THE NEWSLETTER OF ACTIVE MOTIF — OCTOBER 2004 • VOLUME 5 • NUMBER 4

Profile Transcription Factor Phosphorylation Using
New FACE™ NFκB p65 Profiler Kits

Uninduced
Induced

0.8

NFκB Signal (RLU)

Active Motif’s new FACE™ (Fast Activated
Cell-based ELISA) NFκB p65 Profiler Kit
makes it possible to rapidly profile thelevels of three different phosphorylationsites of the transcription factor NFκB
p65 in one simple experiment. Plus, thekit provides an antibody specific for the
total form of the protein, enabling you tocompare phosphorylated to native protein
levels (Figure 1). Because FACE Kits are cellbased ELISAs, you grow, fix and assay yourcells all in the same plate, eliminating theneed to make extracts, run gels or perform
membrane blotting. This makes FACE Kits
faster, more sensitive and easier to use thanother techniques, like Western blot, in-gel
kinase and traditional Sandwich ELISAs.

IN THIS ISSUE

0.6
0.4

1

Profile Transcription Factor
Phosphorylation Using New
FACE™ NFκB Profiler Kits

1

Nuclear Receptor Measurement
Made Easy

2

Complete Kit & ChIP-validated
Antibodies Improve Chromatin
Immunoprecipitation

3

Sensitive, Specific Activation
Assays for GATA-4, GR, Oct-4
and Many More

5

Accurate Monitoring of
Apoptosis Using Mitochondrial
Cytochrome c Levels

5

Reproducible Isolation of
Mitochondrial and Cytoplasmic
Fractions

7

Deliver Active Proteins Directly
Into Mammalian Cells

8

Quantitative DNA Repair ELISAs
for GTBP, Ku and RPA

9GeneDetective™ Generates
Accurate Models for Exploring
Alternatively Spliced Genes

0.2
0

Total-NFκB p65 Phospho-NFκB p65 Phospho-NFκB p65 Phospho-NFκB p65(Ser536)(Ser468)(Ser276)

Figure 1: 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).

continued on page 4

Active Motif offers a variety of nuclear
receptor analysis tools that make studying
the activation of nuclear receptor proteinsboth faster and more accurate than using
traditional technologies. Whether you’re interested in DNA binding activity, activation
state, protein level or agonist/antagonist
effects, Active Motif has the tool for you.
Monitor tissue specific effects with
NR Peptide ELISAs
Upon ligand-receptor binding, nuclear
receptors undergo a conformationalchange, which can result in recruitmentof co-activators (agonist) or co-repressors(antagonist). Depending on the nuclear
receptor and the tissue location, a ligand

ER activation (chemi signal)

Nuclear Receptor Measurement Made Easy
30000

DES
estradioltamoxifen
clomiphene

25000
20000

15000

10000
5000
0

-15

-14

-13

-12

-11

-10

-9

-8

-7

-6

-5

log M (Drug concentration)
Figure 2: 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.

Factor Antibodies
10 Transcription
for Westerns, EMSA & ChIP

11 High-quality Nuclear Extracts
Proteins for
12 RecombinantYour Researchcontinued on page 6

www.activemotif.com

Chromatin Immunoprecipitation

Complete Kit & ChIP-validated Antibodies Improve Chromatin Immunoprecipitation
Chromatin immunoprecipitation (ChIP) is a
powerful tool for analyzing genome regulation because it combines the specificity of
immunoprecipitation with the sensitivity of
PCR. However, ChIP is a complicated, multistep procedure that can be technically challenging and yield results that are difficult
to interpret. To overcome these problems,
Active Motif developed its ChIP-IT™ Kit.
ChIP-IT is a complete solution that contains
all of the reagents required for DNA shearing, “pull down” and purification. Plus, thekit includes positive and negative control
antibodies and PCR primers that help you
interpret your results. With ChIP-IT, you
get a complete package of high-quality,
optimized reagents that make it possible
to successfully obtain and validate yourresults. In addition, Active Motif offers
a number of antibodies that have beentested and shown to work successfully with
ChIP-IT (see table). To get the best results
possible, while saving time and money, try
ChIP-IT and our ChIP-validated antibodies.
Powerful technique
The ChIP technique is used because it canidentify which DNA fragments are boundby a particular protein under specificconditions. A gene’s promoter region typically contains multiple known or putativetranscription factor binding sites that are
involved in regulating the gene’s expression.
Determining if, when and where a specifictranscription factor (or other protein) binds
to the DNA is an important part of understanding the gene’s regulation.
The ChIP process
In the ChIP process, cells are fixed withformaldehyde, which cross-links and therefore preserves the in vivo protein/DNA
interactions. The DNA is then sonicated into
small, uniform fragments and the protein/
DNA complexes are immunoprecipitatedusing an antibody directed against the
DNA-binding protein of interest. Following
immunoprecipitation, the cross-links are reversed and the DNA is screened to determine
which gene or groups of genes were bound by
the protein (Figure 1).

2

Cross-link protein to DNA inliving cells with formaldehyde

TargetDNA

PCR
primers
Induction

Break open cells
and shear DNA

Add pre-blocked
Protein G Agarose Beads

Add primary
antibody of interest

Negative
Control

Reverse cross-links andtreat with proteinase K

Detect and quantify
precipitated DNA through
PCR and hybridization methods

Immunoprecipitate to enrich
for fragments bound byprotein of interest

Figure 1: Schematic of chromatin immunoprecipitation. In ChIP, protein/DNA interactions are fixed, and the DNA is then sheared and precipitated using anantibody. After reversing the cross-links, the DNA is purified and then screened to determine which genes were bound by the protein of interest.

ChIP-validated antibodies available
AML1/Runx1

C/EBPβ

HDAC4

p53

AP-2

E2F-1

HDAC5

Pax-5

ATF-6

E2F-6

HDAC6

Sp1

c-Jun

GATA-1

IRF-3

C/EBPα

HDAC3

JunB

ChIP-IT advantages• Complete solution for ChIP – all critical reagents and controls are supplied• Direct measurement of transcription
factor/DNA interactions or histone
modifications• Compatible with genome-wide profiling or selective PCR-based screening• No need to optimize your own
reagents and protocol
Ensure your success
ChIP-IT simplifies all aspects of chromatin
immunoprecipitation by providing nearly
everything you’ll need to prepare chromatin, optimize shearing conditions, perform

ChIP reactions and analyze ChIP results by
PCR. All buffers (excluding formaldehyde)required for cell fixation, nuclei purificationand chromatin shearing are included. Not
only does this help ensure your results, but
you won’t have to spend hours preparing and optimizing your own buffers andprotocols. Most importantly, the ChIP-IT Kit
provides positive and negative control antibodies and PCR primer sets. These help toimprove result interpretation and also makeit easy for you to validate that your own
antibodies and primer sets function in ChIP.
No other kit or “home grown” method provides such a complete, convenient solution.
For worry-free ChIP, use our ChIP-IT Kit andChIP-validated antibodies.

Product

FormatCatalog No.

ChIP-IT™ Kit

25 rxns

53001

Toll Free — 1 877 222 9543

OCTOBER 2004 • VOLUME 5 • NUMBER 4

Transcription Factor ELISAs

Sensitive, Specific Activation Assays for GATA-4, GR, Oct-4 and Many More
Active Motif’s TransAM™ Kits are DNAbinding ELISAs* that facilitate the study oftranscription factor activation in mammaliantissue and cell culture extracts. The method
is faster and more sensitive than othercommonly used methods, while eliminating
the use of radioactivity. Over 30 different
TransAM Kits are available for studying bothindividual transcription factors as well as
entire transcription factor families. We arepleased this month to announce the introduction of 3 new TransAM Kits for assaying
GATA-4, GR and Oct-4.

����������������������������

Simple, specific assay
Transcription factors bind to their specific
DNA target(s) after they’ve been activated,commonly by phosphorylation. TransAM
Kits take advantage of this property bysupplying a 96-well plate in which multiple
copies of a double-stranded oligonucleotide
are immobilized. When sample is added,
the activated transcription factor binds tothe oligonucleotide at its consensus-binding sequence. Primary antibody directedagainst the bound form of the transcription factor is then added, followed by an
HRP-conjugated secondary antibody (Figure1). The colorimetric change is measured witha spectrophotometer, which provides asensitive, quantitative measurement of theactivated transcription factor (Figure 2). Eachkit also contains wild-type and mutatedcompetitor oligonucleotides that can beused to verify the specificity of the assay(Figure 3, page 6).
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���
���

Add nuclear
extract

Add primary
antibody
1 hr.

Cell extractcontaining activatedtranscription factor

1 hr.

Add anti-IgG
HRP conjugate

Add developing
and stop solution

1 hr.

Oligonucleotide
coated plate

Figure 1: Flowchart of the TransAM procedure.

Improved method
TransAM assays are a marked improvement over other techniques used to study
transcription factor activation. Unlike
gelshift, Western blotting and reporter
gene methods, TransAM does not use
inefficient cloning and cell transfections,time-consuming gel exposures or radioactive probes. Inconsistencies due to variable
reporter plasmid construction and theneed to construct stable cell lines are also
eliminated. TransAM assays are completein less than 5 hours, are more sensitive andprovide quantitative results. Moreover,TransAM can be used to study stimulated
tissue samples.
TransAM assay formats
TransAM Kits are offered in a variety offormats – individual kits assay single transcription factors, while Family Kits make itpossible to profile multiple members of atranscription factor family. And, TransAM
Chemi Kits utilize a luminometer to provide
the maximum sensitivity possible.

���TransAM advantages• Non-radioactive, colorimetric method
provides quantifiable results• Results in less than 5 hours• 10-fold greater sensitivity than gelshift• Simultaneous profiling of multiple
family members• Ability to assay cell and tissue samples
Complete solution for activation assays
TransAM Kits improve transcription factor
activation research because they are faster,
more sensitive and easier to use thanexisting methods. The new GATA-4, GR
and Oct-4 Kits join an already extensive
TransAM product line, including assays for
NFκB p50 and p65, HIF-1, STAT3, c-Jun, as
well as our popular Family Kits. Please visit
www.activemotif.com/transam to learn
which assays are available, and to download
TransAM manuals and a list of publications
that cite the use of TransAM. For a bettermethod for studying transcription factor
activation, try TransAM today.
* Technology covered under AAT-filed patents licensed to Active Motif.

����Product
�����

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Figure 2: Quantitative measurement of GATA-4.
F9 cells are treated with retinoic acid and increasing amounts ofnuclear extract from untreated and treated cells are assayed using theTransAM GATA-4 Kit.

www.activemotif.com

FormatCatalog No.

TransAM™ GATA-4

1 x 96-well plate5 x 96-well plates

46496
46996

TransAM™ GR

1 x 96-well plate5 x 96-well plates

45496
45996

TransAM™ Oct-4

1 x 96-well plate5 x 96-well plates

42496
42996

3

Cell-based ELISAs

continued from page 1 — Profile Transcription Factor Phosphorylation Using New FACE™ NFκB p65 Profiler Kits
NFκB phosphorylation
NFκB p65 plays a pivotal role in the regulation of inflammatory and immune responses. During phosphorylation and degradation of IκBs, NFκB p65 is phosphorylated
on multiple residues, each triggered bydifferent stimuli but essential for maintaining NFκB transcriptional activation (Figure2). Despite the widespread interest in the
study of NFκB, there is a lack of assays
available that are suitable for monitoring
NFκB phosphorylation in living cells. That’s
why Active Motif has developed the new
FACE Profiler Kits. The FACE NFκB p65
Profiler Kit contains antibodies specificfor three phosphorylated sites: Serine 276,
Serine 468 and Serine 536, which enables
you to screen these important sites of
NFκB p65 regulation within a single assay.
The FACE Method
In the FACE method, cells are cultured in
96-well plates and stimulated to induce
the pathway of interest (Figure 3). Following stimulation, the cells are fixed, which
preserves protein modifications, including
phosphorylation. Each well is then incubated with a primary antibody specific forthe activated protein of interest. Subsequent incubation with secondary HRP-conjugated antibody and developing solution
provides an easily quantified, colorimetricor chemiluminescent readout. The signals
can then be normalized for cell number using the provided Crystal Violet solution andplotted (Figure 1, page 1). The entire assay
is complete in about two hours of actual
hands on time – nothing could be simpler.
Two types of detection
For added convenience, all of our FACE
Kits are available in both colorimetricand chemiluminescent formats. The
colorimetric kits detect phosphorylated
and total protein levels using an HRP-

PKA

ser276

P

Phorbol
ester

NFκB p65
P

P

MSK1

ser536

TNF-α

ser468IL-1β

PMA

Figure 2: Multiple phosphorylation sites on NFκB p65.
Transcriptional regulation of NFκB p65 is dependent on the phosphorylation of multiple residues and is controlled by a variety of stimuli.

colorimetric signal at a wavelength of 450
nm and a standard ELISA-plate reader. If
you require maximum sensitivity, takeadvantage of the FACE Chemi Kits. Thesekits use chemiluminescent detection
on a luminometer to accurately monitor even the slightest changes in protein
phosphorylation.

offers you such a quick and convenientsolution. Currently, Active Motif has
developed a number of FACE Kits to study
MAPKs, transcription factors and receptors(see table below). These kits are available
for studying single phosphorylation sites
as well as multiple sites as in the new
FACE Profiler Kits. As we are constantly
adding to the FACE product line, be sureto give us a call or check our website atwww.activemotif.com/face to find out
about new additions to this innovative
product line. To get to the best phosphospecific assay available, order a FACE Kit.

The FACE Family
FACE Kits provide you with a simple, efficient, quantitative method to monitor
proteins activated by phosphorylation.
No other phospho-specific assay available

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 3: Flowchart of the FACE process.
Cells are grown, stimulated and fixed in the same 96-well plate. Addition of primary and secondary antibodies detects phosphorylated or total protein.

FACE Kits available
FACE™ AKTFACE™ c-Jun

FACE™ ErbB-2

FACE™ FKHR (FOXO1)FACE™ JNKFACE™ p38

FACE™ ATF-2

FACE™ c-Src

FACE™ ERK1/2

FACE™ GSK3β

FACE™ MEK1/2

FACE™ PI3 Kinase

FACE™ Bad

FACE™ EGFRFACE™ FAKFACE™ JAK1FACE™ NFκB p65 Profiler

4

Toll Free — 1 877 222 9543

OCTOBER 2004 • VOLUME 5 • NUMBER 4

Cytochrome c & Mitochondrial Fractionation

The FunctionELISA™ Cytochrome c Kit
makes measuring cytoplasmic andmitochondrial cytochrome c levels both
fast and accurate. Unlike other techniques
used for studying proteins, such as Western
blotting, FunctionELISA Cytochrome c is asandwich ELISA-based approach that provides quantitative results in a few hours.

Convenient format saves time
For maximum convenience, the Capture
Antibody in FunctionELISA Kits is suppliedalready immobilized to the wells of the
96-well assay plate. This makes it easy to

Sensitivity counts
Though it can be induced by a wide varietyof events, once the cycle that leads toapoptosis begins, cytochrome c is released
from the mitochondria into the cytoplasm
within one hour. Therefore, assaying thecytochrome c levels in mitochondrial andcytosolic fractions is an extremely usefulway to monitor apoptosis. FunctionELISA
Cytochrome c simplifies this task by
providing everything you need to performsensitive, quantitative measurement of
your samples and generate standard curves(Figure 1).

Absorbance (OD450 nm)

Accurate Monitoring of Apoptosis Using Mitochondrial Cytochrome c Levels

1.5
y = 0.067x - 0.107 r2 = 0.998

1

0.5

0

0

10

20

30

40

50

60

Cytochrome c (ng/ml)
Figure 1: Cytochrome c Standard Curve.
Increasing amounts of recombinant human cytochrome c (0.78-50 ng)
were assayed using the FunctionELISA Cytochrome c Kit.

process multiple samples quickly. Plus, it
saves you the time and inconvenience ofperforming an overnight incubation to attach the antibody yourself.
Complete kit ensures reproducibility
FunctionELISA Cytochrome c contains all of
the reagents required, including recombinant cytochrome c protein, to quickly
quantify cytochrome c in your sample. For
accurate quantification of cytochrome c,try the FunctionELISA Cytochrome c Kit.
Start your experiments out right
To add to the effectiveness of the
FunctionELISA Cytochrome c, Active Motif
recommends its Mitochondrial Fractionation Kit (see below). Use of this kit helps
increase the accuracy of your results by ensuring that your mitochondrial and cytosolic
fractions are properly segregated (Figure 2).

Product
FunctionELISA™ Cytochrome cFormatCatalog No.

1 x 96 well-plate5 x 96 well-plates

48006
48506

Reproducible Isolation of Mitochondrial and Cytoplasmic Fractions
The Mitochondrial Fractionation Kit isolates
highly enriched mitochondrial and cytosolic
fractions from mammalian cell lines. This
simplifies the study of protein translocation

Absorbance (OD450 nm)

0.8
0.6

Cytosolic
Mitochondrial

0.4
0.2
0

Uninduced

16 hr apoptosis

Cellular fractions (2 µg/well)Figure 2: Monitoring cytosolic and mitochondrial cytochrome c.
HeLa cells were grown to 90-95% confluence and treated with 10 nM
Actinomycin D to induce apoptosis. Cell samples were harvested prior
to treatment as well as 16 hours post-treatment. The Mitochondrial
Fractionation Kit was used to isolate cytosolic and mitochondrial
fractions from each sample. Two µg of each fraction was assayedusing the FunctionELISA Cytochrome c Kit to monitor changes in the
mitochondrial and cytosolic cytochrome c levels.

www.activemotif.com

events that occur during apoptosis and inmany other signal transduction pathways.
The kit’s high-quality reagents and optimized
protocol eliminate cross-contamination andproduce high yields of properly segregatedmitochondrial and cytosolic fractions.
Precise fractionation
To demonstrate its utility, Active Motif’s
Mitochondrial Fractionation Kit was used
to isolate mitochondrial and cytosolic fractions from both uninduced and apoptotic
HeLa cells. The FunctionELISA™ Cytochromec Kit (see above) was used to determine thelevels of cytochrome c in the mitochondrialand cytosolic fractions because the releaseof cytochrome c from mitochondria is a key
indicator of apoptosis. As expected, nearly
all cytochrome c in uninduced cells wasfound in the mitochondrial fraction, while
nearly all the cytochrome c in the apoptotic
sample was in the cytosol (Figure 2).
Upstream quality for downstream successUsing the Mitochondrial Fractionation Kit,
with its optimized protocol and uniqueformulation of buffers, you’ll be certain to
get the specifically segregated fractions you
need. For more consistent results, try the
Mitochondrial Fractionation Kit.

Product

FormatCatalog No.

Mitochondrial Fractionation Kit

100 rxns

400155Nuclear Receptor Assays

continued from page 1 — Nuclear Receptor Measurement Made Easy

Perfect method for NR biomarker analysis
The ability to regulate nuclear receptor
protein activity via addition of an inducing
ligand makes nuclear receptors promising targets for development of improved
therapeutic agents. However, one challenge of the drug discovery process is theidentification of an appropriate biomarker
for monitoring drug efficacy. Because theend point of nuclear receptor activation isDNA-binding, monitoring changes in DNAbinding activity of a target nuclear receptor
can serve as an ideal biomarker. Classicalmethods such as radioactive electrophoretic mobility shift assays (EMSA) andtime-consuming reporter gene assays are
not well suited to this application. Active
Motif’s TransAM™ Kits provide an innovative alternative to these classical assays.
TransAM Kits use a combination of DNA
binding and antibody detection to give a
specific, quantitative readout of DNA-binding activity from all sample types (Figure 3).
To learn more about the TransAM method

6and the many different kits available forquantifying DNA-binding activity of various
nuclear receptor and transcription factor
proteins, please turn to page 3.

GR activation (OD450 nm)

may have both agonist and antagonist
properties, resulting in different biologicaloutcomes. As such, studying the agonist/
antagonist effects of target compounds is
an important element of nuclear receptortargeted drug discovery. Active Motif’s NR
Peptide ELISAs are specifically designed tocapture ligand-activated nuclear receptor
and can be used with both cellular extracts
and recombinant proteins. Each NR Peptide
ELISA Kit provides a 96-well plate that iscoated with a Capture Peptide that includes the consensus-binding motif of thenuclear receptor’s co-activator. Additionof sample results in binding of ligand-activated nuclear receptor to the Capture
Peptide. Each well is then incubated witha primary antibody specific for the nuclear
receptor of interest. Subsequent incubation
with HRP-conjugated secondary antibody
and developing solution provides an easily
quantified readout (see Figure 2, page 1).

1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

Wild-type oligonucleotide
Mutated oligonucleotide

No oligo

20X

40X

80X

Excess oligonucleotide

Figure 3: The TransAM method is specific.
Ten µg of nuclear extract from HeLa cells stimulated with 100 nM
Dexamethasone are assayed using TransAM GR in the absence orpresence of oligonucleotides containing either the wild-type ormutated consensus-binding site of GR. The inability of the mutatedcompetitor oligonucleotides to reduce the signal demonstrates thatthe assay is specific for activated GR.

NR antibodies for all applications
Active Motif’s extensive line of antibodies provide superior performance andreliable results. We offer over 200 highly
characterized antibodies directed against
transcription factors and nuclear receptors.
Antibodies for GR, PPARα and γ, PXR, RARβ, -β2 and -γ, RXR-α and -β, and VDR are
all available, with more being added all thetime. These mono- and polyclonal antibodies are suitable for a variety of applications
including Western blotting, EMSA and chromatin immunoprecipitation. Check out our
online search tool at www.activemotif.com
to see if we have the antibody you need.
Product

Extensive line of recombinant NR proteins
Active Motif’s growing line of recombinant
proteins includes an increasing number ofrecombinant nuclear receptor proteins.
Our recombinant proteins are ideal for use
as positive controls, in in vitro screening
studies and in many other applications.
For more details, see page 12 or go to our
website at www.activemotif.com to download complete information.
Coming soon….Simple, fast measurementof nuclear receptor proteins
The new NR ELISAs offer a simple, rapid
method to monitor changes in nuclear
receptor proteins. NR ELISAs utilize a
Sandwich ELISA-based method that is an
improvement over other methods used to
study proteins, such as Western blotting.
Using NR ELISAs means that there is noneed for gels, blotting or long, tedious
incubations. The 96-well format is convenient and sensitive, with only a minimal
amount of material required to give aquantitative readout of nuclear receptor
levels. NR ELISAs are ideal for use on clinicalmaterial and will initially be available for
ERα, PR and AR, with many more to be
added over the upcoming months. Sendin the enclosed reply card or give us a call,
and we’ll be happy to keep you updated of
new NR ELISAs as they become available.
For a complete line of nuclear receptor
analysis tools, there’s no need to lookfurther than Active Motif!
FormatCatalog No.

NR ERα Peptide ELISA

1 x 96-well plate5 x 96-well plates

49096
49596

NR ERα Peptide ELISA Chemi

1 x 96-well plate5 x 96-well plates

49097
49597

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

Toll Free — 1 877 222 9543

OCTOBER 2004 • VOLUME 5 • NUMBER 4

Protein Delivery

Deliver Active Proteins Directly Into Mammalian 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 the ideal
tool for a variety of functional studies.

Deliver biologically active proteins
The ability of Chariot to deliver biologically
active compound was demonstrated using
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.
Figure 3: Staining of HeLa cells.
One µg of β-galactosidase protein, which is included with Chariot for
use as a positive control, was complexed with Chariot for 30 minutes
and delivered into HeLa cells. Cells were fixed and stained 2 hours
post-delivery using Active Motif’s β-Galactosidase Staining Kit.

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).

A

0

G1

10

15

20
(h)

G2

Figure 2: Chariot delivery of functional protein.
HS-68 cells arrested in G0 phase by serum deprivation for 48 hours
were released by addition of serum for 3 hours. Flow cytometry
performed 0, 10, 15 and 20 hours after addition of Chariot alone anda Chariot-p27kip1 complex indicate that 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.

Why use protein delivery?
Direct delivery of active protein makes it
easy to perform studies not even possibleusing DNA transfection and expression.
Successful Chariot delivery of proteins,
peptides and antibodies has been shownin a wide range of cell lines, including
hard-to-transfect neuronal, primary andplant cells. For a list of papers that cite
the use of Chariot, simply return theenclosed reply card or download the list at
www.activemotif.com/chariot.

Deliver large proteins
Chariot can efficiently deliver a broad range ofmacromolecules directly into mammalian cells
and the results can be assayed immediately.
To demonstrate, Chariot was used to deliverfunctional β-galactosidase protein (a tetramer
composed of four identical, 119 kDa subunits)
into HeLa cells (Figure 3).

Chariot delivers results
Chariot speeds and simplifies a variety offunctional studies because it efficiently delivers biologically active proteins, peptides
and antibodies directly into mammaliancells, even hard-to-transfect and non-dividing cells. To learn what your protein is really
doing, study it using Chariot.

B

0
G1

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 protein folding characteristics
of your protein and, ultimately, its function.
Because Chariot forms a non-covalentbond with your protein, it does not affectthe delivered protein’s folding or function.

Advantages• Delivers active protein directly
into living cells — no fixing needed• Up to 95% efficiency in less than
2 hours• Works in a variety of cell lines,
as well as in vivo

10

15

20
(h)

G2

Product

FormatCatalog No.

Chariot™

25 rxns*
100 rxns

30025
30100

β-Galactosidase Staining Kit

75 rxns

35001* A rxn is defined as sufficient Chariot reagent to deliver protein to cells in a 35 mm plate.

www.activemotif.com

7

DNA Repair

Quantitative DNA Repair ELISAs for GTBP, Ku and RPA
Active Motif’s DNA Repair Kits offer abetter alternative to Western blots andgelshift assays for the study of DNA repair
protein activity because their DNA-binding
ELISA format eliminates radioactivity andprovides quantitative results in less than
five hours. The DNA Repair Kits are 10-foldmore sensitive than traditional assays andcan be used with both cell lines and tissues,
providing you with even more flexibility.

GTBP

Absorbance (OD450 nm)

1.25

1
0.75

0.5

0.25
0

Raji

PANC-1

JurkatFigure 2: Measurement of GTBP activity in different cell lines using
Western blot and the GTBP DNA Repair Kit.
Raji, PANC-1 and Jurkat nuclear extracts were assessed for GTBP activity using Western blot (A) and the GTBP DNA Repair Kit (B).

8

1 hr.

Add anti-IgG
HRP conjugate

1 hr.

Cell extractcontaining DNA
repair protein

Add developing
and stop solution

1 hr.

Oligonucleotide
coated plate

Figure 1: Flowchart of the DNA Repair Kit procedure.

More quantitative results
The ability to distinguish between smallchanges in DNA repair protein activity isimportant for understanding DNA damage
and repair protein interactions. Assays like
Western blots are sensitive yet yield morequalitative rather than quantitative results.
Active Motif’s DNA Repair Kits provide
colorimetric readings that can be easily
quantified, enabling the detection of eventhe slightest change in protein activity. To
illustrate, GTBP activity was measured andcompared using Active Motif’s GTBP DNA
Repair Kit and Western blot. The GTBP Kit
results are clearly more quantitative thanWestern blot (Figure 2).
Improved accuracy for better results
In addition to their ease-of-use and increased sensitivity, DNA Repair Kits provide
more accurate measurements because theyuse colorimetric detection, which is linear
over a wider sample range (Figure 3). This
saves you both time and money, as fewer
samples will need to be reassayed to obtainaccurate results. Get the most out of yourexperiments by using the DNA Repair Kits.

A

B

Add anti-DNA
repair antibody

Product

0.8
y = 0.121x + 0.125 r 2 = 0.998
Absorbance (OD450 nm)

How it works
The DNA Repair Kits utilize the DNA-binding properties of DNA repair proteins to
selectively capture activated protein. EachDNA Repair Kit includes a 96-well plate inwhich multiple copies of a specific oligonucleotide have been immobilized. Whencellular extract is added, the repair protein
of interest binds to the oligonucleotide
on the plate. Each well is then incubatedwith a primary antibody that is specific forthe repair protein being studied. Additionof a secondary HRP-conjugated antibody
and developing solution provides an easily
quantified colorimetric readout (Figure 1).
This allows you to easily monitor the stimulation or deficiency of DNA repair proteinsin a particular cell line, animal model ortumor biopsy.

Add extract
to plate

0.6

0.4

0.2

0

0

1

2

3

4

5

6

Raji nuclear extract (µg)
Figure 3: Monitoring Ku70 with the Ku70/86 DNA Repair Kit.
Different amounts of nuclear extract from Raji cells were tested for
activity using the Ku70/86 DNA Repair Kit.

Try one today
Our DNA Repair Kits will simplify the way
you study interactions between damaged
DNA and repair proteins. These easy-to-use
kits are flexible enough to use in a variety
of functional studies, such as examining thespecificity of repair proteins for a particular
type of DNA damage. Don’t waste timewith inefficient blotting and radioactivity,
try a DNA Repair Kit today.
FormatCatalog No.

GTBP DNA Repair Kit

1 x 96 well-plate5 x 96 well-plates

51096
51596

Ku70/86 DNA Repair Kit

1 x 96 well-plate5 x 96 well-plates

51196
51696

RPA DNA Repair Kit

1 x 96 well-plate5 x 96 well-plates

51296
51796

Toll Free — 1 877 222 9543

OCTOBER 2004 • VOLUME 5 • NUMBER 4

Gene Modeling

GeneDetective™ Generates Accurate Models for Exploring Alternatively Spliced Genes
GeneDetective™ provides the computational tools you need for constructing highly
accurate gene models, which are necessaryfor exploring alternatively spliced genes.
The system simplifies the development ofgene models across entire genomes.
GeneDetective is on the case
GeneDetective elucidates intron/exon
structure by comparing your cDNA, EST andprotein sequences to genomic DNA. The
system’s algorithm pipeline reconstructs
splice junctions in order to accurately alignsequences to genomic regions. It identifies
exons as small as 10 bases and can span
introns of 100 Kb in order to deliver highly
accurate gene models. GeneDetective has
demonstrated greater sensitivity1 thanother current gene modeling tools such as
GeneWise2, and is considerably faster thansoftware-only solutions.
GeneDetective advantages• Easy visualization of intron/exon
structure with a zoomable view
(Figure 1)• Rapid searches are processed by theultra-fast DeCypher Engine™
• Utilizes splice site correction andmultiple sequence alignment to
identify correct exons• Produces a predicted transcript foreach gene model for further analysis• Use via web browser or command line
client for scripted batch searches• Easily add additional software modules (BLAST, HMM, Smith-Waterman)
If you’re focused on alternatively-spliced
genes, GeneDetective’s highly accurate
gene models make it a valuable tool foryour genome exploration studies. The
turnkey, single-computer GeneDetective
is an ideal solution for cost-effective genemodeling analyses. Give us a call or returnthe enclosed reply card to learn more.
1. Roland Luethy, Ph.D. (2004) Gene modeling using amino acid togenomic sequence alignments, RECOMB poster.
View at: www.timelogic.com/downloads/genedetective_poster.pdf
2. Birney et al. (2004) Genome Res. 14(5):988-995.

Figure 1: Intron/Exon structure visualization of the human Fatty Acid Synthase (FAS) gene. GeneDetective was used to align human ESTs with genomic regions.
Gene models are valuable for identifying exon-specific primers for experimental validation of a gene. Exon-specific oligos can be used to construct microarraysfor profiling the expression patterns of alternative splice forms and disease-related polymorphisms.

The DeCypher Engine G4 speeds GeneDetective mapping comparisons
GeneDetective is a DeCypher application, so it has the computational horsepower for evenlarge gene modeling projects. When deployed in a single 2-CPU server or workstation, the
DeCypher Engine PCI card(s) easily handle the rigorous sequence comparison phase of thegene modeling pipeline. With GeneDetective, you don’t need the complication of adding
30-60 computing nodes to undertake extensive annotation projects.
GeneDetective can be expanded with additional software modules for high-throughput
BLAST, sensitive Smith-Waterman searches and Hidden Markov Model analysis methods
for elucidating protein family (PFAM) and superfamily assignments. An additional DeCypher
Engine can be installed in minutes to double your analysis throughput.
Software Module

DeCypher Engines

GeneDetective™ (software plus 1 DeCypher Engine*)

1

BLAST
Smith-Waterman
Hidden Markov Model (HMM)———* Start with GeneDetective software and one DeCypher Engine, then add additional software modules and/or DeCypher Engines as your needs evolve.
** Introductory GeneDetective pricing is valid until December 15, 2004.

www.activemotif.com

9

Transcription Factor Antibodies

Transcription Factor Antibodies for Westerns, EMSA & ChIP
Active Motif’s extensive line of antibodies
will provide you with superior performanceand reliable results. We offer over 200 highly characterized antibodies directed against
transcription factors, including members
from the AML/Runx, AP-1, Cell Cycle
Regulator, NFκB/Rel and STAT families.
These mono- and polyclonal antibodies are
suitable for a variety of applications including Western blotting, EMSA and chromatin
immunoprecipitation.
Western blotting – specific and sensitive
Active Motif’s antibodies are guaranteed
to provide you with superior data in yourWestern blotting experiments. Every
antibody has been fully characterizedusing appropriate cellular extracts, andthe specificity of each antibody is verified
by peptide competition (Figure 1). Eachantibody is also accompanied by a detailed
technical data sheet that provides specificrecommendations on appropriate positive
controls, dilutions and incubation conditions. Say goodbye to non-specific bands
and endless optimization – get the results
you need the first time.
A

B

C

D

E

F

G

H

I

116
97
84
66
55
45
36

Figure 1: Western blot with Active Motif antibodies.
Using nuclear extract from Raji cells, antibodies for Sp1 (B), c-Rel (C),YY1 (D) and Pax-5 (E) were tested individually or as a mixture (A) toreveal their respective antigens. The mixed antibodies were simultaneously incubated with one peptide at a time for Sp1 (F), c-Rel (G), YY1(H) and Pax-5 (I), demonstrating specific competition of each antibody
by its respective peptide.

10

1
YY1 Probe
Oligo - Wild-type
Oligo - Mutant
YY1 Antibody
Peptide2345Competition
Competition
Competition

Supershift

Bandshift

Antibodies for ChIP
Chromatin immunoprecipitation is oneof proteomics’ powerful new tools foranalyzing genome regulation (see page 2).
However, one of the drawbacks of performing ChIP is finding antibodies that are provento work in this technique. To help, we are
testing most of our antibodies in ChIP todetermine which ones function in this procedure. Please see the table on page 2 for alist of ChIP-validated antibodies. As this list
will be growing, please be sure to check our
website for up-to-date information.

Free Probe

Figure 2: Highly specific YY1 mobility shift.
The Nushift YY1 Kit (Catalog No. 37043) detects YY1 using nuclear
extracts prepared from K-562 cells. Specificity is determined by useof the wild-type and mutated competitor oligonucleotides andneutralizing peptide.

Nushift™ – for fail-safe supershifts
Locating commercially available antibodies
that have been optimized for performing
Supershifts in EMSA can be a lengthy anddifficult process. Active Motif has removed
the guesswork with its line of Nushift
antibodies. These antibodies are specifically formulated for Supershift experiments
and are available both individually andas complete Nushift Kits (Figure 2). The
Nushift Kits come with everything needed
to successfully perform Supershift experiments including the transcription factor
antibody of interest, neutralizing peptides(if available), wild-type and mutatedoligonucleotides, ready-to-label wild-type
oligonucleotide probe, positive control
nuclear extract, reaction buffers and G-25
purification columns. Don’t waste yourtime and effort developing and optimizing“home grown” kits; purchase a Nushift Kit
for error-free results every time.

Figure 3: Active Motif’s antibody search engine.
Active Motif’s online search engines enable you to find antibodies
by Catalog No., keyword, isotype, species reactivity and application.
You can also generate a list of cell extracts that can be used as apositive control with a particular antibody in Western blotting,gelshift or ChIP.

Find what you need on the web
Finding your antibody of interest and asuitable positive control is easy using ouronline search tools (Figure 3). You canalso download technical data sheets thatprovide complete information on every
antibody and cell extract. With such a largeline of specialized antibodies and extracts
to choose from, chances are we’ll havewhat you’re looking for. Go ahead and test
drive our website today.

Toll Free — 1 877 222 9543

OCTOBER 2004 • VOLUME 5 • NUMBER 4

Nuclear, Whole-cell & Cytoplasmic Extracts

High-quality Nuclear Extracts
Active Motif offers over 100 different
ready-to-use nuclear, whole-cell andcytoplasmic extracts from a variety of celllines and tissue types (see table). Many areprepared from cells that have been treatedto specifically induce activation of hard-todetect transcription factors. Our extracts
are ideal for use as positive controls in
Western blot, EMSAs, ELISAs and more.
They can also be used as a starting point
for isolating and purifying proteins for use
in other downstream applications.
Quality is the key
When used as a positive control, quality
extracts are the key to ensuring that yourapplications are working correctly. All Active Motif extracts are tested for protein
viability and functionality by various methods, such as Western blot, EMSA or ELISA.
Don’t see the extract you need?
Active Motif’s Nuclear Extract Kit makes
it simple for you to isolate high yields ofactive nuclear, whole-cell and cytoplasmic
extracts from your own mammalian celland tissue samples. Its use eliminates theneed to optimize your own reagents andprotocols, so you get error-free results
every time.
Nuclear Extract Kit advantages• Complete kit contains all required
reagents• No need to optimize your procedure• QC’d reagents ensure reproducibility
NFκB activation (OD450 nm)

2.5

Unstimulated
TNF-α stimulated

2.0
1.5

Nuclear, whole-cell & cytoplasmic extracts available
293

HaCat

MCF-7

R2C

3T3

HeLa

MDA-MB-231

Raji

3T6

HeLa S3

MEL c88

Rat Tissue (various)

624 melHep G2

MG-63

Saos-2

A-431

HL-60

MIA PaCa-2

Schneider’s Drosophila L2

AtT-20/D16v-F2

HT-29

Mouse Brain

SK-N-BE (2)B16

Human Tissue (various)

Nb2

SW48

C2C12

JEG-3

NCI-H441

T-47D

Caco-2

Jurkat

NCI-H446

THP-1

CCF-STTG1

K-562

NCI-H661

U-373 MG

CCRF-CEM

Kelly

NIH/3T3

U-87 MG

COS-7

KG-1

NIH:OVCAR-3

U-937

Daudi

L6E9

P19

WI-38

DU 145LA-4

PANC-1

Y-1

F9

LNCaP

PC-3

Y-79

GH3

LoVo

PC-12

ZR-75-1

Results you can rely on
Accurate research often depends on the
precise isolation of proteins from a specificcellular compartment. If your nuclear
extract is contaminated with cytoplasmic
proteins, your results may not be valid. The
Nuclear Extract Kit helps ensure that proteins remain segregated in their particular
fraction. To demonstrate the importance ofusing a quality extraction procedure, nuclear and cytoplasmic extracts were preparedfrom unstimulated and TNF-α-stimulated
HeLa cells using our Nuclear Extract Kit andassayed for activated NFκB p50 using ourTransAM™ NFκB p50 DNA-binding assay(Figure 1). As activated NFκB is not present

in the cytoplasm, only nuclear extract fromthe stimulated cells should test positive.
This is precisely what was seen, demonstrating the specificity of the kit.
Quality in means quality out
Extracts prepared using the Nuclear Extract
Kit are ideal for use in any experiment that
requires nuclear, whole-cell or cytoplasmic
extract. Whether you choose to make yourown extracts using our Nuclear Extract Kit
or to purchase our ready-to-use extracts,
the use of high-quality extracts will
improve the accuracy of your downstream
experiments. For complete details, visit our
website at www.activemotif.com.

1.0
0.5
0.0

0.625

1.25

2.5

5

10

0.625

1.25

2.5

5

10

Cytoplasmic

Nuclear

Product

FormatCatalog No.

Nuclear Extract Kit

100 rxns
400 rxns

40010
40410

All extracts

200 µg***Extract/well (µg)
Figure 1: NFκB p50 activity in nuclear and cytoplasmic extracts.
Increasing amounts of nuclear and cytoplasmic extracts isolated with
the Nuclear Extract Kit are assayed using TransAM™ NFκB p50.

www.activemotif.com* Most human, rat and mouse tissue extracts are supplied as 120 µg aliquots.
** Call us or visit our website for complete information on all of our extracts, including catalog numbers and details on growth & stimulation conditions.

11

Recombinant Proteins

Recombinant Proteins for Your Research
Active Motif offers an ever-growing line ofrecombinant proteins that are ideal for use
in many different biological applications,
including in vitro transcription assays, as
EMSA controls, in protein-protein interaction studies and as protein standards inon the recombinant proteins, including
detailed technical data sheets that specify
protein length, the species it was produced
in, the method used for purification, etc.,
can be found at www.activemotif.com.

Recombinant proteins available

1.5
y = 0.468x - 0.055 r2 = 0.999

Absorbance (OD450 nm)

ELISAs. The c-Fos, c-Jun, c-Myc, CREB, NFκB
p50, NFκB p65, p53, and Sp1 proteins havebeen validated for use in making standard
curves in our TransAM™ Transcription Factor
ELISAs (see page 3). Complete information

1

0.5

0
0

0.5

1

1.5

2

2.5

p50 Recombinant Protein (ng)

3

AKT1

ER

NFκB p65

RARβ

ATF-2

FXR

p53

RARγ

BRCA1

GR

p300

RXRα

c-Fos

IκBα

PPARα

RXRβ

c-Jun

JNK2α1PPARβ(δ)

RXR-LBD

c-Myc

JNK2α2

PPARγ

Sp1

CREB

LXRα

pRB

STAT1

CTF1 (NF-1)

LXRβ

Rad51

TRα1

eIF2α

NFκB p50

RARα

TRβ1

Figure 1: NFκB p50 Standard Curve.
A NFκB p50 standard curve was generated using the recombinant
NFκB p50 protein and the TransAM™ NFκB p50 Kit.

12Toll Free — 1 877 222 9543

CONTACT INFO
U.S.
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

EUROPE
104 Avenue Franklin-Box 25
B-1330 Rixensart, Belgium
Toll Free: +800 222 95 430
Direct: +32 (0)2 653 0001
Fax: +32 (0)2 653 0050
Email: eurotech@activemotif.com

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