THE NEWSLETTER OF ACTIVE MOTIF — September 2006 • volume 7 • number 3

Speciﬁc, Flexible Labeling of Proteins
Active Motif’s LigandLink™* Universal Labeling technology is an innovative tool
for the speciﬁc labeling of proteins in living cells. With LigandLink, it is now
possible to create a single fusion protein that can be labeled with a variety oftags, making it ideal for use in FRET and localization studies.
Improved localization studies
The ability to label proteins in living cells
is key to understanding the dynamics
and functions of proteins. Classically,
these studies have been performed byfusing a gene of interest to a ﬂuorescentprotein, such as green ﬂuorescent pro-

INSIDE THIS ISSUE
Simple, Efﬁcient Study
2 New:of Protein SUMOylation
3

New: FACE™ – Cell-based
ELISAs for EGFR and HSP27

4Complete ChIP-IT™ Kits
Simplify Chromatin
Immunoprecipitation

5

Sensitive, Speciﬁc Transcription
Factor ELISAs

However, protein function is often regulated via protein-protein interactions,7

New: Improved FluorescentLabels for Bioanalysis

continued on page 6

8

New: Accurate Cell-by-Cell
Fluorescent Viability Assaytein (GFP), and transfecting the construct
into cells. These approaches have signiﬁcantly expanded our understanding ofprotein function within the cell.

MethylDetector™ – Efﬁcient DNA Methylation Analysis
Active Motif’s MethylDetector™ Bisulﬁte Modiﬁcation Kit makes DNA
methylation analysis fast and efﬁcient by combining optimized reagents for
performing DNA conversion with time-saving DNA puriﬁcation columns,
along with positive control PCR primers that help you validate your results.
Proven controls ensure success
DNA methylation analysis typicallyinvolves treating DNA with bisulﬁte. This
converts unmethylated cytosines to uracils while leaving methylated cytosines
unchanged. The DNA is then ampliﬁed
by PCR and analyzed by sequencing or
restriction digest. However, because
bisulﬁte conversion can be technicallychallenging, MethylDetector provides
positive control PCR primers that are
speciﬁc for bisulﬁte-converted DNA.
This enables you to conﬁrm that theconversion step was successful before
beginning costly sample analysis, saving
you time, effort and materials (Figure 1).

www.activemotif.com

Advantages• Flexible – use high G/C content
sequences and uncut DNA• Reproducible – 99% conversion
efﬁciency• Efﬁcient – reagents and protocol
with proven controls• Easy – DNA puriﬁcation columns
mean no more precipitations
Try MethylDetector today
MethylDetector will speed and simplify
your methylation analysis. Learn more atwww.activemotif.com.

M

1

2

3

4

5

500 bp400 bp300 bp200 bp100 bpFigure 1: Agarose gel analysis of PCR products generated
with MethylDetector.
Three different DNA conversions were performed(Lanes 1-3) and compared to an unconverted DNA
control (Lane 5) and to a no DNA control (Lane 4). The
presence of PCR product in only the converted samples
demonstrates the conversion efficiency and reproducibility of the MethylDetector Kit.

Product

FormatCatalog No.

MethylDetector™

50 rxns

55001

1SUMOylation

New: Simple, Efﬁcient Study of Protein SUMOylation
The post-translational modiﬁcation known as SUMOylation has been shown
to be a factor in an increasing number of biological processes. In response,
Active Motif developed its SUMOLink™ Kits to ﬁll the need for fast, efﬁcientand robust assays to detect and generate SUMOylated proteins in vitro.
SUMOylation is now widely studied
because it orchestrates a diverse arrayof effects on many different biologicalprocesses including chromosomal organization and function, DNA repair, nuclear
transport and signal transduction.
SUMOLink Kits provide a simple, yeteffective method for performing in vitro
SUMO (small ubiquitin-like modiﬁer)
conjugation and detecting SUMOylated
proteins. With SUMOLink, all required
reagents are provided, including positive
control p53 protein and antibody, which
help to ensure your success.

“The positive control protein andantibody supplied in SUMOLink Kits
help ensure the accuracy of yourin vitro SUMOylation assays.”or SUMO-2/3 are provided so you canmonitor the in vitro SUMOylation ofyour protein of interest compared to thepositive control p53 protein (Figure 1).
SUMOLink applications• Investigating SUMOylation’s effects
on transcription factor activity• Understanding the role of
SUMOylation in the regulation ofcellular processes• Identifying novel proteins that are
targets for SUMO proteins
MW

1

SUMO-12
3

1SUMO-1
2
3

98

98

64

64

50

50

36

36

30

2

Choose from SUMO-1 or SUMO-2/3
SUMOLink Kits are available for either
SUMO-1 detection/conjugation or
SUMO-2/3 detection/conjugation. For
complete information, visit our website
at www.activemotif.com/sumolink.

1

SUMO-22
3

1SUMO-32
31

SUMO-22
3

1SUMO-32
3

30

16

The SUMOLink method
SUMOLink Kits are very easy to use.
Simply add the assay components to a
microcentrifuge tube with your protein
of interest and incubate for 3 hours. To
aid in result analysis, SUMOLink Kits contain both wild-type and mutated SUMO
proteins. After incubation, the wild-type
and mutated reactions are stopped andthe samples analyzed by Western blot.
Antibodies to p53 and either SUMO-1

MW

SUMOLink advantages• Simple, effective method• Positive control p53 protein andantibody to ensure success• Wild-type and mutated SUMO
proteins are provided• Versatile – works with recombinant
proteins and cell extracts• All included antibodies react withhuman, mouse and rat samples

16A

p53 antibody

SUMO-1 antibody

B

p53 antibody

SUMO-2/3 antibody

Figure 1: Speciﬁc protein labeling using SUMOLink.
Western blot analysis of in vitro SUMOylation of p53 protein by wild-type and mutated isoforms of SUMO-1, SUMO-2 and
SUMO-3 proteins. A. SUMO-1 analysis: Western blots incubated with p53 antibody (1:5000 dilution) and SUMO-1 antibody(1 :4000 dilution); B. SUMO-2/3 analysis: Western blots incubated with p53 antibody (1:5000 dilution) and SUMO-2/3
antibody (1:4000 dilution).
Lane 1: Wild-type SUMO protein conjugation reaction.
Lane 2: Mutated SUMO protein conjugation reaction.
Lane 3: No p53 control protein used in the conjugation reaction.

Product

FormatCatalog No.

SUMOLink™ SUMO-1 Kit

20 rxns

40120

SUMOLink™ SUMO-2/3 Kit

20 rxns

40220

Toll Free — 1 877 222 9543

September 2006 • volume 7 • number 3

Protein Phosphorylation

New: FACE™ – Cell-based ELISAs for EGFR and HSP27
To help scientists keep pace with the ever-changing face of signal transduction
pathways, Active Motif continues its development of fast, efﬁcient and robust
assays to measure protein phosphorylation with further additions to the Fast
Activated Cell-Based ELISA (FACE™) Kit family of products.

The FACE method
In FACE, cells are cultured as desired in
96-well plates and stimulated to induce
the pathway of interest. Following stimulation, the cells are rapidly ﬁxed, which
preserves activation-speciﬁc protein
modiﬁcations. Each well is then incubated with a primary antibody speciﬁc
for the protein modiﬁcation of interest.
Subsequent incubation with secondary
HRP-conjugated antibody and developing solution provides a colorimetricor chemiluminescent readout that isquantitative and reproducible. FACE Kits
also contain a primary antibody for thenative non-modiﬁed protein, so that you
can monitor both native and activatedprotein levels in the same experiment(Figure 1).
Reproducible, scaleable assay
Typically, phospho-speciﬁc analyses
are performed on a small number ofsamples because classical methods, suchas Western blot and in-gel kinase assays,
are labor intensive and time consuming. This forces you to run multiple

www.activemotif.com

experiments on different days and thencompare the results. However, as thevariability of these methods is high andthe induction of phosphorylation is low,
this can lead to statistically poor data.
Fortunately, FACE Kits make runningmore samples as simple as adding an
extra tip to your multi-channel pipettor.
And, because FACE is highly reproducible,
you’ll get statistically relevant results.
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FACE Kits provide a simple, efﬁcient,cell-based method to monitor proteins
activated by phosphorylation. FACEELISAs are 96-well, high-throughput
assays that do not require cell extracts,
electrophoresis or membrane blotting.
Unlike Western blotting, FACE enables
modiﬁcation-speciﬁc analysis inside thecell. This eliminates the need to perform cumbersome, low-throughput cell
extractions, saving you time and money.

FACE advantages• Cell-based method – no need forextracts, gels or membrane blotting• Fast – requires less than 2 hours ofhands on time
• Flexible – use suspension or
adherent cells• Total & phospho antibodies –compare both phosphorylated andnative protein levels in the same kit• Quantitative results• Versatile – adaptable for single
reactions and high-throughput
automation
Latest additions to the FACE line
FACE Kits are now available for monitoring
activation of phosphorylation on Tyr845
of EGFR as well as for the detection ofphosphorylation of Ser82 on HSP27.

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Figure 1: Monitoring total- and phospho-HSP27 using FACE.
The FACE HSP27 Kit was used to monitor total andphosphorylated HSP27 (S82) contained within untreatedor Anisomycin-treated (25 µg/ml for 30 min) HeLa cells.

A variety of kits to choose from
FACE Kits are available for many kinases
and receptors, as indicated in the tablebelow. All kits are conveniently provided
in both colorimetric and ultra-sensitive
chemiluminescent formats. For more
information about the FACE Kits, please
visit www.activemotif.com/face.

FACE™ Product Line
FACE™ AKT (S473)

FACE™ EGFR (Y992)

FACE™ GSK3β (S9)

FACE™ PI3 Kinase p85

FACE™ ATF-2 (T71)

FACE™ EGFR (Y1173)

FACE™ HSP27 (S82)

FACE™ STAT2 (Y689)

FACE™ Bad (S112)

FACE™ ErbB-2 (Y877)

FACE™ JAK1 (Y1022/Y1023) FACE™ STAT4 (Y693)

FACE™ c-Jun (S63)

FACE™ ErbB-2 (Y1248)

FACE™ JNK (T183/Y185)

FACE™ c-Jun (S73)

FACE™ ERK1/2 (T202/
Y204 & T185/Y187)

FACE™ MEK1/2 (S217/S221)

FACE™ c-Src (Y418)

FACE™ FAK (Y397)

FACE™ NFκB p65 Proﬁler(S468 & S536)

FACE™ EGFR (Y845)

FACE™ FKHR (T24)

FACE™ p38 (T180/Y182)

FACE™ STAT6 (Y641)

3

Chromatin Immunoprecipitation

Complete ChIP-IT™ Kits Simplify Chromatin Immunoprecipitation
Chromatin immunoprecipitation (ChIP) is a powerful, yet technically challenging
tool used to study protein/DNA interactions. Active Motif’s ChIP-IT™ Kits makeyour ChIP more successful by combining nearly everything you need in a single kit,including positive control antibodies and primers, DNA puriﬁcation columns and acomprehensive protocol.

Coming soon – ChIP-IT Express Kits
Our soon-to-be released ChIP-IT Express
Kits streamline the ChIP procedure, eveneliminating DNA puriﬁcation, making it
possible to perform ChIP in just 1 day. Callus or visit our website to ﬁnd out more.

The ChIP method
In ChIP, intact cells are ﬁxed using
formaldehyde, which cross-links andpreserves protein/DNA interactions. The
DNA is then sheared into small, uniform
fragments. The protein/DNA complexes
are then immunoprecipitated using
antibodies directed against the DNAbinding protein(s) of interest. Following
immunoprecipitation, the cross-links arereversed and DNA fragments are puriﬁed
and screened to determine which gene,or group of genes, was bound by theprotein of interest (Figure 1).

Start your ChIP today
Active Motif’s ChIP-IT Kits provide youwith everything you need to make yourChIP experiments easier and more reproducible. For more information on ChIP-IT
visit www.activemotif.com/chip.

ChIP-IT advantages• Easy to use – all critical buffers,
Protein G beads and DNA puriﬁcation columns are provided• No need to optimize reagents andprotocol• Your choice of enzymatic orsonication shearing• Direct measurement of transcription
factor/DNA interactions or histone
modiﬁcations
Choose enzymatic or sonication shearing
The key to a successful ChIP experiment
begins with correctly shearing the DNA
into 200-1000 bp fragments. This can beachieved by using one of two methods: enzymatic digestion or sonication.
Sonication shearing is an effective
method for shearing DNA but can be
difﬁcult to optimize due to complications from overheating, emulsiﬁcationand dependence on sonicator type. In
contrast, enzymatic digestion can beused to quickly and easily digest DNA

4

into fragments suitable for ChIP. And, as
digestion is dependent only on time andtemperature, very little optimization isrequired. For your convenience, ChIP-IT
Kits are available with your choice ofeither sonication or enzymatic shearing.

“ChIP-IT makes performing ChIP with transcription factors more successful by
combining all of the critical components you need in a single kit.”
Cross-link protein to DNA inliving cells with formaldehyde

Break open cells
and shear DNA

Add primary
antibody of interest

Analyze purified DNA
using PCR or
hybridization methods

Reverse protein/DNA
cross-links, treat withProteinase K, and purify DNA

Collect antibody-boundprotein/DNA complexes using
Protein G agarose beads

Figure 1: Flow chart of chromatin immunoprecipitation.
In ChIP, protein/DNA interactions are fixed, and the DNA is sheared and precipitated using an antibody. After cross-linkreversal, the DNA is purified and screened to determine which genes were bound by the protein of interest.

Product

FormatCatalog No.

ChIP-IT™

25 rxns

53001

ChIP-IT™ w/o controls

25 rxns

53004

ChIP-IT™ Shearing Kit (included in 53001 & 53004)

10 rxns

53002

ChIP-IT™ Enzymatic

25 rxns

53006

ChIP-IT™ Enzymatic w/o controls

25 rxns

53007

Enzymatic Shearing Kit (included in 53006 & 53007)

10 rxns

53005

Toll Free — 1 877 222 9543

September 2006 • volume 7 • number 3

Transcription Factor ELISAs

Sensitive, Speciﬁc Transcription Factor ELISAs
Active Motif’s TransAM™ Kits simplify the study of activated transcription
factors by combining high sensitivity with convenience in a non-radioactive,
ELISA-based assay.
Simple, quantitative assay
In contrast, Active Motif’s TransAM Kits
make it straightforward to measure theactivity of transcription factors. You
can even study multiple members ofa transcription factor family in a single
experiment that is complete in less than
5 hours. In addition to being fast, theTransAM method is non-radioactive andprovides quantitative results (Figure 1).
Plus, TransAM Kits can be used on all
sample types, including cell lines and tissues, giving you unsurpassed ﬂexibility.

Traditionally, transcription factor activity
has been studied using either Electrophoretic Mobility Shift Assays (EMSA),
immunoblotting or reporter gene assays.
However, these methods are quite
time-consuming and, at best, provide
only semi-quantitative results. Moreover,use of these methods to determine theactivity of multiple transcription factor
family members would require the investment of signiﬁcant amounts of bothtime and money; in many cases, it may
not even be possible.

NFκB activation (OD450 nm)“TransAM Kits are sensitive, non-radioactive DNA-binding ELISAs that facilitate
the study of transcription factor activation in cell and tissue samples.”A

2.5

IL-1α stimulated

Unstimulated
IL-1α stimulated

2

0.5

1

5

10

Unstimulated

25

50

0.5

1

5

10

25

50

1.5
1

Flexi or Original format
The Original TransAM Kits offer a fast,
non-radioactive alternative to gelshiftby providing a 96-well plate that isprecoated with oligonucleotide that
contains a consensus-binding site for thefactor of interest. Activated transcription factor binds the oligo and is quantiﬁed on a spectrophotometer using the
supplied antibodies and developing
reagent. This format is convenient formeasuring binding at the consensus
site, but does not enable you to study
alternative sites. For this reason, we developed TransAM Flexi Kits; they containall of the optimized reagents provided
in the Original kits, including antibodies,but give you the ﬂexibility to immobilize
any oligo in the 96-well plate.
TransAM advantages• Non-radioactive, colorimetric method provides quantiﬁable results• Results in less than 5 hours• 10-fold greater sensitivity thangelshift (100-fold with Chemi Kits)• Simultaneous proﬁling of multiple
family members• Assay both cell and tissue samples

0.5
0

0.5

1

2
5
10 25
Cell extract/well (µg)

50B

µg of cell extract

Figure 1: TransAM NFκB is more sensitive than gelshift, and provides more quantitative results.
Human fibroblast WI-38 cells are stimulated with IL-1α for 30 minutes. Increasing amounts of whole-cell extract are assayedusing either the TransAM NFκB p50 Kit (A) or gel retardation (B).

Try sensitive, quantitative ELISAs
TransAM Kits make it simple to quantify
activated transcription factors. Please
give us a call or visit our website to learn
more about the TransAM product line.

TransAM™ Product Line
TransAM™ AP-1 Family

TransAM™ AML-3/Runx2

TransAM™ CREB & pCREB

TransAM™ MEF2

TransAM™ Oct-4

TransAM™ GATA Family

TransAM™ AP-1 c-Fos

TransAM™ Elk-1

TransAM™ MyoD

TransAM™ p53

TransAM™ HNF Family

TransAM™ AP-1 c-Jun

TransAM™ ER

TransAM™ NF-YA

TransAM™ PPARγTransAM™ MAPK Family

TransAM™ AP-1 FosB

TransAM™ FKHR (FOXO1)TransAM™ NFATc1

TransAM™ Sp1 & Sp1/Sp3

TransAM™ Flexi NFκB Family

TransAM™ AP-1 JunD

TransAM™ GATA-4

TransAM™ Flexi NFκB p50

TransAM™ STAT3

TransAM™ NFκB Family

TransAM™ ATF-2

TransAM™ GR

TransAM™ NFκB p50*

TransAM™ STAT Family

TransAM™ c-Myc

TransAM™ HIF-1

TransAM™ Flexi NFκB p65

TransAM™ AML-1/Runx1

TransAM™ C/EBP α/βTransAM™ HNF-1

TransAM™ NFκB p65*

* The Original TransAM NFκB p50 & p65 Kits are offered in both colorimetric and chemiluminescent formats. TransAM Chemi Kits require the use of a luminometer.

www.activemotif.com

5

Protein Labeling & Translocation
continued from page 1 — Speciﬁc, Flexible Labeling of Proteins
Pre-made translocation vectors
The LigandLink Universal Labeling technology is ideal for studying translocation
events because of the highly permeable
nature of the TMP ligand and the simple
way in which proteins are labeled in invivo environments (Figure 2). To make iteven easier, we have already prepareda line of pre-made vectors containing
transcription factors such as NFκB, STAT
and p53 that are ready to transfect into
the mammalian cell line of your choice.

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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. Aftertransfection into cells, the protein of interest is expressed as a fusion to eDHFR. Addition of cell-permeable LigandLink Labelto the medium results in rapid, specific binding of the label’s trimethoprim (TMP) ligand by eDHFR on the fusion protein.

creating the need for multiple labelingsystems. Using conventional approaches,
you would need to clone a series of
constructs with your gene of interest
fused to different ﬂuorescent proteins.
This has made such studies time-consuming and labor intensive. In contrast,
LigandLink makes it possible to create asingle clone that can be labeled with different tags. This makes LigandLink ideal
for FRET and localization, where multiple
labeling colors are required.
Why use LigandLink?
• Spend less time on cloning andclone validation – clone only once,
then obtain different functionalities
by using different LigandLink Labels• Small label – eDHFR is much smaller
than GFP, so is unlikely to interferewith protein functions/interactions• High speciﬁcity – TMP is an antibiotic designed to bind eDHFR, but notinteract with mammalian proteins• Superior ﬂuorescence – syntheticdyes have improved spectral properties vs. naturally ﬂuorescent proteins• Label your protein in living cells –LigandLink Labels are cell permeable
The LigandLink method
In the LigandLink method, the geneof interest is cloned in frame with thegene for E. coli dihydrofolate reductase

6(eDHFR) in the pLL-1 vector. This vector
has a CMV promoter for high-level
expression of eDHFR fusion proteins,with Neomycin for selection of stable
cell lines. The vector is transfected into
mammalian cells and used to express
the fusion protein. Twenty-four hours
post-transfection, the protein of interest
can be labeled simply by adding theLigandLink Label of choice to the cell
medium (Figure 1). Depending on thecell type and the label used, cells can beimaged in as little as 10 minutes.

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Try LigandLink today
LigandLink will improve the way youlabel proteins in living cells. The highly
speciﬁc and stable LigandLink tags are
ideal for use in FRET and localization
research because once a protein of interest is expressed as a LigandLink fusion,
you can easily change the properties of
its tag by adding a different LigandLinklabel to the medium. Find out more
about the new LigandLink system byvisiting www.activemotif.com.

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Figure 2: Labeling of NFκB p65 by LigandLink Fluorescein.
U2OS cells were transfected with the pLL-1-NFκB p65 vector. Two days later, the cells were stimulated by TPA (50 ng/ml)
and CI (0.5 µM) and labeled by addition of LigandLink Fluorescein Label to the media. Prior to stimulation, NFκB p65 is
located in the cytoplasm of the cell. Upon activation, the p65 protein translocates to the nucleus.

Product

FormatCatalog No.

LigandLink™ pLL-1 Kit

1 kit

34001

LigandLink™ pLL-1-AKT1 Kit

1 kit

34002

LigandLink™ pLL-1-AKT2 Kit

1 kit

34003

LigandLink™ pLL-1-NFκB 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* Patent pending.

Toll Free — 1 877 222 9543

September 2006 • volume 7 • number 3

Fluorescent Detection

New: Improved Fluorescent Labels for Bioanalysis
Active Motif’s Chromeo™ Dyes provide you with an improved tool for
ﬂuorescently labeling biomolecules. Their unique spectral properties include
excellent photostability, pH tolerance and low cell toxicity, making them
ideal for any application.
Excellent ﬂuorescent properties
The Chromeo Dyes – Chromeo 494,
Chromeo 546 and Chromeo 642 – exhibit
many key features including superior
luminescence properties and broad
Stokes shifts (Table 1). Chromeo Dyes arealso suitable for use with many common
excitation sources such as diode lasers,
LEDs, tungsten and Xenon arc lamps.

Visit the Active Motif Chromeon website
As a leader in providing ﬂuorescent tools
for bioanalysis, Active Motif Chromeon
also offers several other dyes to meetyour research needs, including RuLabels

Multiple applications
The Chromeo Dyes are available in several formats including as reactive NHSesters that can be used to label aminogroups such as antibodies (Figure 1). They
are also available as carboxylic acids andas biotin and streptavidin conjugates. In
addition, Chromeo 494 has a very large
Stokes shift of 124 nm, making it an ideal
partner for multiplexing with other,
shortshifted 488-excitable dyes.

Figure 1: Chromeo 546 conjugated secondary antibody.
Chromeo 546 was conjugated to anti-rabbit secondary
antibody and used to label tubulin in U2OS cells.

Advantages• Convenient spectral properties• High quantum yields• pH insensitive• Excellent photostability• Available as reactive NHS-Esters,carboxylic acid, biotin or
streptavidin conjugateswww.activemotif.com

and Py-Dyes. RuLabels are phosphorescent ruthenium probes that emit strong
polarized light with a long decay time,
which enhances sensitivity and reduces
background. The Py-Dyes possess unique
physical and spectral properties thatmake conjugation simple, while greatly
reducing background. To ﬁnd out more
about Active Motif Chromeon products
visit www.chromeon.com.

Absorption

Emissionε L/(molcm)

Quantum
Yield (%)*

Stokes
Shift

Chromeo™ 494

494

628

20,00025

124 nmChromeo™ 546

545

561

96,8001016 nm

Chromeo™ 642

642

660

180,000

15

18 nm

Dye

Table 1: Chromeo Dye properties. (* when conjugated to BSA)

Product

FormatCatalog No.

Chromeo™ 494 Carboxylic Acid

1 mg
5 mg

15110
16110

Chromeo™ 494 NHS-Ester

1 mg
5 mg

15111
16111

Chromeo™ 494 Biotin

1 mg
5 mg

15112
16112

Chromeo™ 494 Streptavidin

1 mg
5 mg

15113
16113

Chromeo™ 546 Carboxylic Acid

1 mg
5 mg

15210
16210

Chromeo™ 546 NHS-Ester

1 mg
5 mg

15211
16211

Chromeo™ 546 Biotin

1 mg
5 mg

15212
16212

Chromeo™ 546 Streptavidin

1 mg
5 mg

15213
16213

Chromeo™ 642 Carboxylic Acid

1 mg
5 mg

15310
16310

Chromeo™ 642 NHS-Ester

1 mg
5 mg

15311
16311

Chromeo™ 642 Biotin

1 mg
5 mg

15312
16312

Chromeo™ 642 Streptavidin

1 mg
5 mg

15313
16313

7

Fluorescent Detection

New: Accurate Cell-by-Cell Fluorescent Viability Assay��������������������������

The ToxCount™ Cell Viability Assay provides reagents for a simple two-color
ﬂuorescent cell viability assay. Calcein AM is used to detect live cells, while
ethidium homodimer (EthD-1) detects dead cells within a population.

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The ToxCount assay is ideal for the rapid
screening of drug and small molecule
libraries for cytotoxic effects. ToxCount
is particularly applicable to high-content,
cell-by-cell analysis (Figure 1) using the
BlueShift Biotechnologies IsoCyte™ laser
scanner (www.blueshiftbiotech.com).

ToxCount advantages• Accurate and reproducible• Quick and easy assay• Non-toxic reagents• Economical• High-throughput compatible

Save time and money
ToxCount is less expensive, safer andfaster than other cell viability assays suchas 51Cr-release, LDH release and trypan
blue exclusion, and can be used on multiple ﬂuorescence detection platforms
including multi-well plate scanners, ﬂuorescence microscopy and ﬂow cytometry.

“Use ToxCount for accurate counts oflive/dead cells in a population.”

8

THE NEWSLETTER OF ACTIVE MOTIF — September 2006 • volume 7 • number 3

U.S.
Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351
Email: sales@activemotif.com

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Figure 1: Analysis of ToxCount data using the IsoCyte.
The graph depicted above illustrates the mean intensity
ratio for EthD-1 and calcein AM staining in each cellfrom two separate wells. One well was treated withsaponin (copper dots), resulting in cell death and,
therefore, staining with EthD-1. The second well wasuntreated (purple dots), resulting in calcein AM staining.
Three cells in the untreated well were unhealthy, so
were stained by the EthD-1.

Product

FormatCatalog No.

ToxCount™

20 x 96 rxns

18010

PRSRT STD
US POSTAGEPAID
PERMIT #236

92054