THE NEWSLETTER OF ACTIVE MOTIF — MARCH 2004 • VOLUME 5 • NUMBER 1

Chromatin Immunoprecipitation Made Easy
Active Motif’s ChIP-IT™ Kit is the mosteffective product available today forchromatin immunoprecipitation (ChIP).
The kit contains a comprehensive protocol
and optimized buffers, inhibitor cocktails,
antibodies and controls, all of which havebeen validated in actual ChIP experiments.
Plus, ChIP-IT Kit antibodies are directedagainst general transcription factors, so you
get a more accurate analysis of promoter
activity than is possible with histone-based
ChIP. If you are performing chromatin
immunoprecipitation, using the ChIP-IT Kit
will improve your results and save you time.

IN THIS ISSUE

The ChIP method
In the ChIP-IT method, intact cells are fixedusing formaldehyde, which cross-links andtherefore preserves protein/DNA interactions.
The DNA is then sonicated into small, uniform
fragments and the DNA/protein complexes
are immunoprecipitated using an antibody
directed against a DNA-binding protein ofinterest. Following immunoprecipitation, thecross-links are reversed and the DNA fragments are screened to determine which geneor groups of genes were bound by the protein
of interest (see Figure 1, page 4).

ERα activation (OD450 nm)

a unique peptide-capture system that provides you with quantitative results in about
three hours (Figure 1). And, the method is
flexible enough to use on multiple sample
types, including both cellular samples andrecombinant proteins.
continued on page 6

1

Quickly Quantitate Estrogen
Receptor Activity

2

Deliver Biologically Active
Proteins Into Mammalian Cells

3

The Answer for Assaying Transcription Factor Activation

5

TimeLogic® DeCypherBLAST– The Solution for Fast, Flexible
Genome Analysis

7

Cell-based Method for ErbB-2(Her2/neu), GSK3β and EGFR
Phosphorylation Analysis

8

Sandwich ELISAs for Fast, Accurate Protein Quantification
and Normalization

9

Colorimetric Quantitation of
Replication Protein A

Silica Resin for More
10 UseSpecific Protein Purification

0.8

estradiol
DES
genisteintamoxifen
clomiphene
ICI 182,780

0.6
0.4
0.2
0

Chromatin Immunoprecipitation
Made Easy

continued on page 4

Quickly Quantitate Estrogen Receptor Activity
Active Motif’s new Nuclear Receptor ERαELISAs make it fast and easy to accurately
measure the effects and tissue selectivityof agonists and antagonists on Estrogen
Receptor alpha activity. Unlike traditional
assays, which are time consuming and expensive, the Nuclear Receptor ELISAs utilize

1estradiol

DES

genistein tamoxifen clomiphene ICI 182,780

Drug (25 µM)

Proteins for
10 RecombinantYour Research

11 mTRAP™ Isolates More mRNAcient, Affordable
12 EffiCompetent E. coli

Figure 1: ERα agonism/antagonism response of estradiol, diethylstilbestrol (DES), genistein, tamoxifen, clomiphene and ICI 182,780.
Fifteen µg of nuclear extract from the breast cancer cell line MCF-7 are incubated in the presence of test compounds (25 µM final concentration) in wells of the
Nuclear Receptor ERα ELISA plate. Only ligand-activated ERα can bind to the Capture Peptide that is immobilized in the plate. The bound ERα is specifically detectedwith an ERα antibody. Secondary antibody and detection solution are then used to quantitate ligand-activated ERα. Note that in MCF-7 cells estradiol, DES andgenistein are ERα agonists while tamoxifen, clomiphene and ICI 182,780 are ERα antagonists. (DES, genistein, clomiphene and ICI 182,780 results are provided fordemonstration purposes only; these compounds are not included in the Nuclear Receptor ERα ELISA Kits.)

www.activemotif.com

Protein Transfection

Deliver Biologically Active Proteins 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.
Targeted delivery
Chariot is a peptide that forms a non-covalent complex when combined 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).

Deliver large proteins
Chariot can efficiently deliver a broad rangeof macromolecules directly into mammaliancells. To demonstrate, Chariot was used todeliver functional β-galactosidase protein (atetramer composed of four identical, 119 kDa
subunits) into HeLa cells (Figure 3).

A

A

0

G1

10

15

20
(h)

G2

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.

B

B

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

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.

shown in a wide range of cell lines, including hard-to-transfect neuronal, primary andplant cells. The method has proven to beeffective on both adherent and suspension
cells, as well as in vivo. For a list of papers
that cite the use of Chariot, simply returnthe enclosed reply card or download thelist at www.activemotif.com/chariot.

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; successful delivery ofproteins, peptides and antibodies has been

Advantages• Delivers active protein directly
into 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
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.

Product

FormatCatalog No.

Chariot™

25 rxns*
100 rxns

30025
30100

β-Galactosidase Staining Kit

75 rxns

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

2

Toll Free — 1 877 222 9543

MARCH 2004 • VOLUME 5 • NUMBER 1

Transcription Factor ELISAs

The Answer for Assaying Transcription Factor Activation
NFκB activation (OD450 nm)

TransAM™ Kits are DNA-binding ELISAs thatfacilitate the study of transcription factor
activation in mammalian tissue and cell
culture extracts. The method is faster andmore sensitive than other commonly usedmethods, while eliminating the use of radioactivity. Many different kits are available forstudying both individual transcription factors as well as transcription factor families.

A? How does TransAM work?

2.5
2
1.5
1
0.5
0

?

0.5

1

2
5
10 25
Cell extract/well (µg)

How is TransAM an improvement
over existing methods?

Two commonly used methods to study
transcription factor activation are gelshift/
EMSA and reporter gene assays. TransAM
has a number of advantages over each ofthese procedures. With respect to gelshift,
TransAM assays are 10-fold more sensitive,

5 10 25 50

50

Unstimulated

IL-1α stimulated
0.5 1

Transcription factors bind to their 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 specific double-stranded oligonucleotide are immobilized. When sample
is added, the activated transcription factor
binds to the oligonucleotide at its consensus-binding sequence. Primary antibody
directed against the bound form of thetranscription factor is then added, followedby an HRP-conjugated secondary antibody.
The colorimetric change is measured witha spectrophotometer, which provides asensitive, quantitative measurement of theactivated transcription factor.

types of samples can be? Whatassayed with TransAM?

Unstimulated
IL-1α stimulated

0.5 1

5 10 25 50

TransAM assays are typically used with
nuclear extracts; most commonly, extracts
are made from cells in their normal state
as well as after some form of stimulationthat activates the transcription factor ofinterest. The results are then compared todetermine the extent of activation causedby the treatment. For NFκB proteins,
whole-cell extract can be used. Active
Motif also sells a number of recombinant
proteins that can be used in TransAM assays
to generate a standard curve (see page 10).

B
µg of cell extract
Figure 1: TransAM assays are more sensitive than gelshift andprovide quantitative results.
Human fibroblast WI-38 cells are stimulated with IL-1α for 30 minutes.
Increasing amounts of whole-cell extract are assayed using theTransAM NFκB p50 Kit (A) or gel retardation (B).

so you get more accurate results while
using less sample. TransAM eliminates theuse of radioactivity, as well as the need torun, blot, expose and develop gels. This
saves you a considerable amount of time;
TransAM is complete in less than 5 hours,
while with gelshift you’ll wait days forresults. In addition, the qualitative data provided by TransAM is easier to interpret andcompare than blots (Figure 1). TransAM also
overcomes the problems associated with
the inconsistency of reporter gene assays.
Because transfection is a variable process
and gene expression is subject to many confounding factors, reporter assays must be
repeated many times to obtain statisticallyreliable data. And, unlike TransAM, reporter
assays will not work with tissue samples.

transcription factors can be? Whichstudied with TransAM?
Active Motif has developed a number ofkits to study both individual transcription
factors as well as transcription factor families (see table below). TransAM Family Kits
contain multiple antibodies so that you cansimultaneously profile the activation levelsof several different family members.

? What is in a TransAM Kit?
All kits come with the appropriate 96-wellplate(s) and antibody(ies), as well as all buffers required to assay your sample. Positive
control extract(s) is also included to help
ensure results. For complete details, returnthe enclosed reply card or visit our website
at www.activemotif.com. There, you candownload product manuals, as well as a listof publications citing the use of TransAM.

TransAM Product Line
TransAM™ AP-1 Family

TransAM™ AP-1 c-Fos

TransAM™ CREB

TransAM™ MyoD

TransAM™ p53

TransAM™ GATA Family

TransAM™ AP-1 c-Jun

TransAM™ pCREB

TransAM™ NF-YA

TransAM™ PPARγTransAM™ HNF Family

TransAM™ AP-1 FosB

TransAM™ Elk-1

TransAM™ NFATc1

TransAM™ Sp1

TransAM™ IRF Family

TransAM™ AP-1 JunD

TransAM™ ER

TransAM™ NFκB p50

TransAM™ Sp1/Sp3

TransAM™ MAPK Family

TransAM™ ATF-2

TransAM™ HIF-1

TransAM™ NFκB p50 Chemi*

TransAM™ STAT3

TransAM™ NFκB Family

TransAM™ c-Myc

TransAM™ HNF-1

TransAM™ NFκB p65

TransAM™ STAT Family

TransAM™ C/EBP α/βTransAM™ MEF2

TransAM™ NFκB p65 Chemi** TransAM Chemi Kits require the use of a luminometer

www.activemotif.com

3Chromatin Immunoprecipitation

continued from page 1 — Chromatin Immunoprecipitation Made Easy
Analyze the enhanceosome
Gene promoter regions commonly possess
multiple known or putative transcription
factor binding sites. Using sequential or
multiple ChIP experiments to analyze theactive transcription factor sites and thetranscription factor interactions that occur
within this region can be time consuming
and yield variable, conflicting results. The
ChIP-IT Kit, however, makes enhanceosome
analysis faster and more reproducible.
Each ChIP-IT Kit contains all of the buffers,
inhibitor cocktails, controls and blocking
reagents needed for shearing, pre-clearing, immunoprecipitation, washing, elution
and purification of DNA. This means thatsequential and/or multiple ChIPs can be
performed with ease, using consistent reagents and controls that have been provento work. No other kit available today canoffer you such a complete solution.

Cross-link protein to DNA inliving cells with formaldehyde

PCR
primers
Induction

4

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 then the DNA is 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.

Proven controls ensure your results
Successful ChIP depends on sonicatedDNA of the correct length, antibody thatis effective at immunoprecipitating theprotein/DNA complexes and a PCR primer
set that specifically flanks the region of
DNA bound by the protein of interest.
However, it can be extremely difficult todetermine whether “pull down” of the
DNA or its subsequent PCR screening was
successful or not, unless you already havean antibody and PCR primers that havebeen proven to be effective in ChIP.
To help minimize the time and effort spentvalidating antibodies and PCR primer sets,
the ChIP-IT Kit includes a TFIIB antibody
that has been shown to immunoprecipitate
TFIIB-bound DNA specifically. The kit also
contains positive and negative control PCR
primers directed against DNA that either
is or is not bound by TFIIB (Figure 2). Collectively, the ChIP-IT Kit’s proven reagents
make it faster and easier for you to validate
your own antibodies and primers. For more
successful ChIP, try the ChIP-IT Kit.

TargetDNA

Break open cells
and shear DNA

GAPDH
Negative
Control

TFIIB

Negative Control

PCR primers

Negative
Control

ChIP Antibody

TFIIB

Figure 2: PCR amplification of immunoprecipitated DNA. U-937 cells were fixed for 10 minutes with 1% formaldehyde and then ChIP was performed using the
ChIP-IT Kit. The kit’s GAPDH PCR primers flank the TFIIB binding site in the GAPDH promoter region. These primers give a strong PCR product when used on
templates enriched for the GAPDH promoter region, e.g. DNA immunoprecipitated with TFIIB antibody.
The Negative Control primers flank a region of DNA that should not be bound by TFIIB. These primers are used on immunoprecipitated DNA to confirm that
antibody-enrichment of a target DNA is due to specific immunoprecipitation of the protein target, rather than a non-specific precipitation of total DNA.
Note: the Negative Control primers give an amplification product because chromatin immunoprecipitation is an enrichment of DNA bound by a particular
protein, not a complete purification of the DNA of interest. If enough PCR cycles are used, it is always possible to get a PCR product for a given target locus.
DNA immunoprecipitated with the TFIIB antibody and the Negative Control antibody were used in PCRs performed with the GAPDH primers. As shown, the
GAPDH target was effectively immunoprecipitated by the TFIIB antibody (Lane 3) but not by the Negative Control antibody (Lane 2). This demonstrates thatthe DNA enrichment was due to the TFIIB antibody, and was not simply a result of non-specific sticking to the IgG or Protein G beads.
PCRs performed on these same templates with the Negative Control primers produced an equivalent amount of PCR product (Lanes 4 and 5), demonstratingthat the TFIIB antibody did not simply bind non-specifically to chromatin.
Collectively, these data validate that the ChIPs performed with the ChIP-IT Kit and the TFIIB antibody enable efficient and specific enrichment of TFIIB target
DNA, and they demonstrate that the ChIP-IT Kit reagents are suitable for use with any ChIP-proven antibody.

Product

FormatCatalog No.

ChIP-IT™ Kit

25 rxns

53001

Toll Free — 1 877 222 9543

MARCH 2004 • VOLUME 5 • NUMBER 1

BLAST Enhancement

TimeLogic® DeCypherBLAST™ – The Solution for Fast, Flexible Genome Analysis

The processes used to identify genes
and annotate genomic sequences canbe compute-intensive. Large-scalecomparisons to annotated genomes are
required, and these analyses cannot be
handled by publicly available servers. The
solution is DeCypherBLAST™, which brings
tremendous processing power to yoursequence annotation projects through theuse of software and hardware componentsthat are installed in a single computer.
DeCypherBLAST enables you to:• Process complete genome comparisons without the use of an expensiveto-maintain cluster of computers• Utilize familiar BLAST tools withflexible alignment methods• Configure the results format for easy
data integration and faster analysis• Complete more sequence annotations
per day than other methods
High-performance similarity searching
TimeLogic’s DeCypher System dramaticallyincreases the speed of BLAST analyses. In a
recent study, DeCypherBLAST was utilized
to compare 451 EST sequences (384,000
bases) from wheat (Triticum) with NCBI’s
nt database. The search was completed inunder 20 minutes on the DeCypher System,
but would require approximately 1 day tocomplete on the same 2-CPU computer
without DeCypher.

www.activemotif.com

DeCypherBLAST’s speed advantage is evengreater when performing larger searches (i.e.
100,000 or more sequences). For example,
comparison of human chromosome 19 to thecomplete mouse genome can be completed
in as little as 3 hours on a single server.
More optimal local alignment options
DeCypherBLAST includes a variety ofalignment methods that provide greater
sensitivity and flexibility than NCBI BLAST,
enabling you to:• Create accurate alignment representations across nucleic and protein data
• Optimize short sequence alignments(i.e. proteins)• Span introns across genomic data
• Easily identify sequence patterns bylocking the alignment to the 5´ or 3´
end of your query
Flexible result formats improvedata integration
DeCypherBLAST streamlines your analysis
by providing options for the output ofyour data files. You can specify the fieldorder and generate result files in Tab- orcomma-delimited formats, which speeds
and simplifies importing the data into yourproprietary databases.

Integration with other DeCypher modules
Another advantage of DeCypherBLAST
is its strong integration with otherbioinformatics methods. For example, if
you are performing BLAST but also wish to
do protein annotation, DeCypherBLAST cansimultaneously generate files of sequences
for each “hit” in the BLAST search. Processing these files with CLUSTALW produces
a multiple sequence alignment that canbe imported directly into TimeLogic’s
DeCypherHMM™ to generate a model thatcan be used to search for additional domain family members that may have been
missed by the BLAST search.
Keep pace with the data delugeWith over 800 organisms currently being
sequenced, data available for sequencecomparisons is growing at a tremendous
rate. DeCypherBLAST has been designed
for easy scalability, to effectively utilize
growing resources for a deeper understanding of the genes you study. As your analysis
needs increase, simply add an additional
DeCypher Engine to double your system
performance.
DeCypherBLAST enables you to perform
extensive analyses of your sequence data,
which provides you with greater confidence
in your annotations. Its cost-effective performance, powerful alignment methods andturnkey installation lets you begin exploratory bioinformatics studies immediately.
Solution pricing starts from $40,000 andacademic discounts are available. To learn
more about our DeCypher products for
rapid, low-cost genomic analysis, contact us
today by calling toll-free at 877-222-9543,
returning the enclosed reply card or by logging on at www.timelogic.com.

5Nuclear Receptor Binding Assays

continued from page 1 — Quickly Quantitate Estrogen Receptor Activity
Estrogen Receptor regulation
Activation of ERα is dependent on ligand
binding to the receptor. Agonist and antagonist ligands both bind to ERα but have
opposite effects on conformation. When anagonist is bound, a hydrophobic groove is
exposed that enables binding of co-activator proteins (Figure 2A). In contrast, upon
antagonist binding, co-activators are unable
to bind. As inappropriate activation of ERα is
associated with numerous diseases, there isa need for a convenient assay that examines
the effects and tissue selectivity of various
compounds on ERα activity. To fill that void,
we developed the Nuclear Receptor ELISAs.
The Nuclear Receptor ELISA method
Nuclear Receptor ELISAs are simple andefficient and can be used with both cellular
extracts and recombinant proteins. The
Nuclear Receptor ERα ELISAs are designed
to specifically capture the ligand-activatedform of ERα. Each kit contains a 96-stripwellplate that is coated with a sequence-optimized peptide that includes the consensusbinding motif of the ERα co-activators.
When a sample that contains ERα is added
to a well, the ligand-activated ERα binds
to this Capture Peptide. Each well is thenincubated with a primary antibody that is
specific for ERα. Subsequent incubation
with HRP-conjugated secondary antibody
and developing solution provides an easily
quantified colorimetric or chemiluminescentreadout (Figure 2B).
Improved technique
Nuclear Receptor ERα ELISAs are a marked
improvement over other methods used to
study ERα activation. Unlike cell proliferation or reporter gene assays, Nuclear Receptor ERα ELISAs do not require inefficientcloning or cell transfections. Inconsistencies
due to variable transfection of reporter
plasmids and the need to construct stable
cell lines are also eliminated. Nuclear Receptor ERα ELISAs are complete in mere hours,
rather than days, have increased specificity,and provide quantitative results. Studying
ERα has never been easier.

6

A

AgonistligandAgonist-boundnuclear receptor
Co-activator

B

Co-activator recruitment to agonistbound nuclear receptor

Inactive nuclear
receptor

Add sample
to plate

Capture peptide
coated plate

Add primary antibody
1 hr.

Add detection
solution

Add anti-IgG
HRP conjugate

1 hr.

1 hr.

Figure 2: A. Ligand Activation of Nuclear Receptor in the cell. B. Schematic of the Nuclear Receptor ELISA.

Why use Nuclear Receptor ERα?
• Quantitative analysis of agonist/
antagonists effects• Results in ~3 hours• Specific, antibody-based
measurement• High-throughput compatible• Complete kit with proven controls
Two types of detection
To meet your research demands, theNuclear Receptor ERα ELISAs are available
in both colorimetric and chemiluminescentformats. The colorimetric kit utilizes astandard ELISA-plate reader, while theultra-sensitive Chemi kit requires the
Product

use of a microplate luminometer. The
flexible measurement parameters ofchemiluminescence mean that detection
limits can be adjusted to ensure that thesensitivity is appropriate for the sample
type being assayed.
What’s available
Nuclear Receptor ELISAs are currently
available for studying ERα. Additional kits
for studying other nuclear receptors will
be released throughout the year. Be sure
to check out our website for complete
kit configurations, downloadable manuals
and to learn about new additions to thisinnovative product line.
FormatCatalog No.

Nuclear Receptor ERα ELISA

1 x 96-well plate5 x 96-well plates

49096
49596

Nuclear Receptor ERα Chemi ELISA

1 x 96-well plate5 x 96-well plates

49097
49597

Toll Free — 1 877 222 9543

MARCH 2004 • VOLUME 5 • NUMBER 1

Cell-based ELISAs

Cell-based Method for ErbB-2 (Her2/neu), GSK3β and EGFR Phosphorylation Analysis
Fast Activated Cell-based ELISA (FACE™) Kits
provide a simple, innovative alternative toclassical methods for monitoring protein
phosphorylation. With FACE, modification-specific analysis can be performed
without time-consuming cell extractions,gel electrophoresis or membrane blotting.
Plus, in addition to their convenience, FACE
Kits generate better results.
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
with secondary HRP-conjugated antibody
and developing solution provides a colorimetric or chemiluminescent readout thatis 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 activatedprotein levels in the same experiment(Figure 2).

sive and time-consuming. This forces you to
run multiple experiments on different days
and then to compare the results. However,
because the variability of these methods ishigh and the induction of phosphorylation istypically low, this can lead to statistically poor
data. Fortunately, FACE Kits make runningmore samples as simple as adding an extra tip
to your multi-channel pipettor. And, because
FACE Kits are highly reproducible, you’ll get
results that are more statistically relevant.

Uninduced A-431
EGF Induced A-431

0.4

OD450 nm

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.

0.3

Product

FormatColorimetric Kit
Catalog No.

Chemiluminescent Kit
Catalog No.

FACE™ AKT

1 x 96 rxns
5 x 96 rxns

48120
48620

48220
48720

FACE™ EGFR (Y992)

1 x 96 rxns
5 x 96 rxns

48150
48650

48250
48750

FACE™ EGFR (Y1173)

1 x 96 rxns
5 x 96 rxns

48190
48690

48290
48790

FACE™ ErbB-2 (Y877)

1 x 96 rxns
5 x 96 rxns

48130
48630

48230
48730

FACE™ ErbB-2 (Y1248)

1 x 96 rxns
5 x 96 rxns

48105
48605

48205
48705

FACE™ ERK1/2

1 x 96 rxns
5 x 96 rxns

48140
48640

48240
48740

FACE™ GSK3β

1 x 96 rxns
5 x 96 rxns

48170
48670

48270
48770

FACE™ JNK

1 x 96 rxns
5 x 96 rxns

48110
48610

48210
48710

FACE™ p38

1 x 96 rxns
5 x 96 rxns

48100
48600

48200
48700

0.2
0.1
0

Total ErbB-2

Phospho-ErbB-2 Phospho-ErbB-2(Y1248)(Y877)

Figure 2: Monitoring total- and phospho-ErbB-2 using FACE.
The FACE ErbB-2 (Y1248) and ErbB-2 (Y877) Kits were used to assay thelevels of total and phosphorylated ErbB-2 contained within untreatedor EGF treated A-431 cells.

Reproducible, scaleable assay
Typically, phospho-specific analyses are performed on small sample numbers because theclassical methods of study, such as Western
blot and in-gel kinase assays, are labor inten-

www.activemotif.com

Latest additions to the FACE line
FACE Kits are now available for monitoring
activation of two different phosphorylationsites on ErbB-2 (Her2/neu) and EGFR,
as well as one site on GSK3β. Existing
FACE Kits include those for monitoring
phosphorylated p38, JNK, AKT and ERK1/2,
with many other kits on the way. To obtainan up-to-date list of available FACE Kits,
return the enclosed reply card or visit our
website at www.activemotif.com.

7

Sandwich ELISAs

Sandwich ELISAs for Fast, Accurate Protein Quantification and Normalization
Both Active Motif’s FunctionELISA™ Kits andits Normalization Kits utilize the Sandwich
ELISA technique to capture and accurately
quantify the amount of a specific protein inyour sample. This method is faster and simpler to perform than other techniques used
to study proteins, like Western blot. And,
it enables you to make quantitative rather
than just qualitative protein measurements.
Specific, quantitative assay
Sandwich ELISAs utilize two antibodies thatrecognize different epitopes on the protein
to be measured. The first antibody, termed
the Capture Antibody, binds the protein
of interest from the cell lysate. A Detecting Antibody is then used to bind to thecaptured protein. Accurate quantification
can be made by comparing the sample to a
standard curve made using the recombinant
protein included in each kit (Figure 1).
y = 0.015x + 0.032 r=0.999

0.6

0.4

0.2

0

0

10

20

30

40

50

60

Convenient format saves time
The FunctionELISA and Normalization
Kits are faster and simpler than Western
blots because your samples are read on aspectrophotometer (or on a luminometer
for IκBα). This means that you don’t needto run, blot and develop gels. In just hours,
you’ll have accurate, quantitative results thatare easier to compare and interpret thanblots (Figure 2). Plus, the 96-well format
makes it possible to process multiple samples quickly, at your level of throughput.

8

1

2
3
time (hours)

46OD450 nm

0.3
0.2
0.1
0

B

0

1

2

3
4
time (hours)

5

6

7

Figure 2: Time course of TRAIL induction. Jurkat cells treated with 1 nM TNF-α were harvested at various time points. Cell extracts were assayed by Western
blot analysis (A) using TRAIL mAb (Cat. No. 40966) and through the use of FunctionELISA TRAIL Kit (B).

Verify the integrity of your samples
The Normalization Kits can be used toverify your sample’s integrity (Figure 3) andto reduce inaccuracies in other downstream
assays. By normalizing for the levels of thehousekeeping proteins GAPDH and Lamin
B1, you can be sure that you’re comparing
equivalent amounts of your samples.
0.5

0.25

0

GAPDH Control protein (ng)Figure 1: Production of a GAPDH Standard Curve.
The GAPDH Whole-cell Normalization Kit was used with known
amounts of the GAPDH Control protein to generate a standard curve.

00.4

Absorbance (OD450 nm)

Absorbance (OD450 nm)

0.8

A

20

10

5

Nuclear

2.5

20

10

5

Cytoplasmic

2.5

NIH/3T3 extracts (µg)
Figure 3: Assaying cytoplasmic and nuclear extracts for Lamin B1.
The Nuclear Extract & Lamin Normalization Kit was used to assay
for Lamin B1 in extracts from NIH/3T3 cells. As Lamin B1 is a nuclear
protein, its absence verifies the integrity of the cytoplasmic extract.

Advantages• Quantitative results in just hours• No need to run, blot and develop gels
• Easily analyze multiple samples
Complete kits ensure your success
FunctionELISA and Normalization Kits contain all of the reagents required to rapidly
quantify your protein, including recombinant protein for producing a standard
curve. For your convenience, the Capture
Antibody in these kits is supplied immobilized in a 96-well assay plate, so you won’t
waste time with overnight incubations. In
addition, the Normalization Kits containthe same components found in our popular
Nuclear Extract Kit, so you can prepare
quality extracts and normalize their results.
For complete details on these kits, please
return the enclosed reply card or log on to
our website at www.activemotif.com.

Product

FormatCatalog No.

FunctionELISA™ Cytochrome c

1 x 96 rxns
5 x 96 rxns

48006
48506

FunctionELISA™ IκBα

1 x 96 rxns
5 x 96 rxns

48005
48505

FunctionELISA™ TRAIL

1 x 96 rxns
5 x 96 rxns

48010
48510

Nuclear Extract & Lamin Normalization Kit

1 x 96 rxns
5 x 96 rxns

48008
48508

GAPDH Whole-cell Normalization Kit

1 x 96 rxns
5 x 96 rxns

48007
48507

Toll Free — 1 877 222 9543

MARCH 2004 • VOLUME 5 • NUMBER 1

DNA Repair

Colorimetric Quantitation of Replication Protein A

Why study RPA?
RPA is composed of three subunits, RPA70,
RPA32 and RPA14, which bind to ssDNA
during DNA replication, recombination
and repair. Once bound, RPA organizes andprotects the ssDNA. Deficiencies in RPA
activity can lead to cell cycle arrest andapoptosis. Despite the growing interest in
studying this important regulator of DNA
metabolism, there is a lack of convenient,sensitive assays that are suitable formodern high-throughput research. That’s
why the scientists at Active Motif havedeveloped the new RPA DNA Repair Kits.
Advantages• 10X more sensitive than EMSA
• Works in human, hamster and yeast• Two primary antibodies to study
RPA32 and RPA70• Linear over a broad sample range
The DNA Repair Kit method
Each RPA DNA Repair Kit includes a 96-wellplate in which multiple copies of a singlestranded oligonucleotide have been immobilized. When nuclear extract is added, theProduct

Cell extractcontaining DNA
repair protein

Oligonucleotide
coated plate

1 hr.

Add developing
and stop solution

Add anti-IgG
HRP conjugate
1 hr.
1 hr.

Figure 1: Flowchart of the RPA DNA Repair Kit procedure.

RPA protein binds to this oligonucleotide.
Each well is then incubated with a primary
antibody that is specific for either RPA32 or
RPA70. Addition of a secondary HRP-conjugated antibody and developing solution
provides an easily quantified colorimetricreadout (Figure 1). The easy-to-follow protocol makes it simple for you to monitor both
the RPA32 and RPA70 subunit activity under
different growth and stimulation conditions.
Flexible format, accurate results
RPA DNA Repair Kits are accurate overa wider range of sample concentrations
than other methods because they utilize
colorimetric detection, which is linear overa broader sample range (Figure 2). This saves
you time and money, as fewer samples willneed to be diluted and reassayed to obtainaccurate results. Plus, the kit comes in a convenient 96-stripwell format, which provides
you with the flexibility to use from 1 to 96
wells at a time, depending on the needs ofyour experiment.
FormatCatalog No.

RPA DNA Repair Kit

1 x 96 well-plate5 x 96 well-plates

51296
51796

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

www.activemotif.com

Add anti-DNA
repair antibody

Add cell extract

1.2
y = 0.0979x + 0.0595 r2 = 0.995

1
RPA70 binding (OD450 nm)

Active Motif’s RPA DNA Repair Kit makes
quantitation of Replication Protein A (RPA)
activity easy to perform. That’s because our
DNA Repair Kits are DNA-binding ELISAs
that eliminate radioactivity and provide
quantitative results in less than five hours.
This makes studying RPA regulation fasterand more sensitive than traditional assays
like gelshifts and Westerns. Plus, the DNA
Repair Kits can be used on all sample types,
including cell lines and tissues, giving you
unsurpassed flexibility.

0.8

0.6

Raji
Jurkat

0.4
0.2
0

0

2

4

6

8

10

12

Nuclear Extracts (µg/well)Figure 2: Quantitation of RPA70 with the RPA DNA Repair Kit.
Various amounts of nuclear extract from Raji and Jurkat cells wereassayed for RPA70 activity using the new RPA DNA Repair Kit.

A complete solution
Active Motif’s RPA DNA Repair Kits comecomplete with optimized reagents including a positive control nuclear extract
to simplify the way you study interactions between damaged DNA and repair
proteins. Plus, the kit supplies antibodies
against both RPA32 and RPA70 so you can
examine the differences between these
two subunits side by side. Don’t waste timewith inefficient Western blots — use RPA
DNA Repair Kits today.

9

6xHis-Tagged Protein Purification / Recombinant Proteins

Use Silica Resin for More Specific Protein Purification
Specific protein purification is simple with
Active Motif’s Ni-TED™ Protein Purification
System. Unlike traditional resins madeof agarose, Ni-TED uses a silica resin thatmakes purification of 6xHis proteins better
than ever before.

1

2

3

4

5

6

7

8

9

250
LacZ
98
64
50

Stop non-specific binding
Protein purification using agarose-based
nickel columns is frequently plagued by theco-purification of untagged proteins. This iscaused by their non-specific binding to the
agarose matrix. In contrast, Ni-TED resin is
made of silica, which has fewer side chains
than agarose. This increases the selectivityof Ni-TED and reduces the non-specificbinding of cellular proteins. The result is amuch cleaner purification of your target
protein (Figure 1).

36
30

eGFP

16

Figure 1: Dual expression in the Cascade™ Expression System.
lacZ and eGFP were cloned into the pCHROMO and pCHROMO2
vectors, respectively, and integrated into TAP E. coli. Five hours after
induction (2 mM salicylate), the two proteins were co-purified using
Ni-TED Silica, then run on a 4-20% Tris-Glycine gel.
Lane 1:
Protein standard (kDa)
Lane 2:
LysateLane 3:
Flow-throughLanes 5:
WashesLanes 6-9: Elutions

Product

FormatCatalog No.

Ni-TED™ Spin Columns

25 rxns
100 rxns

97025
97100

Ni-TED™ Spin Columns (w/solutions)

25 rxns
100 rxns

98025
98100

Ni-TED™ Silica (0.3 g resin in prepacked columns)

5 columns
20 columns

97005
97020

1g

97001

Ni-TED™ Silica

One-step protocol
Purification using Ni-TED is complete in one
simple step and works for 6xHis-tagged
proteins produced in any expression system.
You can elute your proteins under both
native and denaturing conditions using
either competition with imidazole or by asimple reduction in pH.
Flexible formats
Great for small- or large-scale purifications,
Ni-TED is available in a variety of formats
to meet your purification needs. Ni-TED
Spin Columns for protein isolations fromsmall samples (0.1-10 ml of culture). The Spin
Columns feature a silica-based membranewith a binding capacity of 50 µg that is ideal
for proteins or peptides of < 100 kDa. For
larger-sized samples, Ni-TED Silica is suppliedin prepackaged columns or in bulk for
purifications from culture volumes of up to
100 ml. Ni-TED Silica will bind up to 3 mg of
His-tagged protein per gram of resin and issuitable for isolating proteins of any size. For
quick, efficient purification, try Ni-TED today.

Recombinant Proteins for Your Research
Active Motif now offers a complete line ofrecombinant proteins that are ideal for use
in many different biological applications.
The NFκB p50, NFκB p65, p53 and CREB

proteins are currently optimized for use in
our TransAM ELISAs (see page 2). Complete
information on the recombinant proteins,
including detailed technical data sheetsthat specify protein length, the species it
was produced in, method of purification,etc., can be found at www.activemotif.com.

Recombinant Protein Product Line
Recombinant AKT1 protein

Recombinant CTF1 (NF-1) protein

Recombinant LXR protein

Recombinant RAD51 protein

Recombinant ATF-2 protein

Recombinant eIF2α protein

Recombinant NFκB p50 protein

Recombinant RAR protein

Recombinant BRCA1 protein

Recombinant ER protein

Recombinant NFκB p65 protein

Recombinant RXR protein

Recombinant BRCA2 protein

Recombinant FXR protein

Recombinant p300 protein

Recombinant RXR-LBD protein

Recombinant c-Fos protein

Recombinant GR protein

Recombinant p53 protein

Recombinant Sp1 protein

Recombinant c-Jun protein

Recombinant IκBα protein

Recombinant p53 R273H protein

Recombinant STAT1 protein

Recombinant c-Myc protein

Recombinant JNK2α1 protein

Recombinant PPAR protein

Recombinant TR protein

Recombinant CREB protein

Recombinant JNK2α2 protein

Recombinant pRB protein

10Toll Free — 1 877 222 9543

MARCH 2004 • VOLUME 5 • NUMBER 1mRNA and Total RNA Isolation

mTRAP™ Isolates 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

Competent E. coli

Efficient, Affordable Competent E. coli
RapidTrans™ are high-efficiency competentE. coli supplied in a convenient, 96-tube
tray. Ideal for cloning, plasmid preparationand library construction, RapidTrans cells
are affordably priced and packaged in aformat that provides maximum flexibility
while eliminating waste. Each tube contains
50 µl of cells for one transformation reaction. In contrast to other 96-well formats
that require thawing all 96 reactions at one
time, RapidTrans enables the use of as few
or as many reactions as needed, without
thawing the other cells. This eliminates the
reduced efficiencies and waste caused byrepeated freeze/thaw cycles.
Strain & competency availability
RapidTrans TAM1 competent E. coli can bepurchased with or without the F´ and λ pir+genotype modifications depending on yourneeds. For applications that require higher
efficiencies, some RapidTrans strains areavailable in an Extra Competent format that
supplies an efficiency of > 3 x 108 cfu/µg.

12Advantages• Freedom to use anywhere from 1 to 96
reactions eliminates wasted reactions• Choice of > 1 x 108 or > 3 x 108 cfu/µg• Economical pricing
Additional information
To learn more about RapidTrans Competent E. coli, including specific genotype
information, please return the enclosed
reply card or log on to our website atwww.activemotif.com/rapidtrans.
Product

FormatCatalog No.

RapidTrans™ TAM1-F´ Competent E. coli

1 x 96 rxns
5 x 96 rxns

10096
10596

RapidTrans™ TAM1-F´ Extra Competent E. coli

1 x 96 rxns
5 x 96 rxns

10099
10599

RapidTrans™ TAM1 Competent E. coli

1 x 96 rxns
5 x 96 rxns

11096
11596

RapidTrans™ TAM1 Extra Competent E. coli

1 x 96 rxns
5 x 96 rxns

11099
11599

RapidTrans™ TAM1 λ pir+ Competent E. coli

1 x 96 rxns
5 x 96 rxns

11097
11597

Toll 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: tech_service@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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US POSTAGEPAID
PERMIT #236
92054