung fibroblast

95%

Sensory neurons (DRG)
Thyrocytes
WI-38

G1

10 15

20
0
G1

G2

10

15

20

G2

FIGURE 1:
Cell cycle arrest by delivered peptide.
p27kip1 is a 27 kDa cyclin-dependent kinase inhibitor that causes cell-cycle
arrest in G1 phase. 100 µl of 50 nM p27kip1 protein was complexed with 5 µl of
Chariot for 30 minutes. Chariot alone, p27kip1 alone and the Chariot-p27kip1
complex were added to HS-68 cells. Flow cytometry was performed
0, 10, 15 and 20 hours post-delivery. Cells that received Chariot alone (A)and p27kip1 alone (data not shown) were able to progress into G2 phase.
Over 90% of the cells that received the Chariot-p27kip1 complex (B) remainedin G1 phase. Data generously provided by Dr. Gilles Divita, CNRS, France.

FIGURE 2:
Targeted protein delivery.
50 ng of a 10 kDa nuclear protein labeled with Lucifer yellow at its C-terminus
was complexed with Chariot and delivered into HS-68 cells. Unfixed cells
were observed 90 minutes post-delivery.

TA B L E 1 :Delivery efficiencies of Chariot into specific cell types.
Chariot has been used to deliver proteins, peptides and antibodies into a variety
of different cell types, some of which are shown above. Data was provided to
Active Motif by users of Chariot, who completed our online Chariot survey.
For complete results, please visit our website.

b-Galactosidase Staining Kit
Need a control for Chariot? The b-Galactosidase Staining Kit
provides an easy-to-use and efficient method to determine thepercentage of cells expressing lacZ following transient or stabletransfection. When the positive control b-galactosidase protein
is used in conjunction with Chariot for protein delivery, theb-Galactosidase Staining Kit enables visualization of theefficiency of direct b-galactosidase delivery by Chariot.

FIGURE 3:
Delivery of fluorescent antibody.
A 1:1,000 dilution of an anti-actin antibody was used to label actin filaments.
Unfixed cells were observed 2 hours post-delivery.
REFERENCES

Product

FormatCat. No.

Chariot™

25 rxns
100 rxns

30025
30100

b-Galactosidase Staining Kit

75 rxns35001

www.activemotif.com

JAPAN +81 (0)3 5225 3638

1. Morris, M. et al. (2001) Nature Biotech. 19: 1173-1176.
2. Morris, M. et al. (1999) J. Biol. Chem. 274(135): 24941-2494.
3. Capecchi, M.R. (1980) Cell 22: 479-488.
4. Graham, F.L. and van der Eb, A.J. (1973) Virology 52: 456-467.
5. Felgner, P.L. et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7417.
6. Cepko, C.L. et al. (1984) Cell 37: 1035-1062.
7. Neumann, E. et al. (1982) EMBO J. 7: 841-845.
8. Aoshiba, K. et al. (2003) Amer. J. Resp. Cell Mol. Biol. 28: 555-562.
9. Schwarze, S.L. et al. (2000) Trends Cell Biol. 10: 290-295.

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Protein Labeling & Translocation
LigandLink™ Universal Labelingspecific, flexible labeling of proteins in living cells
Use of green fluorescent protein (GFP) and its derivatives has become commonplace in modern cell biology. However, although
they are widely used, biologically fluorescent proteins (FPs) arelimited by their inherent properties. For example, FPs have arelatively low quantum yield, and it is difficult to engineer theirspectral properties to suit specific applications.
Fluorescent dyes are a potential alternative to biological FPs as
they are available in a broad variety of formats and can be engineered easily to ensure desirable spectral properties. However,
because fluorescent dyes are synthetic molecules, it has notbeen possible to use them as a general tool for labeling specificproteins within a cell.

Active Motif’s LigandLink™ Universal Labeling* technology
overcomes these limitations by providing a small ligand that can
carry a variety of functional tags, including fluorescent dyes.
Expression of your protein of interest as a LigandLink fusion
enables you to specifically label the protein in vivo simply byadding one of the LigandLink Labels to the medium. To change
the properties of your tag, all you need to do is add a different
LigandLink Label. This means that it is now possible to create asingle protein fusion that can be labeled with a variety of tags,
depending on the needs of your experiment.
* Patent pending.

The LigandLink method
In the LigandLink method, the gene of interest is cloned in framewith the gene for E. coli dihydrofolate reductase (eDHFR) in the
pLL-1 vector (Figure 4). The vector is then transfected into mammalian cells and used to express the fusion protein. Twenty-fourhours after transfection, the protein of interest can be labeledsimply by adding the LigandLink Label of choice to the cell medium (Figure 1). Depending on the cell type and the characteristicsof the label, cells can be imaged in as little as 10 minutes.

affinity for endogenous DHFR (KI = ~4 µm). This is because TMP isan antibiotic that was designed to specifically inhibit the bacterial
enzymes responsible for the production of folic acid while notinteracting with mammalian proteins. Because there is minimalbinding of LigandLink Labels to non-tagged mammalian proteins,
the result is extremely low background. Moreover, TMP can bederivatized to carry a number of tags without substantiallyaltering its affinity and specificity for eDHFR.

The eDHFR protein was chosen as a fusion partner because it
is a relatively small, monomeric protein (18 kDa vs. 27 kDa for
GFP) that has been shown to have a high affinity for the ligand
trimethoprim (TMP). TMP binds with a high specificity to theE. coli form of DHFR (KI = ~1 nm), and a substantially lower

Thus, the LigandLink Universal Labeling technology uses eDHFR
and a number of TMP derivatives as a ligand-receptor pair toprovide a variety of functionalities.1 Cell-permeable LigandLink
Labels are available with red and green fluorescent dyes
(Figures 2 & 3).

Why use LigandLink?
• Label your protein in living cells – LigandLink Labels arecell permeable

E?)• Spend less time cloning – rather than using multiple
vectors, clone only once; obtain different functionalities
using different LigandLink Labels

E?)• Small label – LigandLink Labels are unlikely to alter thecharacteristics of your protein of interest

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LigandLink Applications• Protein:protein interactions• Protein capture

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FIGURE 1:
Specific protein labeling using LigandLink.
The gene of interest is cloned into pLL-1 in frame with the vector’s E. coli
dihydrofolate reductase (eDHFR) gene. After transfection into cells, the protein
of interest is expressed as a fusion to eDHFR. Addition of cell-permeable
LigandLink Label to the medium results in rapid, specific binding of the label by
the fusion protein.

• Protein localization & trafficking

8

F:?*

E

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Protein Labeling & Translocation
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lI

FIGURE 2:
Labeling of nuclear-localized eDHFR by LigandLink Fluorescein.
A nuclear localization sequence was cloned into pLL-1 and transfected into
CHO cells. Twenty-four hours post-transfection, 5 µM LigandLink Fluorescein
was added to the cells; 10 minutes later, the above image was taken.

d III
Hin
I
e

tI
No

Nh

ri
Co
U
p

eo
S V 4 0 o ri

P

FIGURE 4:
The LigandLink pLL-1 vector.

User-friendly vector
The LigandLink vector, pLL-1, was designed for convenience ofuse. It features a CMV promoter for high-level expression ofeDHFR fusion proteins, with Kanamycin for selection of stable
cell lines. The multiple cloning site (MCS) was designed tofacilitate cloning, whatever method you use. In addition to many
popular restriction sites, the MCS includes three blunt-cutting
restriction enzymes toward the 3´ end, each in a different reading frame with the eDHFR gene. This makes possible a numberof PCR and restriction enzyme cloning strategies.

FIGURE 3:
Membrane-localized eDHFR labeling by LigandLink Hexachlorofluorescein.
The myristoylation/palmitoylation sequence from Lyn was cloned into pLL-1
and transfected into CHO cells. Twenty-four hours post-transfection, 10 nM
LigandLink Hexachlorofluorescein was added to the cells; 2 hours later, theabove image was taken.

Pre-made translocation vectors

Product

FormatCat. No.

Signal transduction pathways typically involve the movement ofproteins throughout the cell in response to activation by someparticular stimulus. The LigandLink Universal Labeling technology
is ideal for studying such translocation events because it makes
it simple to label the proteins in vivo. To make it even easier, wehave already cloned a number of transcription factors into the
pLL-1 vector, such as NFkB p65, which are ready to transfect into
the mammalian cell line of your choice.

LigandLink™ pLL-1 Kit

1 kit

34001

LigandLink™ pLL-1-NFkB p65 Kit

1 kit

34004

LigandLink™ pLL-1-p53 Kit

1 kit

34005

LigandLink™ pLL-1-STAT1 Kit

1 kit

34006

LigandLink™ Fluorescein Label

300 rxns

34101

LigandLink™ Hexachlorofluorescein Label

300 rxns

34104

Ideal for multi-image capture experiments

REFERENCE

Analysis of co-repressor and co-activator interactions is an
important part of understanding protein function. However,analyzing interactions across multiple proteins can be time consuming and expensive. This is because each interacting partnermust either be cloned into a different FP fusion vector, or primary antibodies must be labeled with multiple dyes. To overcome
this problem, LigandLink Label comes in multiple formats thatcan be easily interchanged, depending on the FP partner of theinteracting protein of interest. This saves you time and money.
www.activemotif.com

JAPAN +81 (0)3 5225 3638

1. Miller, L.W. et al (2005) Nature Methods 2(4): 255-257.

To learn more about LigandLink and theavailable labels, please visit us atwww.activemotif.com/ligandlink.

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9

Deubiquitination
DUB-Detector™
simple fluorescent assay to detect deubiquitinating enzyme activity
Ubiquitination is a reversible, post-translational modification inwhich one or more ubiquitin molecule is added to proteins by
the sequential action of three enzymes: E1 ubiquitin-activating
enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase.
The group of enzymes responsible for the removal of ubiquitinfrom proteins are known as deubiquitination enzymes (DUBs),
of which the human genome encodes nearly 100, making them
the largest family of enzymes in the ubiquitin system. DUBs are
responsible for ubiquitin precursor processing, ubiquitin recycling,

trimming of ubiquitin chains, as well as other diverse roles in cell
growth and differentiation, development, DNA damage, disease
pathways, transcriptional regulation and chromatin remodeling.
Active Motif’s DUB-Detector™ Deubiquitination Assay provides
a simple fluorescent solution for screening the cysteine proteaseclass of DUBs from cell extracts or purified recombinant proteins.
The assay can be used for either endpoint or kinetic analysis of
enzyme activity, or to screen for DUB inhibitors.

How does DUB-Detector work?

+''''

(+''

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*''''
),'''
(%),«^'%-)«^'%*(«^
9XZb^ifle[(%),«^"@e_`Y`kfi'%-)«^"@e_`Y`kfi'%*(«^"@e_`Y`kfi

)''''
(,'''
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J_c[c_dkj[iFIGURE 1:
Kinetic analysis of DUB activity in HeLa nuclear extract.
HeLa nuclear extracts were assayed at 0.313, 0.625 and 1.25 µg per reaction in thepresence or absence of 1 µM inhibitor for 20 minutes. Following the incubation,
Fluorescent Substrate (100 nM) was added to each well and the fluorescentintensity was measured every two minutes.

(-''

()''

,'gDLJG(,'gDLJG(-"@e_`Y`kfi('''),gDLJG(),gDLJG(-"@e_`Y`kfi/''()%,gDLJG(()%,gDLJG(-"@e_`Y`kfi-''

9XZb^ifle[+''

9XZb^ifle["@e_`Y`kfi)''

Why use DUB-Detector?
• Complete assay with optimized buffers for enhanced
enzymatic activity• Includes both positive control extract and a universal
DUB inhibitor• Fast procedure can be completed in less than 1 hour

'
'

('

)'

*'

+'

,'

-'

J_c[i[YedZi

FIGURE 2:
Recombinant USP16 deubiquitination activity.
The DUB-Detector assay was used to evaluate the activity of recombinant
USP16 at 12.5, 25 and 50 pM per reaction in the presence or absence of 100 nM
inhibitor for 20 minutes. Following the incubation, Fluorescent Substrate(100 nM) was added to each well and fluorescent intensity was immediately
measured every minute with a total reaction time of 60 minutes. Data shown
are the results from duplicate wells.

10

*,'''

<bkeh[iY[dj?dj[di_joH<K

Despite the differences in their cellular roles and molecularsizes, the deubiquitinating enzymes all appear to hydrolyze theirsubstrates through a common mechanism. Active Motif’s
DUB-Detector Assay uses a universal ubiquitin substrate todetect enzymatic activity or screen for potential inhibitors.
The fluorescent substrate is based on a C-terminal derivativeof ubiquitin which is hydrolyzed by the cysteine protease classof enzymes to release a fluorescent signal proportional to theamount of enzyme activity. The fluorescence can be detectedusing a microplate reader with excitation of 485 nm and emissionof 535 nm. For added convenience, the assay also includes HeLa
nuclear extract and an inhibitor to all classes of deubiquitinating
enzymes as positive and negative controls (Figure 1).

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• Great for either endpoint or kinetic analysis• Fluorescence can be detected with an excitation
wavelength of 485 nm and an emission wavelengthof 535 nm

Product

FormatCat. No.

DUB-Detector™ Kit

1 x 96 rxns

40110

JAPAN +81 (0)3 5225 3638

www.activemotif.com

SUMOylation
SUMOlink™
simple, effective in vitro SUMOylation of proteins with proven controls
Post-translational modifications can alter protein function bymodifying activity or intracellular localization. Small ubiquitinrelated modifiers (SUMOs) are ubiquitin-like polypeptides thatcovalently bind to specific lysine residues on the target protein
via isopeptide bonds. Mammalians express three major SUMO
paralogs: SUMO-1, SUMO-2 and SUMO-3. SUMO-2 and SUMO-3
are approximately 96% identical to each other and have yet tobe functionally differentiated, so they are often referred to as
SUMO-2/3. There is only 45% identity shared between SUMO-1
and SUMO-2/3, which may explain the increasing evidence thatthese SUMO modifications have distinct cellular functions. The
pathway of SUMO conjugation affects many biological processes including chromosomal organization, gene transcription, DNA

replication and repair, nucleocytoplasmic transport and signaltransduction pathways.
Active Motif’s SUMOlink™ Kits provide a fast, simple method forgenerating SUMOylated proteins in vitro using the E3-independent SUMOylation enzyme cascade. SUMOlink Kits provide all
the reagents required for performing 20 wild-type and 20 mutated in vitro SUMOylation reactions. The Kits also include positive control p53 protein and antibody to help ensure success.
Applications of the kit include the investigation of SUMOylation
on enzyme function, understanding the role of SUMOylationin the regulation of cellular processes and the identification ofnovel proteins as targets for SUMO.

DN

The SUMOlink advantage

Active Motif’s SUMOlink Kit is fast and easy to use. Simply
combine your protein of interest with the provided E1 and E2
enzymes, SUMO-1 or SUMO-2 & SUMO-3 protein(s) and buffers.
After a 3-hour incubation, the reaction is stopped by adding
SDS-PAGE Loading Buffer. SUMOylation can then be analyzed byWestern blot with the provided SUMO-1 or SUMO-2/3 antibodies to determine the extent to which your target protein has
been SUMOylated. p53 protein and antibody are also included in
the kits for use as a positive control (Figure 1).

(

)

*

(

)

*

0/
-+
,'
**'

Why use SUMOlink?

(-• Simple, effective method for SUMO conjugation anddetection of SUMOylated proteinsf+)Wdj_XeZo• Study either SUMO-1 or SUMO-2 & SUMO-3 activity• Positive control p53 protein and antibody provided toensure success• Wild-type and mutated SUMO proteins are provided• Versatile – study SUMOylation of cell or tissue extracts,or recombinant protein

Product

FormatCat. No.

SUMOlink™ SUMO-1 Kit

20 rxns

40120

SUMOlink™ SUMO-2/3 Kit

20 rxns

40220

www.activemotif.com

JAPAN +81 (0)3 5225 3638

IKCE#'Wdj_XeZo

FIGURE 1:
Specific labeling of p53 with SUMO-1 by SUMOlink.
Western blot analysis of in vitro SUMOylation of p53 protein with wild-type
and mutated isoforms of SUMO-1: The two Western blots were incubatedwith p53 antibody (1:5,000 dilution) and SUMO-1 antibody (1 :4,000 dilution).
The p53 protein is SUMOylated only by the wild-type SUMO-1 protein.
Lanes 1: Wild-type SUMO-1 protein conjugation reaction.
Lanes 2: Mutated SUMO-1 protein conjugation reaction.
Lanes 3: No p53 control protein used in a wild-type SUMO-1 protein
conjugation reaction.

To learn more about SUMOylation or to see additionaldata, please visit us at www.activemotif.com/sumolink.

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11

DNA Repair
DNA Repair Protein Kits
quantitative ELISAs for measuring DNA repair protein activity
Active Motif’s DNA Repair Protein Kits are DNA-binding ELISA
assays that facilitate the detection of DNA repair protein activity in both mammalian tissue and cell culture extracts. These kits
combine a fast, user-friendly format with a sensitive, specific assaythat is 10-fold more sensitive than traditional gelshift whileeliminating the use of radioactivity. Quantitative results areachieved in approximately 3.5 hours.

DNA repair proteins are critical for maintaining genome integrity,and deficiencies in their activity are linked to the development ofmany diseases, including cancer. Traditionally, techniques such as
gelshift and immunoblotting have been used to study DNA
damage and repair protein interactions. Although these methods
are sensitive, they are typically more qualitative rather thanquantitative tools (Figure 1).

The DNA Repair Protein Kit advantage

A

GTBP
A

1.25
1
OD450 nmActive Motif’s DNA Repair Protein Kits provide a quick and easy
method for sensitive DNA repair protein detection. Each kit contains a 96-well plate with multiple copies of an immobilized oligonucleotide containing a DNA lesion. Cell extract is added to eachwell and the repair protein of interest binds specifically to theoligonucleotide on the plate. Each well is then incubated with a
primary antibody directed against the repair protein being studied.
Addition of a secondary HRP-conjugated antibody and developing
solution provides an easily quantified colorimetric readout.

Why use the DNA Repair Protein Kits?

0.75
0.5
0.25

• Sensitive, colorimetric readout easily quantified byspectrophotometryB• ELISA format eliminates gels, blotting and radioactivity• From cell extract to completed assay in less than 3.5 hours• Up to 10-fold greater sensitivity than gelshift assays• Compatible with high-throughput automation

0

Raji

PANC-1

JurkatB

FIGURE 1:
Measurement of GTBP activity across multiple cell lines.
Jurkat, PANC-1 and Raji nuclear extracts were assessed for GTBP activity using
Western blot (A) and the GTBP DNA Repair Protein Kit (B). The DNA Repair
Protein Kit is clearly more quantitative and sensitive.

• Ability to assay both cultured cells and tissue samples

Product

FormatCat. No.

GTBP DNA Repair Protein Kit

1 x 96-well plate5 x 96-well plates

51096
51596

Ku70/86 DNA Repair Protein Kit

1 x 96-well plate5 x 96-well plates

51196
51696

RPA DNA Repair Protein Kit

1 x 96-well plate5 x 96-well plates

51296
51796

Please visit us at www.activemotif.com/dnarepair to learn more.

12

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FIGURE 2:
Flow chart of the DNA Repair process.
DNA repair proteins bind to oligonucleotides with specific DNA lesions
that are immobilized in the well. Incubation with primary and secondary
antibodies measures DNA repair protein activity.

JAPAN +81 (0)3 5225 3638

www.activemotif.com

GTPase Activation Assay
Ras GTPase Chemi ELISA Kits
fast, sensitive analysis of activated GTPases
Small GTPases, also known as GTP-binding proteins, are a familyof enzymes that serve as molecular switches to regulate many
important signal transduction pathways by alternating between
active (GTP-bound) and inactive (GDP-bound) states. Because
GTPases are involved in cell growth, apoptosis and differentiation,
understanding the mechanisms that regulate their activation is the
subject of intense investigation. However, the more traditional
methods used to monitor GTPase activation, such as Western blot
or pull-down assays, are tedious, time consuming and not suitablefor high-throughput analysis.

In contrast, Active Motif’s Ras GTPase Chemi ELISA Kit is thefirst ELISA-based kit designed to detect and quantify activatedRas. Because the assay is an ELISA, sensitivity is improved overpull-down/Western methods, enabling detection of low levels ofprotein. In addition, ELISAs are more quantitative than Westerns,
and eliminate the need to run and develop gels. And, as the assay
uses a 96-well plate made up of 12-well strips, it is convenient to
run anywhere from 1 to 96 samples.
Licensed to Active Motif under U.S. Patent No. 6,300,081. Purchase includes the right to use forbasic research only. Other-use licenses available, please contact Technical Services.

The Ras GTPase Chemi ELISA advantage/'''''

8Zk`mXk\[IXjJ`^eXcICL

In the Ras GTPase Chemi ELISA Kit method, a Raf-RBD binding domain protein that has been fused to GST is added to a
96-well, glutathione-coated assay plate. Addition of sample tothe plate results in the binding of activated Ras to the Raf-RBD
binding domain protein on the plate, while inactive Ras does
not bind. Next, a primary antibody specific for H-Ras in mouseand H- & K-Ras in human samples is added. Addition of an HRPconjugated secondary antibody and developing reagent provides
a sensitive readout that can be measured with a luminometer(Figure 1).

Lejk`dlcXk\[<>=jk`dlcXk\[-'''''+''''')'''''
'

-%),

()%,
),
:\cc\okiXZk«^&n\cc,'

FIGURE 1:
Quantification and comparison of Ras activation in HeLa cell extracts.
Increasing amounts of whole-cell extracts from HeLa cells that were either
unstimulated or had been stimulated with 5 ng/ml of EGF for 2 minutes wereassayed for Ras activation using the Ras GTPase Chemi ELISA Kit.

Why use Ras GTPase Chemi ELISAs?
• More sensitive – uses 20-fold less sample thanpull-down/Westerns• Better results – quantitative readout lets you
easily compare samples• Less effort – no need to run gels or develop Westerns• Save time – obtain results in less than 5 hours

45 min45 min• Versatile – study cell or tissue extracts,or recombinant protein

Product
Ras GTPase Chemi ELISA Kit

FormatCat. No.

1 x 96-well plate

52097

For complete details on Active Motif’s Ras GTPase Kit,
please visit us at www.activemotif.com/gtpase.

www.activemotif.com

Add developing
solutions

Add anti-IgG
HRP conjugate

Add primary
antibody

Add cell
extract

JAPAN +81 (0)3 5225 3638

45 minCell extractcontaining small
GTPase protein

GST-RBD
coated plate

FIGURE 2:
Flow chart of the GTPase ELISA process.

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13

Nitric Oxide Quantitation
Nitric Oxide Quantitation Kit
faster, more accurate measurement of nitric oxide production
The Nitric Oxide Quantitation Kit is faster and easier-to-use thanexisting methods for measuring the production of NO in biological samples. The kit employs an innovative cofactor technologythat reduces the time and number of steps required, while increasing the dynamic range of NO measurement. The NO Quantitation
Kit can be used with a wide variety of samples, including cell or
tissue extract, urine, plasma, serum and saliva.
Because NO has an extremely short half-life (< 10 seconds), it
is difficult to detect and study directly. However, because NO
naturally metabolizes to nitrite and nitrate, quantitation of thesestable anions is often used to indirectly determine the amount ofNO originally present in a sample.

Typically, NO assays are performed using either a two-step assay
or a three-step lactate dehydrogenase (LDH) assay. In both methods, the first step is the reduction of nitrate into nitrite by nitrate
reductase. Griess Reagent is then used to convert the nitrite intoa purple-colored azo compound, which is quantitated by spectrophotometer at A540 (Figure 1). However, excess NADPH, an essential
cofactor in nitrate reduction, interferes with the Griess reaction,
limiting the sensitivity of the two-step assay. One method toremedy this is to add a third step, whereby lactate dehydrogenase(LDH) eliminates excess NADPH prior to the Griess reaction. While
this additional step increases assay sensitivity, it also increases theamount of time required and the level of complication.

NO Quantitation Kit advantages

Active Motif’s Nitric Oxide Quantitation Kit offers advantagesin time, ease-of-use and accuracy over the methods describedabove. It employs an innovative cofactor technology that accelerates the conversion of nitrate to nitrite while simultaneously
degrading NADPH. Thus, the time required for the reductase
reaction is reduced to only 30 minutes, and sensitive colorimetric quantification can be performed without the need for LDH
treatment. Moreover, the Active Motif method is linear overa broader dynamic range, which increases the range of sample
concentrations that can be measured accurately (Figure 2). This
saves time and money as fewer samples will need to be diluted
and re-assayed to obtain accurate results. To make it easier toprocess large numbers of samples, the NO Quantitation Kit
is provided in a convenient 96-well format that facilitates
high-throughput automation.

E`kiXk\E`ki`ZFo`[\E`ki`k\8jjXplj`e^>i`\jjI\X^\ek
FIGURE 1:
Measurement of nitric oxide.
Nitric oxide metabolizes rapidly into nitrate and nitrite, which are stable. In NO
assays, the nitrate is converted to nitrite, then assayed with Griess Reagent.

1.2
1.0
OD540 nmWhy use the NO Quantitation Kit?
• Cofactor technology accelerates nitrate reduction• Assay enables high sensitivity in fewer steps• Linear results over a wider range of concentrations

E`kiXk\I\[lZkXj\

Active Motif's NO Kit
LDH method

0.8
0.6
0.4
0.2

• 96-well format enables high-throughput automation

0.0

0 5 10 15 20 25 30 35 40 45 50 55 60

Concentration of Nitrate (µM)

Product

FormatCat. No.

Nitric Oxide Quantitation Kit

2 x 96 rxns

40020

FIGURE 2:
Dynamic range of nitrate standard curves.
Nitrate standard curves produced using the Nitric Oxide Quantitation Kit anda conventional Three-Step LDH-method kit.

For more information on Active Motif’s Nitric Oxide
Quantitation Kit, please visit www.activemotif.com/no.

14

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Gene Silencing
gripNAs™
stronger sequence specificity for superior silencing
Active Motif’s custom gripNAs™* synthesis service provides abetter alternative to more traditional gene inhibition techniques.
gripNA oligonucleotides are a novel form of negatively charged
Peptide Nucleic Acids,1-3 which have a number of properties
that make them ideal for use in gene silencing. These uniquemolecules display superior sequence specificity compared toconventional gene silencing reagents. Plus, gripNAs are resistantto nuclease degradation and delivery is simple with Chariot™ II,a unique formulation of Active Motif’s protein delivery reagent(see page 6).

While gripNAs hybridize tightly to sequences with exact
complementarity, their binding affinity is significantly reduced(or completely eliminated) if there is even a single base-pairmismatch (Table 1). This strong sequence discrimination minimizes non-specific gene interactions, which can greatly improve thequality of your gene silencing experiments by eliminating theoccurrence of unintended phenotypes.4* Covered under U.S. Patent No. 6,962,906. Purchase includes the right to use forbasic research only. Other-use licenses available, please contact Tech Services.

gripNAs advantages
The large number of publications utilizing gene silencing todetermine protein function illustrates the importance of thisstrategy in modern cell biology. Inhibition of gene expression
has primarily been performed using antisense DNA oligos, smallinterfering RNAs (siRNAs) or morpholinos. However, despite theirpotential, the control of gene expression using such methods isoften confounded by non-specific interactions with other genes,5which leads to the occurrence of unintended, mutant phenotypes. gripNAs have been shown to be highly effective in genesilencing experiments in mammalian cells, zebrafish, Xenopus andin in vitro translation systems.

Delivery of inhibitory molecules into the cell is also a key limitation for any gene silencing experiment. Classical delivery mechanisms such as microinjection and DNA/RNA transfection can betime consuming and inefficient. However, their biggest limitationis that they induce cytotoxic responses within the target cells.
Delivery of gripNA oligonucleotides using Chariot II is simple andgives high transfection efficiencies with minimal cytotoxicity.
These characteristics make gripNAs the tool of choice for yourgene silencing studies.

DNA/DNAWhy use gripNAs?

gripNA/DNA• Unsurpassed sequence specificity
eliminates non-specific gene interactions

Tm (°C)DTm (°C)

Tm (°C)DTm (°C)

DNA oligonucleotide sequence

No mismatches

63.9

–

58.6

–

5´-CAC-TGA-CTT-GAG-ACC-A-3´• Resistant to nuclease degradation

Mismatch A

57.0

6.9

41.3

17.3

5´-CAC-TGA-GTT-GAG-ACC-A-3´• Easy delivery with Chariot II (see page 6)

Mismatch B

49.7

14.2

No Tm

–

5´-CAC-TGA-GTG-GAG-ACC-A-3´• Flexible synthesis modifications

Mismatch C

61.8

2.1

54.6

4.0

5´-CAC-TGA-CTT-GAG-ACG-A-3´• Highly soluble

Mismatch D

56.7

7.2

No Tm

–

5´-CGG-TGA-CTT-GAG-ACC-A-3´

Mismatch E

53.6

10.3

No Tm

–

5´-CAC-TGA-CGT-GAG-ACC-A-3´

Mismatch F

56.2

7.7

–

5´-CAC-TGA-CTG-GAG-ACC-A-3´

Complete information online

No Tm

Mismatch G

54.2

9.7

42.4

16.2

5´-CAC-TGA-CAT-GAG-ACC-A-3´

More complete information on gripNAs,
including experimental data, structure
information of the PNAs and ordering
options for gripNAs, is available online atwww.activemotif.com/gripnas.

TA B L E 1 :Stringent binding specificity of gripNAs.
Identical 16-mer gripNA and DNA probes with the sequence 5´-TGG-TCT-CAA-GTC-AGT-G-3´
were synthesized. These were annealed to a complementary DNA oligo (5´-CAC-TGA-CTT-GAGACC-A-3´) in Hybridization Buffer, heated to 90°C for 3 minutes, then cooled gradually to room
temperature. The samples were then heated at a rate of 1°C per minute from 20°C to 100°C using athermal control unit linked to a spectrophotometer. Changes in A260 were recorded and a melting
temperature (Tm) was calculated for each DNA/DNA and gripNA/DNA duplex. The experiment wasthen repeated by hybridizing the gripNA and DNA probes to a series of DNA oligos with one- or
two-base mismatches (shown above as bold and underlined). Tm values were measured for each
DNA/DNA and gripNA/DNA duplex and a DTm was calculated by subtracting the difference in themelting temperatures of the complementary and mismatched probes. Samples that are unable toform a stable duplex generate a “No Tm” value in this assay, so have no DTm value.

REFERENCES

1. Efimov, V.A. et al. (1998) NAR 26: 566-575.
2. van der Laan et al. (1996) Tetrahedron Lett. 37: 7857-7860.
3. Efimov, V.A. et al. (1999) NAR 27: 4416-4426.
4. Urtishak, K.A. et al. (2003) Developmental Dynamics 228: 405-413.
5. Stein, C.A. (1999) Nature Biotech 17: 209.

www.activemotif.com

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15

mRNA Isolation

mTRAP™
isolate more mRNA with gripNAs
mTRAP™ Kits isolate high yields of quality mRNA from mammalian cells, tissue and total RNA. mTRAP Maxi and Midi Kits
optimize isolation from different sample sizes, while mTRAP
Total isolates mRNA from total RNA. All mTRAP Kits utilize
Active Motif’s Poly T gripNA™ Probe*, which has a higher affinity
and specificity for mRNA than oligo dT. The result is more mRNAper sample, with reduced ribosomal RNA and genomic DNA
contamination.

Peptide Nucleic Acids (PNAs) are DNA analogs in which thenucleosides are attached to an N-(2-aminoethyl)glycine backbone instead of to deoxyribose, as in DNA.1-4 However, poor
water solubility and a tendency to self-aggregate have limited
the utility of traditional PNAs. Active Motif has overcome theseshortcomings by developing negatively charged gripNAs.5-7 This
class of PNAs has a number of properties that make them ideal
for mRNA isolation.
* Covered under U.S. Patent No. 6,962,906. Purchase includes the right to use forbasic research only. Other-use licenses available, please contact Tech Services.

1 kb

The mTRAP advantage

Another benefit of gripNAs is that they have a higher affinity
for DNA than for RNA, making possible selective elution of themRNA. While non-complementary nucleic acids are removed
during the wash steps, DNA with homology to the Poly T gripNA
Probe remains bound, even during the 75°C mRNA elution step.
mTRAP Kits utilize this high-temperature elution step to remove
all mRNA from the probe, which greatly increases the yield.
For absolute certainty that no genomic DNA is co-isolated, an
optional DNase step can be performed during isolation because
gripNAs, unlike oligo dT, are resistant to enzymatic degradation.

Q

AM

I

250
mRNA isolated (µg)

gripNAs bind to nucleic acids with high affinity and specificity(see page 15). This enables mTRAP Kits to employ low-salt Lysis,
Wash and Elution Buffers that destabilize mRNA secondary
structures as well as any weak, non-specific interactions that may
have formed between nucleic acids and proteins with the Poly
T gripNA Probe. The result is significantly higher yields of puremRNA with lower levels of rRNA contamination compared tooligo dT-based methods (Figure 1). The higher affinity of Poly T
gripNA Probe also enables isolation of mRNA that have shorter
poly A tails, resulting in a more representative sample. This makes
mTRAP-isolated mRNA ideal for production of cDNA probes for
microarrays, cDNA libraries or in Northern blots.

A

2 x 108 cells
1 x 108 cells

200
150
100
50
0

Company A

Company Q

Active Motif

Company I

FIGURE 1:
Higher yields of pure mRNA.
mRNA was isolated from 1 and 2 x 108 HeLa cells using mTRAP Maxi and threeother suppliers’ kits. Eluted mRNA was quantified by spectrophotometer andplotted. 2 µg of mRNA from each 1 x 108 sample was run on a 0.8% agarose gel.
mTRAP-isolated mRNA shows no genomic DNA and less rRNA.
Lane 1:
Lane 2:
Lane 3:
Lane 4:Lane 5:

1 kb DNA LaddermRNA isolated using Company A’s kit
mRNA isolated using Company Q’s kit
mRNA isolated using mTRAP Maxi Kit
mRNA isolated using Company I’s kit

Why mTRAP and negatively charged gripNAs?

Complete product details are available
online at www.activemotif.com/mtrap.

• Isolates substantially more mRNA than oligo dT methods• Reduces ribosomal RNA and genomic DNA contamination

REFERENCES• Captures mRNA with secondary structure and short tails

1. Nielsen, P.E. et al. (1991) Science 254: 1497-1500.
2. Egholm, M. et al. (1992) J. Am. Chem. Soc. 114: 1895-1897.
3. Harvey, J.C. et al. (1992) Science 258: 1481-1485.
4. Buchardt, O. et al. (1993) Trends Biotechnol. 11: 384-386.
5. Efimov, V.A. et al. (1998) NAR 26: 566-575.
6. van der Laan et al. (1996) Tetrahedron Lett. 37: 7857-7860.
7. Efimov, V.A. et al. (1999) NAR 27: 4416-4426.

16

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• Purifies a more representative mRNA population• Includes TouchDown™ for precipitating small quantities ofnucleic acids

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mRNA Isolation

mTRAP ™ Total• Add Poly T
gripNA Probe• Chaotropic agent• Cells• Detergent
AA• Protease• TissuemTRAP ™

A

AA

A AA
AAA

AAA

AAA

AA

AA

AAA

A

A

AAA

AA

A

AAA

A

AA

• Add Streptavidin
Beads

A

A

AA

• Elute in waterat 75°C• mRNA dissociates
and is collected• Wash Buffer• Transfer to microcentrifuge tubes

Flow chart of the mTRAP procedure.

mTRAP Maxi and mTRAP MidimTRAP Total

The mTRAP Maxi Kit provides all reagents and plasticware
required for 6 large-scale mRNA isolations (Figure 1). Each reaction can isolate mRNA from 0.5-2.0 x 108 cells or 0.4-1 gram oftissue and will yield 100-200 µg mRNA, depending on cell type.

The mTRAP Total Kit was created for single-pass enrichment ofmRNA from total RNA (Figure 2). Reagents and plasticware areincluded for 12 mRNA isolations. Each reaction can isolate mRNA
from up to 500 μg total RNA and will yield 10-15 μg mRNA.

The mTRAP Midi Kit provides all reagents and plasticware
required for 24 medium-sized mRNA isolations. Each reaction
can isolate mRNA from 0.5-1.0 x 107 cells or 50-200 mg tissue andwill yield 10-20 µg mRNA, depending on cell type.

FIGURE 2:
Pure mRNA from total RNA in one passage.
mRNA was isolated from 500 µg of total RNA in duplicate
using mTRAP Total, quantified by spectrophotometry andrun on a 0.8% agarose gel.
Lane 1:
1 kb DNA Ladder
Lanes 2-3: 2 µg mRNA isolated in one pass		with the mTRAP Total Kit.

mTRAP-related Products
Poly T gripNA™ Probe has been optimized to ensure efficient
recovery of mRNA from cell lysates and tissue homogenates. It isavailable in amounts comparable to those provided in the mTRAP
Maxi and mTRAP Midi Kits and is stable for 6 months at -20°C.
Streptavidin Beads are stable, uniform, non-porous spheres
bound with highly active streptavidin. They are RNase- andDNase-free for direct addition to cell or tissue lysates. The
one-micron diameter beads can bind 10 pmol biotinylated
gripNA-analog probe per 1 µl of beads and are supplied at~1% solids in 50 mM Tris, pH 8, 150 mM NaCl, 0.05% sodium
azide and 0.1% stabilizing detergent.
TouchDown™ Precipitation Reagent enables precipitation of any
nucleic acid without the addition of salt, mussel glycogen orother carrier. Precipitation can be carried out at +4°C, eliminating the need to freeze samples at -70°C. TouchDown Precipitation Reagent is ideal for precipitating small quantities of DNA or
mRNA. TouchDown is supplied as a powder that is resuspended
in 20 ml ethanol. Use 3-5 volumes per precipitation. Enoughreagent is provided for 40 x 500 µl precipitations.

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Product

FormatCat. No.

mTRAP™ Maxi

6 rxns5 x 6 rxns

23006
23506

mTRAP™ Midi

24 rxns
5 x 24 rxns

23024
23524

mTRAP™ Total

12 rxns
5 x 12 rxns

23012
23512

Poly T gripNA™ Probe (from mTRAP™ Maxi)

6 rxns29007

Poly T gripNA™ Probe (from mTRAP™ Midi)

24 rxns

29008

Streptavidin Beads

1.6 ml
3 ml

29010
29009

Touchdown™ Precipitation Reagent

400 mg80280

mTRAP™ Lysis Buffer

100 ml29011

Protease

20 mg

29012

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17

Nuclear & Cytoplasmic Extraction
Nuclear Extract Kit
high yields of specifically segregated extracts
The Nuclear Extract Kit is ideal for the preparation of nuclear,whole-cell and cytoplasmic extracts from mammalian cells andtissues. The resultant high-quality extracts may be used with Active Motif’s TransAM™ and DNMT Activity / Inhibition Kits or ingelshift assays, Western blots, DNA footprinting or as a starting
point for transcription factor purification.

The Nuclear Extract Kit eliminates the need to optimize reagentsand ensures consistently high yields. The detailed protocol helps
ensure that your extract is not contaminated with proteins fromother cellular compartments (Figure 1).

The Nuclear Extract Kit advantage
In the Nuclear Extract Kit, cells are collected in ice-cold PBS in
the presence of phosphatase inhibitors to limit further protein
modifications. Next, the cells are resuspended in hypotonic
buffer to swell the cell membrane. Addition of detergent causes
leakage of the cytoplasmic proteins into the supernatant. After
collection of the cytoplasmic fraction, the nuclei are lysed andthe nuclear proteins are solubilized in lysis buffer in the presence of the protease inhibitors. Whole-cell extracts can also be
prepared by collecting the cells in the PBS/phosphatase inhibitors solution and lysing in lysis buffer. Solubilized proteins are
separated from the cell debris by centrifugation. The concentration of protein in the cell extract can then measured by Active
Motif’s ProStain™ Protein Quantification Kit (see page 20) or witha Bradford-based assay.

Why use the Nuclear Extract Kit?
• Specific segregation reduces the risk of inconsistent
results from cross-contamination• Optimized reagents and protocol ensure
reproducible results

FIGURE 1:
Specific extraction of nuclear and cytoplasmic extracts.
Nuclear and cytoplasmic extracts were prepared using the Nuclear Extract Kit
from HeLa samples unstimulated or stimulated with TNF-a for 30 minutes andassayed using the TransAM™ NFkB p50 Kit. Because activated NFkB translocatesto the nucleus, only nuclear extract from stimulated cells should contain activated NFkB.

• Includes phosphatase and protease inhibitors topreserve protein modifications and prevent degradation• Prepare nuclear, cytoplasmic or whole-cell extracts
with one kit• Ability to prepare extracts from both cultured cells
and tissue samples

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Product

FormatCat. No.

Nuclear Extract Kit

100 rxns
400 rxns

40010
40410

ProStain™ Protein Quantification Kit

1000 rxns

15001

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Mitochondrial Separation & Staining
Mitochondrial Fractionation Kit
isolate highly enriched mitochondrial and cytosolic fractions
The Mitochondrial Fractionation Kit isolates highly enrichedmitochondrial and cytosolic fractions from mammalian cell lines,
simplifying the study of protein translocation events that occur
during apoptosis and in many signal transduction pathways. The

procedure is simple to perform and does not require ultracentrifugation or the use of any toxic chemicals. Isolated fractions canbe used to detect the onset of apoptosis in Western blots or inother assays.

Why use the Mitochondrial Fractionation Kit?
• Eliminates cross-contamination of fractions• Quality-controlled reagents ensure reproducibility• No need to optimize your own reagents or protocol

The Mitochondrial Fractionation Kit advantage
The Mitochondrial Fractionation Kit’s high-quality reagents andoptimized protocol eliminate cross-contamination and produce
high yields of properly segregated mitochondrial and cytosolic
fractions (Figure 1). This helps ensure that you get accurate,
reproducible results.
Product

FormatCat. No.

Mitochondrial Fractionation Kit

100 rxns

40015

FIGURE 1:
Location of cytochrome c in HeLa cells.
The Mitochondrial Fractionation Kit was used to isolate cytosolic (Lanes 1 & 2)
and mitochondrial (Lanes 3 & 4) fractions from HeLa cells. Twenty µg of eachfraction was assayed by Western blot using the cytochrome c oxidase IV (COX
IV) antibody at a 1:500 dilution. In non-apoptotic cells, cytochrome c is found in
the mitochondria only. The Western blot results reveal that the Mitochondrial
Fractionation Kit is specific for isolating segregated mitochondrial and cytosolic
fractions and that there is no leakage between the two cellular compartments.

Chromeo™ Live Cell Mitochondrial Staining Kit
Mitochondria play an important role within living cells. They are
involved in the generation of cellular energy, the regulation ofcell growth and differentiation, and also play a critical role in the
induction of the apoptotic process and various signaling cascades.
Diseases of the mitochondria result in underproduction of energy
within the cell, causing cell injury and even cell death.

Active Motif’s Chromeo™ Live Cell Mitochondrial Staining Kit
provides a non-toxic, water-soluble, membrane-permeable dyethat can be used to stain the mitochondria of live or fixed cells.
Because of the dye’s excellent retention within the cell and low
toxicity, the stain is an ideal tool for long-term labeling of cells
and cellular tracking.

Chromeo Live Cell Mitochondrial Stain advantages
The Chromeo™ Mitochondrial Cell Stain is a unique dye that does
not fluoresce until it has covalently reacted with an amino groupin the cell, eliminating background fluorescence. The stain can be
excited between 470 nm and 550 nm, enabling the use of common lasers and light sources. Additionally, the provided Hoechst
Nuclear Stain can serve as a nuclear counter-stain.
Product

FormatCat. No.

Chromeo™ Live Cell Mitochondrial Staining Kit

1 kit

15005

www.activemotif.com

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FIGURE 1:
Staining of living HeLa cells with Chromeo Live Cell Mitochondrial Stain Kit.
The mitochondria (in red) and the nuclei (in blue) of live HeLa cells were stainedusing the Mitochondrial Stain and Hoechst Nuclear Stain, respectively.

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19

Protein & Extract Quantification
ProStain™ Protein Quantification Kit
simple, sensitive fluorescent quantification of cell extracts
ProStain™ simplifies the determination of cell extract protein
concentrations by providing highly sensitive detection reagentsin a convenient and easy-to-use format. The fluorescent dye
in ProStain is not effected by many commonly used buffer
components such as detergent and salt (Figure 1), which can skew
the results of other protein quantification systems. And, thespectral properties of ProStain change greatly after it is bound,
effectively eliminating any background from unbound dye (Table1). These characteristics make ProStain a significant improvement
over other detection systems such as the Bradford or BCA.
Determining the concentration of cell extracts is an essential
step in many cell biology procedures, such as Western blot

and ELISA. Not surprisingly, there are numerous methods used.
Photometric methods have traditionally been based either on
intrinsic absorbance at 280 nm or on reagent-based assays suchas Bradford. However, these classical approaches tend to be time
consuming, have limited sensitivity and may be influenced by
the presence of contaminating substances.
Fluorescent-based detection methods, while generally moresensitive, have also tended to suffer from limitations such as
the requirement for toxic co-factors, high rates of hydrolysis
or unsuitable spectral properties. In comparison, Active Motif’s
ProStain Kit offers significant improvements in sensitivity, assay
robustness and convenience.

The ProStain advantage

Relative fluorescence

80
70
60
50

BSA alone
BSA + 20 mM NaCl
BSA + 2 mM MgCl2
BSA + 10 mM Tris
BSA + 5 mM (NH4)2SO4
BSA + 0.02% SDS

10
3

Free
Conjugated

612 nm503 nm

665 nm
600 nme L/(mol-cm) Quantum Yield
60,00024,000< 1%~50%

TA B L E 1 :Properties of ProStain dye.
The background of ProStain is extremely low because its absorption max shifts
by over 100 nm after the dye is bound, and because bound dye has a 50-foldgreater quantum yield than free dye.

absorb (503 nm)

emit (600 nm)

absorb (612 nm)

1.0

Unbound
Conjugated

0.8
0.6
0.4
0.2

emit (665 nm)

Wavelength (nm)30

2Emission

FIGURE 2:
Absorption and emission spectra of free vs. bound dye.
Normalized absorption and emission spectra of free (solid, purple lines) andconjugated dye (dotted, copper lines) in phosphate buffer of pH 7.2. As free
dye and bound dye absorb at different wavelengths (612 nm vs. 503 nm) and thequantum yield of bound dye is 50-fold greater than that of free dye, backgroundfrom free dye is effectively eliminated.

20

1

Absorption

0
400 450 500 550 600 650 700 750 800

40

0

Dye State

Fluorescence excitation

A disadvantage of many protein quantification methods, suchas the Bradford assay, is that the absorbance spectra of the freeand conjugated forms of the dye partially overlap. This causes
non-linear protein measurement, as the free dye is excited by
the same wavelength of light used to excite the bound dye. In
contrast, the free versus conjugated absorbance maxima of
ProStain dye are separated by > 100 nm (Table 1 and Figure 2).
Nearly all free ProStain dye is hydrolyzed during the conjugation
reaction, which improves the linearity and accuracy of the assay
because excitation of the sample at 488 nm can only excite dyethat is conjugated to protein. The small amount of free dye thatremains is not excited at this wavelength, so background is completely eliminated. Finally, ProStain conjugation is fast and easy;
simply resuspend the dye, add it to the wells of a microplate,then add a serial dilution of the BSA standard, along with yoursample. After just 30 minutes, the fluorescence is read and theprotein concentrations are easily quantified.

4

5

BSA (µg)
FIGURE 1:
Linear quantification in the presence of common buffer components.
Increasing amounts of BSA protein were quantified using the ProStain Protein
Quantification Kit in the presence of a variety of buffer components.

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Product

FormatCat. No.

ProStain™ Protein Quantification Kit

1000 rxns

15001

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Albumin Quantification
Albumin Blue Fluorescent Assay Kit
sensitive, quantitative determination of albumin in biological samples
Active Motif’s Albumin Blue Fluorescent Assay Kit is a quantitative assay designed to measure albumin levels in biologicalsamples including serum and urine. The kit combines unmatchedsensitivity and high specificity with a fast, simple protocol toprovide an effective assay for studying albumin.
Albumin, produced only in the liver, is the major plasma proteinthat circulates in the bloodstream. Albumin plays a significant
role in the maintenance of colloid osmotic pressure and thebinding of long-chain fatty acids, bile acids, bilirubin, haematin,
calcium and magnesium. It also acts as a carrier for nutritional
factors and drugs. Because serum albumin is a reliable prognostic indicator for morbidity and mortality, liver disease, kidneydisease and malnutrition, efficient study methods are in highdemand. The Albumin Blue Fluorescent Assay Kit from Active
Motif provides a simple tool for highly selective and sensitive
albumin detection, and comes complete with everything
required for performing the assay.

The Albumin Blue advantage

Each kit contains enough dye for 250 reactions in 96-well plates,
but the assay can also be easily adapted for use in smaller orlarger formats, such as 384-well plates or cuvettes. The kit also
includes human serum albumin (HSA) that can be used to makestandard curves for use in quantifying the amount of albuminin your samples (Figure 2). Due to the high amino acid homology and tertiary structural similarities of albumin across species,
the Albumin Blue Reagent can be used for detection of albuminfrom various species.

Why use Albumin Blue?
• Quick and easy to use

Bound dyeabsorb (590 nm)
emit (620 nm)

Fluorescence excitation

0.6

0.4
Free dyeabsorb
emit

0.2

0
500

550

600

650

700

750

Wavelength (nm)
FIGURE 1:
Absorption and emission spectra of dye that is free or bound to albumin.
Normalized absorption and emission spectra of free (copper curves) andconjugated dye (purple curves) in the Albumin Blue Assay.
+,'''<d`jj`fe@ek\ej`kpF;-)'ed

Currently, assays suitable for determining low concentrations ofalbumin (< 100 mg/L) are nonspecific, and merely quantify total
protein content. In contrast, Active Motif’s Albumin Assay Kit issensitive, easy to use and specific for albumin. Simply add the
Diluent Buffer and Dye Reagent to the standards and samples,
then read the fluorescence. The intensity of the fluorescentsignal is directly proportional to the albumin concentration of
the sample. And, as albumin-bound dye has a greatly increased
excitation, the background caused by the emission of any free
dye is minimal (Figure 1).

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+''''
*,'''
*''''
),'''
)''''
(,'''
(''''
,'''

• Unmatched precision''• Sensitive detection limit (< 0.5 mg/L in urine
and serum samples)

,'

(''

(,'

)''

),'

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FIGURE 2:
Human Serum Albumin Standard Curve.
A HSA standard curve generated using the Albumin Blue Fluorescent Assay Kit.

• Highly selective – no interference fromother proteins or lipids• High-throughput compatible

Product

FormatCat. No.

Albumin Blue Fluorescent Assay Kit

1 kit

15002

www.activemotif.com

JAPAN +81 (0)3 5225 3638

For more information about the Albumin Blue Fluorescent
Assay, please visit us at www.activemotif.com/albumin.

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21

Live Cell Membrane Staining
LavaCell™
rapidly stain cellular membranes in live or fixed cells
Active Motif’s LavaCell™ is a naturally occurring compound fromthe fungus Epicoccum nigrum with a molecular weight of
410 Daltons. It is a small, non-toxic molecule that readily
diffuses into live or fixed cells and can be used to fluorescently
stain cell membranes and lipophilic organelles within minutes.
Cells require no pre-treatment or permeabilization for staining,and LavaCell does not stain nucleic acids or adversely affect cell
growth. Once inside the cell, LavaCell binds to primary aminegroups instantly to produce bright red fluorescent compounds.

Unbound LavaCell has a weak green fluorescence in water(520 nm), but following the reaction with primary amine groups
on proteins, the fluorescence shifts to red (610 nm). LavaCell canbe excited at 405, 488 and 532 nm, enabling the use of common filters or lasers. Because unbound LavaCell fluoresces at adifferent wavelength than the excitation wavelength, there islittle background and no need to wash after protein binding.
The staining does not self-quench, and can be multiplexed witha range of yellow-, green- and blue-emitting compounds due to
LavaCell’s long Stokes shift of at least 100 nm.

The LavaCell advantage
The LavaCell stain is a unique, non-toxic, cell-permeable dye that
is ideal for studying cell proliferation, cell counting, chemotaxis,
cell morphology or any application that requires cellular imaging.
LavaCell is simple to use; just add it to your cell culture, wait
30 minutes, then analyze. The dye readily diffuses into live or
fixed cells to produce bright fluorescence on the plasma andinternal membranes, such as those of the nucleus, Golgi and ER.
Because the emission of unbound LavaCell is 520 nm while thatof bound LavaCell is 610 nm, and because LavaCell does noteffect cell growth, there is no need to wash away unbound dye.

FIGURE 1:
LavaCell stains the plasma membrane and internal membranes of CHO cells.

Why stain with LavaCell?
• Water soluble fluorescent cell stain• Fluoresces only on interaction with proteins;no need to wash away unbound stain• Readily diffuses into live or fixed cells• No effect on cell growth or viability• 405, 488 and 532 nm excitable• Non-fluorescent prior to cellular internalization• Ideal for multiplexing with other fluorophores
due to its long Stokes shift
FIGURE 2:
Live mouse macrophage cells stained with LavaCell, calcein AM and Hoechst.

Product

FormatCat. No.

LavaCell™

200 µg15004

To learn more, please visit us at www.activemotif.com/lavacell.

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Cell Viability Assay
ToxCount™
convenient, accurate cell viability assay
Active Motif’s ToxCount™ Cell Viability Assay is a simple twocolor fluorescence cell viability assay that is easy to use, andrequires only 30 minutes of hands-on time. The ToxCount assay
is based on the simultaneous determination of live and deadcells using two probes, calcein AM and ethidium homodimer(EthD-1). These probes recognize parameters of cell viability
including intracellular esterase activity and plasma membrane
integrity, respectively.
Calcein AM is a non-fluorescent cell permeable dye that converts to green fluorescent calcein in live cells after acetoxymethyl ester hydrolysis by intracellular esterases. Ethidium

homodimer (EthD-1) is a red fluorescent nuclear and chromosome counterstain. EthD-1 enters cells with damaged membranes and binds to nucleic acids, where it emits a bright red
fluorescent signal. Because both dyes used in ToxCount are notfluorescent before interacting with cells, background fluorescence levels are very low. ToxCount can be used on multiple
fluorescence detection platforms, including multi-well platescanning, fluorescence microscopy or flow cytometry. The assay
is suitable for use with many adherent cell lines, primary cell
types and small organisms. It is less expensive, safer and fasterthan traditional cell viability assays such as 51Cr-release, LDH
release and trypan blue exclusion.

The ToxCount advantage:XcZ\`e8D

Traditional methods for measuring cell viability can frequently
be time-consuming and require the use of radioactive or toxic
reagents. In contrast, the ToxCount method is safe and easy to
perform. Simply grow cells directly on a microtitre plate andtreat with the drug of interest. After a 30-minute incubation,
cells are ready for imaging and analysis (Figure 1). As the dyes are
non-fluorescent in solution, they can be used in homogenous
protocols, making ToxCount ideal for the rapid screening ofdrug and small molecule libraries for cytotoxic effects.

<k_;$(

C`m\

C`m\

;\X[

;\X[

9i`^_k=`\c[

FIGURE 1:
ToxCount staining using a fluorescence microscope.
CHO cells were treated with calcein AM (0.5 mM) and EthD-1 (0.5 mM), thenimaged using a fluorescence microscope. Untreated, live cells are shown in theupper images while dead cells that had been treated with 70% methanol are
shown in the bottom series of images. Images were acquired for 1 second in thecase of calcein AM and for 2 seconds in the case of EthD-1.

Why use ToxCount?
• Dependable, reproducible assay• Quick and easy protocol• Non-toxic reagents• High-throughput compatible

Product

FormatCat. No.

ToxCount™

20 x 96 rxns

18010

For more information about Active Motif’s ToxCount Assay,
please visit us at www.activemotif.com/toxcount.

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23

Competent E. coli
RapidTrans™
efficient, affordable competent E. coli in a convenient format
RapidTrans™ are high-efficiency competent E. coli supplied in aconvenient 96-tube tray. Ideal for cloning, plasmid preparationand library construction, RapidTrans cells are affordably priced
and packaged in a format that provides maximum flexibility
while eliminating waste. Each tube contains 50 µl of cells for one
transformation reaction. Unlike other 96-well formats that require

thawing all 96 tubes at one time, RapidTrans enables the use of as
few or as many reactions as needed, without thawing the othercells. This eliminates the reduced efficiencies and waste causedby repeated freeze/thaw cycles. For your convenience, the cells
are provided with SOC media, supercoiled pUC19 DNA and sterile
reservoirs for use with multi-channel pipettors.

The RapidTrans Kit advantage• Consistent high-quality, high-efficiency E. coli
• Freedom to use anywhere from 1-96 reactions eliminates
wasted reactions• Efficiency of > 1 x 108 cfu/µg• Economical pricing• Adaptable to high-throughput use

Strain and genotype information
TAM1 E. coli have been genetically modified for use in cloning,
plasmid preparation and library construction. The transformation
efficiency is > 1 x 108 cfu/µg supercoiled pUC19 DNA.
TAM1:

mcrA ∆(mrr-hsdRMS-mcrBC) Φ80lacZ∆M15 ∆lacX74 recA1
araD139 ∆(ara-leu)7697 galU galK rpsL endA1 nupG

Product

FormatCat. No.

RapidTrans™ TAM1 Competent E. coli

1 x 96 rxns
5 x 96 rxns

11096
11596

Please visit us at www.activemotif.com/rapidtrans to learn more.

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Fluorescent Dyes, Antibodies & Labeling
Fluorescent Dyes & Conjugated Secondary Antibodies
easy-to-use, effective fluorescent labels for bioanalysis
The Chromeo™ product line is a complete toolset for fluorescence applications like fluorescence microscopy or fluorescentplate-based assays. Providing sensitivity and flexibility, Active
Motif Chromeon dyes and reagents exhibit superior luminescence properties, including a broad range of fluorescence excitations and emissions, large Stokes shifts, limited photobleaching
and a broad pH tolerance. We identified the need for improved

Conjugated Secondary Antibodies
For the utmost in convenience, our dyes are all available alreadyconjugated to high-quality goat anti-mouse and goat anti-rabbit
secondary antibodies. Because of the wide range of dyes offered(see Table 1), you can use multiple secondaries at the same time
for multi-color staining (Figure 1). HRP conjugates are also available. For details, please visit www.activemotif.com/secondary.

Chromeo™ Dyes
Active Motif’s Chromeo™ Dyes are also offered ready to use tocovalently label biomolecules. For added flexibility, the dyes areoffered in 6 different formats: as amine-reactive NHS-esters,
which are useful to label amino groups on peptides, proteinsor amino-modified DNA; as azides and alkynes for use in click
chemistry; as carboxylic acids, and as biotin and streptavidin
conjugates. For details, please visit www.activemotif.com/dyes.

Chromeo™ Py-Dyes
Py-Dyes are a set of 5 fluorescent chameleon-type dyes thatare ideal for labeling proteins; they undergo a large shortwave
spectral shift, as well as a large increase in quantum yield, uponreacting with primary amines. This eliminates background fromany unbound dye, so there is no need for purification following
the simple one-step, room-temperature labeling reaction. For
more information, please visit www.activemotif.com/pydye.
Histone H2AX phospho Ser139 pAb+
Chromeo™ 642 anti-rabbit 2°alpha Tubulin mAb+
Chromeo™ 488 anti-mouse 2°

fluorescence-based assays and developed some unique tools like
Py-Dyes, which are chameleon-type pyrylium dyes that change
their color upon binding to proteins.
The fluorescent dye products shown below are but a small subset of those offered by Active Motif. For more comprehensive
information, please visit www.activemotif.com/fluor.

Get 12-fold Greater Resolution Using STEDThe Abbe Law of Diffraction Limiting Resolution restricts
the ability of classical confocal microscopy to visually resolve
objects separated by less than ~200 nm. Leica Microsystems’
STimulated Emission Depletion (STED) microscopy, however,improves resolution by up to 12-fold, enabling separation of
sub-cellular structures that previously could not be resolved.
Leica recommends Active Motif’s Chromeo™ 488 and 494
dyes and secondary antibody conjugates, and its fluorescent
ATTO (STED) secondary antibody conjugates for use in STED.
For more details, please visit www.activemotif.com/sted.
Dyee L/(mol-cm) Stokes Shift

Absorption

Emission

Chromeo™ 488

488 nm

517 nm73,00029 nm

Chromeo™ 494

494 nm

628 nm55,000134 nmChromeo™ 505

505 nm

628 nm70,00021 nm

Chromeo™ 546

545 nm561 nm

98,80016 nm

Chromeo™ 642

642 nm

660 nm180,000

18 nm

ATTO 594

601 nm

627 nm

120,000

26 nm

ATTO 647N (STED)

644 nm

669 nm150,000

25 nm

ATTO 655 (STED)

663 nm

684 nm

125,000

21 nm

TA B L E 1 :Fluorescent properties of dyes when conjugated to secondary antibodies.

DAPI Nuclear Stain

All 3 images merged

FIGURE 1:
Concurrent staining using Chromeo 488 and Chromeo 642 fluorescent secondaries.
HeLa cells were treated with 100 µM etoposide for 6 hours, prior to fixation with methanol. The histone variant H2AX was then stained with
Histone H2AX phospho Ser139 rabbit pAb (Cat. No. 39117) and Chromeo 642 goat anti-rabbit IgG (Cat. No. 15044), while tubulin was visualizedusing alpha Tubulin mouse mAb (Cat. No. 39527) and Chromeo 488 goat anti-mouse IgG (Cat. No. 15031). The nuclei were stained using DAPI andthe three separate images were merged.

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U.S.
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008 USA
Toll Free: 1 877 222 9543
Direct: 1 760 431 1263
Fax: 1 760 431 1351
Email: sales@activemotif.com
EUROPE
104 Avenue Franklin Roosevelt – Box 25
B-1330 Rixensart, Belgium
Germany Free Phone: 0800/181 99 10
France Free Phone: 0800/90 99 79
UK Free Phone: 0800/169 31 47
Other countries, Direct: +32 (0)2 653 0001
Fax: +32 (0)2 653 0050
Email: eurotech@activemotif.com
JAPAN
Azuma Bldg. 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
Email: japantech@activemotif.com