THE NEWSLETTER OF ACTIVE MOTIF — JUNE 2005 • VOLUME 6 • NUMBER 2

Look Inside For
Hot New
Technologies

IN
THIS
ISSUE

2 Your Source for Studying the NFκB Pathway

7 New: Complete ChIP Kit with Enzymatic Shearing

3 New: TransAM™ Flexi Kits Enable Profiling of
NFκB Binding at Any DNA Sequence

8 Sensitive Quantification of Activated Ras GTPase

4 Chariot™ Delivers Functional Protein into Living Cells

9 A Complete Solution for Studying
Nuclear Receptor Protein Activity

5 Improved Gene Silencing ThroughHigher Specificity Binding

10 Faster, More Accurate Measurement of
Nitric Oxide Production

6 Simple, Efficient Analysis of Protein Phosphorylation

11 mTRAP™ Kits Isolate More mRNA

T o o l s f o r t h e N F κB P a t h w a yYour Source for Studying the NFκB Pathway

NFκB activation (OD450 nm)

TransAM™ measures DNA-binding activity
In order to initiate transcription, transcription factors such as NFκB must first bindto their DNA target(s). TransAM Kits takeadvantage of this property by using a
combination of DNA binding and antibody
detection to give a specific, quantitative
readout of DNA-binding activity fromall sample types (Figure 1; see page 3 formore details). TransAM Kits are sensitive,
quantitative and fast, making them an ideal
replacement for classical methods such asradioactive gelshifts and time-consuming
reporter gene assays.
2

HeLa
HeLa (TNF-α)
JurkatJurkat (TPA + CI)
Raji

1.5
1
0.5
0

p65

p50

c-Rel
p52
NFκB family members

RelB

Figure 1: NFκB family profiling of DNA binding.
Nuclear extracts prepared from HeLa, HeLa treated with TNF-α,
Jurkat, Jurkat treated with TPA + calcium ionophore (CI) and Raji cells
were assayed at 10 µg/well for p65, p50, c-Rel, p52 and RelB activity
using the TransAM NFκB Family Kit.

Detection of phospho-proteins
Because many of the proteins involved
with the NFκB pathway are activatedby phosphorylation, it is important tobe able to quickly and easily quantitate
phosphorylation events. FACE™ Kits are cellbased ELISAs that offer a convenient alternative for monitoring the phosphorylationof the NFκB pathway members AKT, JNK,
PI3K, GSK and NFκB p65. FACE Kits enable2

analysis directly within the cell, which
eliminates the need for preparing extracts,running gels and performing Western blots.
All FACE Kits provide both a total and aphosphorylated antibody for the protein
being studied, and all kits are available inboth colorimetric and chemiluminescentformats (Figure 2). See page 6 for more
information on FACE Kits.

Figure 2: Monitoring phosphorylation of NFκB p65 at multiple sites.
The FACE NFκB p65 Profiler Kit was used to assay levels of total andphosphorylated NFκB p65 in uninduced and TNF-α + Calyculin A
induced HeLa cells. Data was plotted after correction for cell number(performed through use of the kit’s Crystal Violet reagent).

In addition, Active Motif offers a Sandwich
ELISA to study IκBα phosphorylation.
FunctionELISA™ IκBα provides a quantitative
method for monitoring phosphorylation of
the low-level IκBα protein in a sensitive
chemiluminescent ELISA (Figure 3).

0Achemiluminescence

NFκB is an important cellular regulator thathas also been shown to play a role in many
disease states such as inflammation, cancer,
apoptosis and autoimmune response. To
make the study of NFκB easier and more
efficient, Active Motif has developed anumber of innovative products that are
sure to suit your needs.

B

Monitor gene promoter regulation
Chromatin immunoprecipitation (ChIP) is a
powerful tool used to study protein/DNA
interactions. To make ChIP experiments
more successful, Active Motif offers its
complete line of ChIP-IT™ Kits. Dependingon your needs, you can buy all of the critical components you need in a single kit, including antibodies, buffers and PCR primer
controls that have been proven to workin ChIP. Or, if you are a more experienced
user, you can use ChIP-IT Kits that do notcontain control antibodies and PCR primers. This makes our ChIP-IT product line themost effective and versatile available. Lookon page 7 to learn about our exciting new
products for ChIP.
Antibodies & recombinant proteins
Active Motif’s extensive line of antibodies
for NFκB, toll-like receptors (TLRs) and IKK
provide superior performance and reliable
results. In addition, Active Motif offers anumber of recombinant proteins for use as
positive controls, in in vitro screening studies and as protein standards in our ELISAs.
To find out more about Active Motif’s tools
for studying the NFκB pathway, simply visit
www.activemotif.com.

5
10
30
time (minutes)

60

200000160000120000
80000
40000
0

0

10

20

30
40
time (minutes)

50

60

70

Figure 3: Induction of IκBα phosphorylation.
Jurkat cells were treated with 1 nM TNF-α and harvested at the indicated time points. Whole-cell extracts were assayed in Western blot analysis (A) using
Phosphorylated-IκBα mAb (Cat. No. 40904) and the FunctionELISA IκBα Kit (B).

Toll Free — 1 877 222 9543

JUNE 2005 • VOLUME 6 • NUMBER 2

DNA-binding ELISAs

New: TransAM™ Flexi Kits Enable Profiling of NFκB Binding at Any DNA Sequence

Flexi or Original TransAM?
The original TransAM Kits offer a fast,
non-radioactive alternative to gelshift
assays and provide a 96-well plate that ispre-coated with the consensus bindingsite for the factor of interest. This originalformat is convenient for measuring binding
activity at the consensus site but does notenable you to study alternative sites. That’s
why we’ve developed the new Flexi Kits; weprovide pre-optimized reagents including
proven antibodies, controls and reaction
buffers, while you have the flexibility toimmobilize any oligo in the 96-well plate.
How do TransAM Flexi Kits work?
In order to measure binding at your sites ofinterest, you first design biotinylated oligos
or PCR products that contain the bindingsites you wish to study. Each biotinylated
oligo is then incubated with nuclear
extract that has been treated to activate
the transcription factor of interest. The
extract/oligo mixture is then transferred toa 96-well, streptavidin-coated plate, where
the bound, biotinylated oligonucleotide iscaptured. A primary antibody specific forthe activated transcription factor is added,
followed by HRP-conjugated secondary
antibody and developing reagent. The
Product

Add primary
antibody

Add the extract-DNA
cocktail to plate

1 hr.

1 hr.

Add developing
and stop solution

1 hr.

Streptavidin
coated plate

Incubate biotinylated DNA with cell extractcontaining activated transcription factor
Figure 1: Flowchart of the TransAM Flexi procedure.

levels of DNA-bound transcription factor
are then read on a spectrophotometer,
which provides a quantitative, readout oftranscription factor activation (Figure 1).
TransAM Flexi advantages• Monitor binding at any sequence• High-throughput 96-stripwell format• Sensitive & quantitative• Results in less than 5 hours• Assay cell or tissue samples
Check binding affinities at multiple sites
The new Flexi Kits make it easy to quantitate the binding affinity of a given NFκB
Family member at any binding site. To
demonstrate, three different oligos weresynthesized that included the natural binding sites of promoters regulated by NFκB:
Igκ, IL-8 and uPA. The binding affinity of
NFκB p65 was then tested at these sites.
The results demonstrate that NFκB p65
FormatCatalog No.

TransAM™ Flexi NFκB Family

2 x 96-well plates

43298

TransAM™ Flexi NFκB p50

1 x 96-well plate

41098

TransAM™ Flexi NFκB p65

1 x 96-well plate

40098

TransAM™ NFκB Family

2 x 96-well plates

43296

TransAM™ NFκB p50

1 x 96-well plate

41096

TransAM™ NFκB p50 Chemi

1 x 96-well plate

41097

TransAM™ NFκB p65

1 x 96-well plate

40096

TransAM™ NFκB p65 Chemi

1 x 96-well plate

40097

www.activemotif.com

Add anti-IgG
HRP conjugate

had its weakest affinity for the oligo that
contained the natural binding site of the
IL-8 promoter (Figure 2).
Order one today
TransAM Flexi Kits make it fast and easy for
you to study transcription factor bindingat any DNA sequence you choose. A Kit
for the NFκB Family (p50, p65, p52, c-Rel
and RelB) and individual kits for NFκB p50
and p65 are now available, with more on
the way. For complete details, visit us atwww.activemotif.com/transam.
NFκB activation (OD450 nm)

Active Motif’s new TransAM™ Flexi NFκB
Family Kit enables you to profile thebinding of the NFκB Family members p65,
p50, p52, c-Rel and RelB to any DNA-binding site. Flexi Kits are the latest version of
the original TransAM Kits, which are themethod of choice for studying transcription factor binding activity.

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

Untreated
Treatedconsensus
control

Igκ

IL-8

uPA

50 bp
5´

TF Binding site

3´BIgκ - GGAAAGTCCC
IL-8 - GGAAATTCCA
uPA - GTACTTTCCC

Figure 2: Measuring affinity of NFκB p65 for different binding sites.
Five µg of nuclear extract from untreated and TNF-α-treated HeLa
cells were used to assay the binding affinity of NFκB p65 for four different biotinylated 50-mer oligonucleotides. Each of the 3 test oligos(Igκ, IL-8 and uPA) contained the wild-type binding site of a promoter
that is regulated by NFκB. The binding affinities of these natural binding sites were compared to the consensus binding site control that isprovided in TransAM Flexi Kits.

3

Protein Delivery

Chariot™ Delivers Functional Protein into Living Cells
The Chariot™ delivery reagent efficiently
transports biologically active proteins,
peptides and antibodies directly into
cultured mammalian cells. Delivery is complete in less than two hours and provides
efficiencies of 65-95%. After delivery, living
cells can be assayed immediately to determine the effects of the introduced material. These features make Chariot an ideal
tool for a variety of functional studies.
Targeted delivery
Chariot is a peptide that forms a non-covalent complex when incubated with yourpurified protein, peptide or antibody for
30 minutes at room temperature. Additionof the complex to cells results in its rapid
internalization. Once inside the cell, thecomplex dissociates and Chariot is transported to the nucleus, while the delivered
protein is biologically active and free toproceed to its cellular target (Figure 1).

Figure 1: Targeted protein delivery.
50 ng of a 10 kDa nuclear protein labeled with Lucifer yellow at the
C-terminus was complexed with Chariot and delivered into HS-68
cells. Unfixed cells were observed 90 minutes post-delivery.

Non-covalent delivery
Many protein delivery systems requirethat you begin by fusing a carrier protein
to your macromolecule. However, this canchange the folding characteristics of yourprotein and, ultimately, its function. Because Chariot forms a non-covalent bondwith your protein, it does not affect thedelivered protein’s folding or function.
Deliver biologically active proteins
The ability of Chariot to deliver biologically
active compound was demonstrated using

4

Figure 2: Chariot delivery offunctional protein.
HS-68 cells arrested in G0 phaseby serum deprivation for 48 hours
were released by addition ofserum for 3 hours. Flow cytometry
performed 0, 10, 15 and 20 hours
after addition of Chariot alone anda Chariot-p27kip1 complex indicatesthat cells receiving Chariot alone
progressed into G2 phase (A), while
over 90% of the cells receiving theChariot-p27kip1 complex remained in
G1 phase (B). Data generously provided by Dr. Gilles Divita, Biophysics
Dept., CNRS, Montepellier, France.

p27kip1, a 27 kDa cyclin-dependent kinase inhibitor that causes cell-cycle arrest in G1 phase.
Over 90% of cells receiving a Chariot-p27kip1
complex were unable to progress beyond G1
phase (Figures 2A & 2B), demonstrating theefficient delivery of active p27kip1.
Advantages• Delivers active protein directly
into living cells• Up to 95% efficiency in less than
2 hours• Works in a variety of cell lines,
as well as in vivo
• Study living cells – no fixing needed

What can Chariot do for you?
Direct delivery of active protein makes
it easy to perform many studies that are
not possible using DNA transfection andexpression. Chariot results have been
extensively published (see table below);
successful delivery of proteins, peptides
and antibodies has been shown in a wide
range of cell lines, including hard-totransfect neuronal, primary and plant cells.
The method has proven to be effective
on both adherent and suspension cells, aswell as in vivo. For a more extensive list of
papers that cite the use of Chariot, simply
return the enclosed reply card or download
the list at www.activemotif.com/chariot.

Cell/Tissue Type

Delivered

Citation

Mouse Lung Tissue

Protein

Aoshiba, K. et al. (2003) Amer. J. Resp. Cell Mol. Biol. 28:
555-562.

Neuronal Growth Cones

Protein

Jurney, W. et al. (2002) J. Neuroscience. 22(14): 6019-6028.

Primary Mouse Hepatocytes

Protein

Bardag-Gorce, F. et al. (2003) Exp. Mol. Pathology 74:160-167.

Hodgkin’s and B Cells

Smac protein and Smac peptide

Kashkar, H. et al. (2003) J. Expr. Med. 198(2): 341-347.

Human Primary T cells

Unc119 protein

Gorska, M. et al. (2004) J. Expr. Med. 199(3): 369-379.

Rat DRG Axons

Dynamitin protein

Heerssen, H. et al. (2004) Nature Neuro. 7(6): 596-604.

Chicken Embryo Retina

XAC (various mutations)Gehler, S. et al. (2004) J. of Neuro. 24(47): 10741-10749.

Mouse Photoreceptor cells

PeptideKoulen, P. et al. (2005) Invest. Ophth. Vis. Sci. 46: 287-291.

Mouse Adrenal Gland slices

PP-19 peptide

Chan, S. et al. (2005) J. of Neurophysiology 10.1152/jn.01213.2004

Rat Lung Tissue

PKA holoenzyme

Maron, M. et al. (2005) Amer. J. Physiol. Lung Cell Mol. Physiol.
10.1152/ajplung.00134.2005

Product

FormatCatalog No.

Chariot™

25 rxns*
100 rxns

30025
30100

* A rxn is defined as sufficient Chariot reagent to deliver protein to cells in a 35 mm plate.
** Chariot is covered under U.S. Patent No. 6,841,535. Purchase includes the right to use for basic research purposes only. Other-use licenses are available,please contact Active Motif Technical Services for additional information.

Toll Free — 1 877 222 9543

JUNE 2005 • VOLUME 6 • NUMBER 2

Gene Silencing

Improved Gene Silencing Through Higher Specificity Binding
Active Motif’s custom gripNAs™* offer ahighly effective alternative to traditional
antisense- and siRNA-based gene silencing.
gripNA probes utilize a modified antisense
approach that offers high-affinity binding
with unsurpassed sequence discrimination. This minimizes non-specific interactions that can cause unintended, mutant
phenotypes. Plus, delivery is simple using
either a lipid-based approach or Chariot™ II.
gripNAs have proven to be effective at silencing genes in Zebrafish, mammalian cells
and Xenopus. And because gripNAs areresistant to nuclease degradation, they arestably maintained in the cell, enabling themto silence genes over a prolonged period.
New antisense for specific gene silencing
The use of antisense molecules has long
been viewed as a promising strategy forgene-specific silencing. However, successful
gene silencing using classical DNA-antisense
technology has been limited by the rapid
degradation of probes by intracellular
nucleases, insufficient target affinity andnon-specific side effects. In order toovercome these limitations, several novel
oligonucleotide chemistries have beendeveloped. Peptide nucleic acids (PNAs) area novel form of DNA-analogs that possess
several characteristics that seemed to makethem ideal for gene silencing. PNAs are resistant to nucleases and display high-affinity and sequence-specific binding. However,
PNAs have proven to be inefficient tools
for gene silencing, due mostly to their lackof cell permeability, tendency to self-aggregate and poor solubility. To capitalize on
the promise of PNAs, Active Motif developed its gripNAs. gripNAs are a novel formof negatively charged PNA that overcome
the limitations of classical PNAs. The inclusion of a negative charge in gripNAs has
eliminated the solubility and aggregation
issues associated with PNAs, while retaining
their high affinity and specificity. gripNAs
can be delivered efficiently using multiple
approaches and have been shown to behighly effective at gene silencing (Figure 1).

www.activemotif.com

Chariot II/gripNA

Chariot II/PNA

A

B
1

2

3

4

5

6

7

1

2

3

4

5

6

7

Cyclin B1
CdK2

Figure 1: Inhibition of expression by gripNAs and PNAs directed against cyclin B1.
Increasing concentrations (lane 3: 50 nM, lane 4: 100 nM, lane 5: 500 nM, lane 6: 1 µM, lane 7: 2 µM) of anti-cyclin B1 gripNA (A) or PNA (B) were incubated with
Chariot II at a molar ratio 1:25 at 37°C for 1 hour, then overlaid onto cultured HS-68 cells. Cyclin B1 protein levels were analyzed by Western blotting after 24hours. Cdk2 protein was used as a control to normalize protein levels. The level of endogenous cyclin B1 in untreated cells is shown in lane 1 of each panel,
while lane 2 shows addition of gripNA or PNA alone, without Pep-2. Data generously provided by Dr. Gilles Divita, Biophysics Dept., CNRS, Montpellier, France.1

Why choose gripNAs?
• Unsurpassed sequence specificity
reduces unintended phenotypes• Resistant to nucleases• Flexible synthesis modification• Multiple methods for gripNA delivery• Convenient online ordering
Hit your target only
A recurring problem of many gene silencing
reagents is a lack of specificity. This cancause unintended phenotypes because
these reagents may bind and silence
expression of more than just the intended
target. In vivo mismatch discrimination experiments have shown that gripNA binding
is reduced or eliminated by the presence ofeven a single base-pair mismatch. Recent
experiments performed in Zebrafish embryos indicate that this specificity can improve your results. While wild-type gripNAand morpholino probes were comparable inpotency at silencing the chordin, uroDand no tail genes, the intentional inclusionof 2 and 4 base-pair mismatches in themorpholino probes caused non-specificeffects that were not triggered by comparable gripNA probes.2
Order anytime
Active Motif’s custom gripNA synthesis
service can provide you with better materials for your gene silencing experiments. All
the information needed to design, orderand use gripNA probes can be accessed atwww.activemotif.com/gripna. Our simple,
online ordering system makes it easy toplace your gripNA order 24 hours a day, 7days a week. gripNA probes can be orderedwith or without Chariot II. In addition, you
can add a 3´ modification (Biotin, FITC or
primary amine) to your probe. Each gripNA
is verified by mass spectrophotometry andsupplied with a fluorescently labeled positive control for human CREB. Log on now
and order your custom gripNA today.
* Patent pending. Purchase includes all rights for research use. Other-use
licenses are available, please contact Technical Services for details.
1. Morris et al. (2004) Gene Therapy 11: 757-764.
2. Urtishak et al. (2003) Developmental Dynamics 228: 405-413.

Product

FormatCustom gripNA™ Probe

200 nmol

24001

Custom gripNA™ Probe w/Chariot™ II

200 nmol

24002

Primary Amine Modification

Catalog No.

24004

Biotin Modification

24005

Fluorescein Modification

24006

Chariot™ II

96 rxns

24008

gripNA™ Human CREB Positive Control

25 nmol

24007

gripNA™ Chordin Positive Control

5 nmol24009

5

Cell-based ELISAs

Simple, Efficient Analysis of Protein Phosphorylation
As interest in kinase activation and theireffect on cellular regulation increases, sodoes the need for fast, efficient and robust
assays to measure protein phosphorylation.
That is why Active Motif developed its
Fast Activated Cell-based ELISA (FACE™)
Kits. FACE Kits provide a simple, sensitive, cell-based method for monitoring
protein phosphorylation. They enable youto perform modification-specific analysis
directly within the cell, without the needfor time-consuming cell extractions, gelelectrophoresis or membrane blotting, allof which saves you time and money.
The FACE method
In the FACE method, cells are cultured in
96-well plates and stimulated to induce thepathway 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 withsecondary HRP-conjugated antibody and developing solution provides a colorimetric or
chemiluminescent readout that is quantitative and reproducible (Figure 1). FACE Kits also
contain a primary antibody for the native
non-modified protein, so you can monitor
both native and activated protein levels inthe same experiment (Figure 2).
0.6
OD450 nm0.5

Uninduced
Induced

0.4
0.3
0.2
0.1
0

Total-AKT

Phospho-AKT

Figure 2: Measurement of phosphorylated and total AKT.
NIH/3T3 cells were cultured in 96-well plates and serum-starved for
16 hours. Cells were then treated with 50 ng/ml PDGF for 5 minutes
and fixed. Total and phospho-AKT were each assayed in triplicate
using the phospho and total AKT antibodies from the FACE AKT Kit.

6

Add anti-IgG
HRP conjugate

Add primary
antibody
3 hrs.

1 hr.

Cells arefixed following
stimulation

P

P

P

Add developing
and stop solution

Stimulatecells
Cells cultured in
96-well plate
Figure 1: 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.

FACE advantages• Simple, quantitative method• Fixing cells preserves protein
activation state• Cell-based assay eliminates extractions and gel electrophoresis• Fast – requires less than 3 hours ofhands on time
A variety of kits for all your needs
Many cellular kinase cascades containcritical control points, where multiple downstream components can bephosphorylated by a single kinase. The
phosphorylation of one or other substrate
can cause significant shifts in the cellularresponse, which means that monitoring
phosphorylation events across multiple
proteins can become commonplace.
However, trying to monitor this type of“signaling switch” using Western blotting
or classical radioactive kinase assays is alabor intensive and expensive undertaking,especially if there are multiple treatments

to be measured. To overcome this problem,
using FACE can be as simple as an extra
pipetting step. This is because in the FACE
method, cells are cultured and analyzed
within the same well and therefore do not
require the use of multiple culture plates,
extract preparations, gels, membranes,
etc. And, because you can easily increasesample throughput, your results will bestatistically more relevant.
Prevents unwanted sample modifications
When analyzing protein phosphorylation
via Western blotting or ELISA, it is necessary to prepare cellular extracts. However,additional protein modifications can occur
during the extraction process, which
may alter your results. To eliminate thisproblem, FACE Kits use a fixation step that
“freezes” the cellular state and prevents
further protein modifications. This enables
detection of the exact protein state in thecell at a chosen time point, which provides
you with more accurate results. So, for abetter phospho-specific assay, try FACE.

FACE™ Product Line
FACE™ AKTFACE™ c-Jun (S73)
FACE™ ErbB-2 (Y877)
FACE™ FKHR (FOXO1)FACE™ MEK1/2
FACE™ STAT2

FACE™ ATF-2
FACE™ c-Src
FACE™ ErbB-2 (Y1248)
FACE™ GSK3β
FACE™ NFκB Profiler
FACE™ STAT4

FACE™ Bad
FACE™ EGFR (Y992)
FACE™ ERK1/2
FACE™ JAK1FACE™ p38
FACE™ STAT6FACE™ c-Jun (S63)
FACE™ EGFR (Y1173)
FACE™ FAKFACE™ JNKFACE™ PI3 Kinase p85

Toll Free — 1 877 222 9543

JUNE 2005 • VOLUME 6 • NUMBER 2

Chromatin Immunoprecipitation

New: Complete ChIP Kit with Enzymatic Shearing
Active Motif is excited to announce
the release of a complete chromatin
immunoprecipitation (ChIP) kit that
includes all of the reagents for performing
ChIP with enzymatic shearing. The new
ChIP-IT™ Enzymatic Kits combine Active
Motif’s innovative Enzymatic Shearing Kit
with our popular complete ChIP-IT Kit.
Together, this combination provides you
with the simplest solution for performing
ChIP experiments.

���� ����� ����� ��
�

�

�

�

�

Figure 1: Analysis of DNA sheared using the Enzymatic Shearing Kit.
HeLa cells were fixed for 10 minutes with 1% formaldehyde and thenchromatin was prepared using the Enzymatic Shearing Kit protocol.
Chromatin was sheared with the enzymatic shearing cocktail for 5, 10& 15 minutes and the reaction was stopped.
Lane 1: 100 to 1000 bp ladder.
Lane 2: Unsheared HeLa DNA.
Lane 3: HeLa DNA treated for 5 minutes.
Lane 4: HeLa DNA treated for 10 minutes (optimized).
Lane 5: HeLa DNA treated for 15 minutes.

Enzymatic vs. Sonication Shearing
The starting point for any ChIP experiment is the preparation of chromatin. This
critical step must be optimized to ensure
the chromatin is sheared to 200-1000 bp
fragments. Typically, chromatin shearing is
performed by using sonication. Althoughsonication is an effective method for shearing DNA, it can also be time consuming anddifficult to optimize due to complications
arising from emulsification and overheating during the sonication process. Also,
because the quality of your sheared sample
depends greatly upon the quality of yoursonicator, it may be necessary to purchasean expensive, “high-end” sonicator to get
reproducible shearing. In contrast, theChIP-IT Enzymatic and Enzymatic Shearing
Kits utilize a proprietary enzymatic shearing
cocktail that quickly and easily shears DNA
into 200-1000 bp fragments (Figure 1). And,because enzymatic shearing is dependent
only on time and temperature, all of the
problems associated with sonication are
eliminated, which greatly improves downstream ChIP results.

ChIP Product Line
Complete ChIP-IT Kits
Active Motif’s ChIP-IT Kits are themost effective products available
for performing ChIP. The kits containnearly everything you need to perform
ChIP including a comprehensive protocol and optimized buffers, inhibitor
cocktails, DNA purification columns
as well as positive control primers andantibodies, all of which have been validated in actual ChIP experiments. The
ChIP-IT Kits are available with either
sonication and enzymatic shearing.
Pre-validated ChIP antibodies
Finding a ChIP validated antibody canbe difficult. Active Motif is working
diligently to validate our extensive
antibody list for use in ChIP. Below
is a list of antibodies that have been
positively tested in ChIP to date, which
are ideal when used with our ChIP-IT
w/o controls Kits. Be sure to keep up todate on new ChIP-validated antibodies
by visiting our website.
Antibody

How do I order one
The Enzymatic Shearing Kit is sold separately and also as part of the new ChIP-IT
Enzymatic Kits. The ChIP-IT Enzymatic Kits
are available with and without positive control antibodies and primers. Want to learn
more? Give us a call or download a manualat www.activemotif.com/chip.

Cat. No.

AP-2 pAb

39304

c-Jun pAb

39309

C/EBPα pAb

39306

DNMT1 mAb

39204

DNMT3A mAb

39206

DNMT3B mAb

39207

E2F-1 pAb

39313

E2F-6 mAb

39509

GATA-1 pAb

39025

HBP-1 mAb

39511

Product

FormatCatalog No.

HDAC3 pAb

40968

ChIP-IT™ Enzymatic

25 rxns

53006

HDAC4 pAb

40969

ChIP-IT™ Enzymatic w/o controls

25 rxns

53007

HDAC5 pAb

40970

HDAC6 pAb

40971

IRF-3 pAb

39033

Enzymatic Shearing Kit

10 rxns

53005

ChIP-IT™

25 rxns

53001

JunB pAb

39326
39328

ChIP-IT™ w/o controls

25 rxns

53004

JunD pAb

ChIP-IT™ Shearing Kit

10 rxns

53002

p53 pAb39334

Pax-5 pAb

39336

www.activemotif.com

PPARγ pAb

39338

RNA pol II mAb

39097

Sp1 pAb

39058

TRF2 pAb

39223

7

Small GTPase Activation

Sensitive Quantification of Activated Ras GTPase

Ras GTPase is a much-studied regulator
Small GTPases, also called GTP-binding proteins, are a family of enzymes that serve as
molecular switches in regulating a numberof signal transduction pathways including
growth, apoptosis and differentiation. Ras
is of particular interest to researchers because aberrant regulation by Ras has been
implicated in a number of disease states.
Mutant, constitutively active forms of Ras
are estimated to be present in ~30% of all
human cancers.
The Ras GTPase Chemi ELISA method
Because activated Ras binds specifically to
the Ras-binding domain (RBD) of the Raf
effector protein, Raf-RBD is used as a probe
to isolate activated Ras. The Ras ELISA Kit
contains a Raf-RBD protein fused to GST
and a 96-well, glutathione-coated assay
plate. GST-Raf-RBD is first incubated on
Product
Ras GTPase Chemi ELISA Kit

8

Add anti-IgG
HRP conjugate

Add primary
antibody
45 min45 min45 minAdd developing
solution, thenquantify

Add cell
extract toplate

Cell extractcontaining activatedRas GTPase

GST-Raf-RBD
coated plate

Figure 1: Flowchart of the Ras GTPase Chemi Kit.
Cell extract is added to a glutathione-coated plate that contains immobilized GST-Raf-RBD protein. Activated Ras in the extract binds to the Raf-RBD protein.
Addition of primary & secondary antibodies and developing solution followed by reading on a luminometer enables sensitive quantification of activated Ras.

the plate for one hour to immobilize thecapture protein. Addition of sample to theplate results in the binding of activated Ras
to the Raf-RBD. A primary antibody specificfor Ras is then added, followed by an HRPconjugated secondary antibody and developing reagent (Figure 1). The plate is thenread on a luminometer, which provides asensitive, quantitative chemiluminescentreadout of activated Ras (Figure 2).
Ras ELISA advantages• More sensitive – assay uses only 25 µgof extract, which is 20-fold less thanpull-down/Western methods• Better results – quantitative readouts
make it easier to compare results• Less effort – no need to run gels ordevelop Western blots• Save time – results in less than 5 hours• Versatile – assay activated extracts
from cells or tissue samples, or study
recombinant Ras protein
FormatCatalog No.

1 x 96-well plate

52097

Activated Ras Signal (RLU)

Active Motif’s new ELISA-based kit forstudying activated Ras GTPase is a marked
improvement over other commerciallyavailable kits. All other products utilize
immunoprecipitation of Ras followedby Western blotting. In contrast, Active
Motif’s Ras GTPase Chemi ELISA Kit uses
a combination of Raf-RBD protein andantibodies in a 96-well format to provide afaster and more sensitive alternative. This
enables you to use less of your precious
sample and still detect low-level events.
In addition, because ELISAs provide morequantitative results than Westerns, yourdata is more meaningful. And, as the Ras
ELISA Kit uses a 96-well plate made up of
12-well strips, you can run anywhere from
1 to 96 samples at a time, which lets you
accommodate whatever throughput yourexperiment requires.

800000

Unstimulated
EGF stimulated

600000400000200000
0

6.25

12.5
25
Cell extract (µg/well)50

Figure 2: Quantification of activated Ras in stimulated HeLa cells.
Increasing amounts of whole-cell extract from HeLa cells that had
been stimulated with 5 ng/ml of EGF for 2 minutes were assayedusing the Ras GTPase Chemi ELISA Kit.

Try sensitive, quantitative ELISAs
The Ras GTPase Chemi ELISA Kit makes
it simple to quantify activated Ras. It is
ideal for studying signaling pathways that
activate Ras. Please give us a call, returnthe enclosed reply card or log on towww.activemotif.com/gtpase for complete
information. To get to the only ELISA-based
Ras activation assay, order the Ras GTPase
Chemi ELISA Kit.

Toll Free — 1 877 222 9543

JUNE 2005 • VOLUME 6 • NUMBER 2

Nuclear Receptor Assays

A Complete Solution for Studying Nuclear Receptor Protein Activity

Monitor ligand activation with NR Peptide
Studying the agonist/antagonist effects
of potential drug targets is an importantelement of nuclear receptor-targeted drug
discovery. Active Motif’s NR Peptide ELISAs
are specifically designed to capture ligandactivated nuclear receptor and can be usedwith both cell extracts and proteins. EachNR Peptide ELISA Kit provides a 96-wellplate that is coated with a Capture Peptidethat includes the consensus-binding motifof the nuclear receptor’s co-activator.
Addition of sample results in binding ofligand-activated nuclear receptor to the
Capture Peptide. Each well is then incubated with a primary antibody specific forthe nuclear receptor of interest, followedby an HRP-conjugated secondary antibody
and developing solution to provide aneasily quantified readout. This enables you
to quickly and quantitatively measure theagonist/antagonist effects of target compounds on the binding of ligand-activatednuclear receptors (Figure 1).

Assess DNA-binding activity of NRs
Inappropriate nuclear receptor signaling is associated with numerous diseases
including cancer, asthma and arthritis,which makes NRs promising drug targets.
Because the end point of nuclear receptor activation is DNA binding, monitoringchanges in the DNA-binding activity ofa target nuclear receptor can serve as an
ideal biomarker. Classical methods suchas gelshifts and reporter gene assays are
not well suited to this application. Active
Motif’s TransAM™ Kits provide an innovative
alternative by using a combination of DNA
binding and antibody detection to give a
specific, quantitative readout of DNA-binding activity from all sample types.
Quantify total NR with Sandwich ELISAs
In order to fully examine the activation ofa given nuclear receptor, it is important
to be able to quantify the total levels of agiven nuclear receptor within a sample. The
new NR Sandwich ELISAs offer a simple,
rapid method to quantify the total amountof nuclear receptor protein present inboth cell and tissue samples. NR Sandwich
Kits utilize the Sandwich ELISA-based
method that is an improvement over othermethods used to study proteins, such asWestern blotting. The 96-well format isconvenient and sensitive, with only a miniProductFigure 1: ERα agonism/antagonism dose-response curves.
Nuclear extracts from the breast cancer cell line MCF-7 are incubatedin wells of the NR Peptide ELISA ER plate in the presence of 100-foldserial dilutions (from 10-6 to 10-14 M) of the agonist compounds
diethylstilbestrol (DES) and estradiol, and the antagonist compounds
tamoxifen and clomiphene. Only ligand-activated ER protein can bindto the Capture Peptide immobilized in the plate. Bound ER is specifically detected with ERα antibody and quantified using a secondary
antibody and Detection Solution.

www.activemotif.com

mal amount of material required to givequantitative readout of nuclear receptor
levels (Figure 2).
AR detection (OD 450nm)

Active Motif offers a variety of nuclear
receptor analysis tools that make studying
nuclear receptor proteins both faster andmore accurate than using traditional methods. Active Motif has products for monitoring DNA-binding activity, activation state,
protein level or agonist/antagonist effects.

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

LNCaP (AR+ve)
PC-3 (AR-ve)
DU 145 (AR-ve)

10

5

2.5

1.2

0.62

extract/well (µg)
Figure 2: Monitoring expression levels of AR using NR Sandwich AR.
Different amounts of nuclear extracts from three human prostatecancer cell lines, LNCaP, PC-3 and DU 145, were analyzed for levels of
AR protein using the NR Sandwich AR Kit.

A collection of NR antibodies & proteins
Active Motif offers over 200 highly
characterized antibodies directed against
transcription factors and nuclear receptors,
including antibodies for GR, PPARα and γ,
PXR, RAR-β, -β2 and -γ, RXR-α and -β,
and VDR. In addition, Active Motif offers anumber of recombinant nuclear receptor
proteins that are ideal for use as positive
controls, in in vitro screening studies and inmany other applications.
Get the tools you need for NR research
To get complete information on all ofActive Motif’s tools for studying nuclear
receptors, please give us a call, visit our
website or send in the enclosed reply card.
FormatCatalog No.

NR Peptide ERα

1 x 96-well plate5 x 96-well plates

49096
49596

NR Peptide ERα Chemi

1 x 96-well plate5 x 96-well plates

49097
49597

NR Sandwich AR

1 x 96-well plate5 x 96-well plates

49196
49696

NR Sandwich ERα

1 x 96-well plate5 x 96-well plates

49296
49796

NR Sandwich PR

1 x 96-well plate5 x 96-well plates

49396
49896

TransAM™ ER

1 x 96-well plate5 x 96-well plates

41396
41996

TransAM™ GR

1 x 96-well plate5 x 96-well plates

45496
45996

TransAM™ PPARγ

1 x 96-well plate5 x 96-well plates

40196
40696

9

Nitric Oxide Quantitation

Faster, More Accurate Measurement of Nitric Oxide Production

Nitric oxide is a key signaling molecule that,either directly or indirectly, stimulates hostdefenses in the immune system, maintains
blood pressure in the cardiovascular system
and modulates neural transmission in thebrain. Consequently, NO has become thesubject of extensive research. However,
because NO has an extremely short halflife (< 10 seconds), it is extremely difficult to
detect and study unless you have expensive, specialized equipment.
NO Quantitation Kit advantages• Increased sensitivity• Fast and easy to use• Wider dynamic range than LDH assays• Works with a variety of sample types
Indirect detection of NO
Because NO is rapidly metabolized to nitrite
and nitrate, quantitation of these stableanions can be used to indirectly measure
the amount of NO originally present in asample. The best index of total NO is thesum of the nitrite and nitrate in the sample,
which are commonly quantified in a Two-

10

Step assay. The first step is conversion of nitrate to nitrite using an NADPH-dependent
nitrate reductase. The nitrite is then converted into a purple-colored azo compoundby adding Griess Reagent. Quantitation ofthis azo compound by spectrophotometry
provides an accurate measurement of
the original NO concentration (Figure 1).
However, the sensitivity of conventional
Two-Step assays have been limited because
NADPH, which is an essential cofactor instep one, interferes with Griess Reagent
in step two. The sensitivity of Two-Step
assays can be increased by the addition of athird step, in which lactate dehydrogenase(LDH) is used to eliminate excess NADPH
prior to the Griess reaction. However, while
inclusion of this additional step increases
assay sensitivity, it also increases the timerequired to perform each assay.
NitrateNitric Oxide

Nitrate Reductase

decreased to only 30 minutes and there’s
no need for a time-consuming third step,saving you both time and effort.
Improved accuracy for better results
In addition to its increased speed and user
friendliness, the Nitric Oxide Quantitation
Kit provides more accurate measurement
over a wider range of sample concentration than possible with other currently
used methods. The improved linearity overa broader dynamic range (Figure 2) meansthat your results will be more precise withless optimization and repetition than ispossible with any other kit.
1.2
1.0
OD540 nmActive Motif’s Nitric Oxide Quantitation Kit
is a faster, more sensitive method for measuring the production of nitric oxide (NO)in your samples. The kit employs an innovative cofactor technology that reduces
the time and number of steps needed tomeasure NO levels. Plus, the Nitric Oxide
Quantitation Kit has a wider dynamic rangeof NO measurement than other methods,
which makes your results more accurate.
And, the kit can be used with a large varietyof sample types, including plasma, serum,
saliva, urine, cell lysate, tissue homogenate
and tissue culture medium.

Assay Using
Griess reagent

LDH method

0.8
0.6
0.4
0.2
0.0

NitriteActive Motif's NO Kit

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

Concentration of Nitrate (µM)

Figure 1: Measurement of nitric oxide by Griess Reagent.
Nitric oxide is converted to nitrite, then assayed using Griess Reagent.

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

Save time and money
Active Motif’s Nitric Oxide Quantitation Kit
offers a faster, easier-to-use alternative toconventional Two- and Three-Step NO assays. The kit contains a unique formulationof cofactors that accelerate the conversionof nitrate to nitrite, while simultaneously
degrading NADPH. The result is that thetime required for the reductase step isGet better results today
The Nitric Oxide Quantitation Kit makes
NO measurement faster, more sensitiveand easier to perform than other, moreconventional methods. In addition, its
96-well format enables straightforward
high-throughput automation. For a bettermethod of measuring NO production, try
the Nitric Oxide Quantitation Kit today!

Product

FormatCatalog No.

Nitric Oxide Quantitation Kit

2 x 96 rxns

40020

Toll Free — 1 877 222 9543

JUNE 2005 • VOLUME 6 • NUMBER 2mRNA and Total RNA Isolation

mTRAP™ Kits Isolate More mRNA

The mTRAP advantage
gripNAs are a novel class of negatively
charged peptide nucleic acids (PNAs) thatbind to nucleic acids with high affinity
and specificity. This enables mTRAP Kits toemploy 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 issignificantly higher yields of pure mRNA
with lower levels of rRNA contaminationcompared to oligo dT-based methods (Figure 1). The higher affinity of Poly T gripNA
Probe also enables isolation of mRNA thathave shorter poly A tails, resulting in amore representative sample. This makes
mTRAP-isolated mRNA ideal for production of cDNA probes for microarrays, cDNA
libraries or in Northern blots.
Advantages• Isolates more mRNA per sample thanoligo dT-based methods• Reduces ribosomal RNA and genomic
DNA contamination• Captures mRNA with secondary
structure and shorter tails, for a more
representative mRNA population• Higher purity mRNA means better
cDNA probes for microarray analysis

www.activemotif.com

1 kb

A

Q

AM

I

250

mRNA isolated (µg)

mTRAP™ Kits isolate high yields of quality
mRNA from mammalian cells, tissue andtotal RNA. mTRAP Maxi, Midi and 96 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 formRNA than oligo dT. The result is more
mRNA per sample, with reduced ribosomal
RNA and genomic DNA contamination.

2 x 108 cells

1 x 108 cells

200
150

Lane 1:
Lane 2:
Lane 3:
Lane 4:Lane 5:100
50
0Figure 1: Higher yields of pure mRNA.
mRNA was isolated from 1 and 2 x 108 HeLa cells using mTRAP
Maxi and three other suppliers’ kits. Eluted mRNA wasquantified by spectrophotometer and plotted. Two µg ofmRNA from each 1 x 108 sample was run on a 0.8% agarosegel. mTRAP-isolated mRNA shows no genomic DNA and lessrRNA contamination, with a far greater percentage of thesample being actual mRNA.

Company A

Company Q

Active Motif

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

Company I

mTRAP Maxi and mTRAP Midi
The mTRAP Maxi Kit provides 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 of tissue and will yield 100-200µg mRNA, depending on cell type.
The mTRAP Midi Kit provides 24 mediumsized mRNA isolations. Each reaction canisolate mRNA from 0.5-1.0 x 107 cells or
50-200 mg tissue and will yield 10-20 µg
mRNA, depending on cell type.
mTRAP 96
The mTRAP 96 Kit is supplied in a 96-wellformat designed for high-throughput usage
and is ideal for use with arrays and RT-PCR.
Each reaction can isolate mRNA from 0.5-1.0
x 106 cells or 10-20 mg tissue and yields 100300 ng mRNA, depending on cell type.
mTRAP Total
The mTRAP Total Kit was created for singlepass enrichment of mRNA 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.

1 2 3

Figure 2: Pure mRNA from total RNA in one passage.
mRNA was isolated from 500 µg of total RNA in duplicate usingmTRAP Total, quantified by spectrophotometry and run on a 0.8%
agarose gel.
Lane 1:
1 kb DNA Ladder
Lanes 2-3: 2 µg mRNA isolated in one passwith the mTRAP Total Kit.

Complete package
mTRAP Kits come complete with all of
the plasticware and reagents needed toperform your mRNA isolations. And, withincreased yields and purity, you’ll havemore confidence using your mRNA in
microarrays, Northerns, RPA and RT-PCR
applications. For higher yields of cleaner
mRNA, switch to mTRAP today.

Product

FormatCatalog No.

mTRAP™ Maxi

6 rxns5 x 6 rxns

23006
23506

mTRAP™ Midi

24 rxns
5 x 24 rxns

23024
23524

mTRAP™ 96
mTRAP™ 96 w/MAG-96
MAG-96 magnetic stand3 x 96 rxns
3 x 96 rxns
1 stand

23096
23097
90096

mTRAP™ Total

12 rxns
5 x 12 rxns

23012
23512

11

Contact Info

Active Motif Offices
U.S.

EUROPE

JAPAN

1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351
Email: sales@activemotif.com

104 Avenue Franklin Roosevelt–Box 25
B-1330 Rixensart, Belgium
Germany Free Phone: 0800 181 99 10
France Free Phone: 0800 90 99 79
United Kingdom Free Phone: 0800 169 31 47
Other countries, Direct: +32 (0)2 653 0001
Fax: +32 (0)2 653 0050
Email: eurotech@activemotif.com

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

Distribution Network
Active Motif is represented in the
countries listed to the right throughour network of distributors. For
additional contact and ordering
information, please visit the
distributor page on our website:www.activemotif.com/distributors.

12

Australia

Austria

CanadaChina

Denmark

Finland

Hungary

India

Israel

Italy

Japan

Korea

Spain

SwedenTaiwanTurkeyNew Zealand Portugal

Toll Free — 1 877 222 9543
PRSRT STD
US POSTAGEPAID
PERMIT #236
92054