THE NEWSLETTER OF ACTIVE MOTIF — MAY 2004 • VOLUME 5 • NUMBER 2

Faster Phospho-specific Analysis
using Cell-based ELISAs

1

Faster Phospho-specific Analysis
using Cell-based ELISAs

1

Simple, Efficient Delivery ofProteins into Cells

2Complete Solution forMore Successful Chromatin
Immunoprecipitation (ChIP)

Phospho-c-Jun(Ser73)3

Sensitive, Specific Transcription
Factor Analysis

Figure 1: Monitoring c-Jun phosphorylation using FACE.
The FACE c-Jun (S63) and c-Jun (S73) Chemi Kits were used to assay thelevels of total and phosphorylated c-Jun contained within uninduced
and anisomycin-induced NIH/3T3 cells.

5High-Quality Transcription
Factor Antibodies for Westerns,
EMSA & ChIP

78

More Effective Gene Silencing

9

Quickly Quantitate Estrogen
Receptor Activity

c-Jun Chemi Signal (RLU)

Active Motif’s Fast Activated Cell-based
ELISA (FACE™) Kits enable you to detect
phosphorylation events faster and easierthan ever before. Conventional techniquessuch as in-gel kinase assays and Western
blotting are costly, time-consuming andprovide you with only semi-quantitative
results. Moreover, because additional
protein modifications can occur during cell
extraction procedures, results obtained using these techniques may not reflect actualin vivo conditions. FACE cell-based assays
eliminate the need for extracts, radioactivity, electrophoresis and membrane blotting,providing you with quantitative results inrecord time.

IN THIS ISSUE
Uninduced
Anisomycin Induced

200000160000120000

80000
40000
0

Total-c-Jun

Phospho-c-Jun(Ser63)

continued on page 4

Simple, Efficient Delivery of Proteins into Cells
Chariot™ is a revolutionary delivery reagent
that transports biologically active proteins,
peptides and antibodies directly into mammalian cells in about 2 hours with a typicalefficiency of 65-95%. After delivery, living
cells can be assayed to determine theeffects of the introduced molecules. This
makes Chariot ideal for a wide variety offunctional studies, including delivery of
inhibitory peptides, organelle labeling,
screening peptide libraries and transientcomplementation studies.

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.

Figure 2: Delivery of Actin antibody.
Chariot was complexed with a 1/1000 dilution of Actin antibody
and used to label Actin filaments in human fibroblast (HS-68) cells.
Unfixed cells were observed 2 hours post-delivery.

Fast, Quantitative Measurementof DNA Repair Protein Activity

DeCypher® Helps
10 TimeLogic®
Unlock Key Bioinformatics
Questions

11 Rapid Gene Isolation using RecA
Proteins for
12 RecombinantYour Researchcontinued on page 6

www.activemotif.com

Chromatin Immunoprecipitation

Complete Solution for More Successful Chromatin Immunoprecipitation (ChIP)
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 tointerpret. To help eliminate these problems,
Active Motif’s ChIP-IT™ Kit combines nearly
everything needed to successfully perform
ChIP. The ChIP-IT Kit includes antibodies,
controls, reagents and a comprehensive
protocol that have been proven to work in
ChIP. This provides a complete solution thatmakes it possible to successfully obtain andvalidate your results.

? What is ChIP; why is it used?
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).
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.

Cross-link protein to DNA inliving cells with formaldehyde

PCR
primers
Induction

TargetDNA

Break open cells
and shear DNA

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.

factors are important when
? Whatperforming ChIP?

does the ChIP-IT Kit make
? How
ChIP more successful?

Successful ChIP depends on a variety
of different factors: proper fixation of
the protein/DNA interactions, preparing sheared DNA of the correct length,
and having both an antibody that works
in immunoprecipitation as well as a PCR
primer set that specifically amplifies theregion of DNA bound by the protein ofinterest. A variety of specialized buffers,
protease inhibitor cocktails and blocking
reagents are also needed to ensure that theprotein/DNA interactions are preserved
and to minimize non-specific enrichment
and reduce background. ChIP experiments
are likely to be unsuccessful should any ofthese steps or solutions fail. However, using
the ChIP-IT Kit means you won’t have toworry about this, as all reagents are rigorously QC’d and proven to work in ChIP.

The ChIP-IT Kit simplifies all aspects ofchromatin immunoprecipitation by providing nearly everything you’ll need to preparechromatin, optimize shearing conditions,perform ChIP reactions and analyze ChIP
results by PCR. All buffers (excluding formaldehyde) required for cell fixation, nuclei
purification and chromatin shearing are included. Not only does this help ensure yourresults, but you won’t have to spend hours
preparing and optimizing your own buffers and protocols. Most importantly, theChIP-IT Kit provides positive and negative
control antibodies and PCR primer sets.
These help to improve result interpretation
and also make it easy for you to validatethat your own antibodies and primer setsfunction in ChIP. No other kit or “homegrown” method provides such a complete,convenient solution. For complete details,
call us today or download the comprehensive ChIP-IT manual from our website.

Product

FormatCatalog No.

ChIP-IT™ Kit

25 rxns

53001

2

Add pre-blocked
Protein G Agarose Beads

Toll Free — 1 877 222 9543

MAY 2004 • VOLUME 5 • NUMBER 2

Transcription Factor ELISAs

Traditionally, transcription factor activity
has been studied using either Electrophoretic Mobility Shift Assays (EMSA),
immunoblotting or reporter gene assays.
These methods are time-consuming and, atbest, provide only semi-quantitative results.
Moreover, use of these methods to determine the activity of multiple transcription
factor family members would require theinvestment of significant amounts of bothtime and money; in many cases it may noteven be possible. TransAM™ Kits, however,make it straightforward to measure theactivity of entire families of transcription factors in one simple experimentthat is complete in less than 5 hours. Plus,
TransAM Kits can be used on all sample
types, including cell lines and tissues, giving
you unsurpassed flexibility.
Assay more relevant sample types
Many important disease states are known
to be associated with aberrant changes
in cellular gene expression. Consequently,
transcription factors, which regulate gene
expression, are of great interest both fortheir potential as therapeutic targets andas biological markers of disease states.
Reporter gene assays expressed transiently
or from a stable cell line are commonlyused as a primary screen in drug discovery
and biomarker research. However, because
it can be difficult to relate cell culturebased data to actual in vivo experiments,
the value of reporter assays is limited. And,
reporter assays rely on the intracellulardelivery of vectors, which can be time-consuming and inefficient. In contrast, TransAM
Kits can selectively bind activated tran-

NFκB activation (OD450 nm)

Sensitive, Specific Transcription Factor Analysis
2.5
2

0.5

1

5

10

25

50

0.5

1

5

10

25

50

1.5
1
0.5
0

A

Unstimulated

IL-1α stimulated

Unstimulated
IL-1α stimulated

0.5

1

2
5
10 25
Cell extract/well (µg)

50B

µg of cell extract

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

scription factors in cellular extracts isolated
from either tissue or cell culture samples.
This means that TransAM Kits are ideal foruse in both cell culture-based screening
and in more biologically relevant, in vivo
experiments. Why use reporter assays
when you can access the most versatiletranscription factor assay available?
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
A better transcription factor assay
EMSA has long been considered the “goldstandard” for measuring transcription
factor activation. However, EMSAs are time
consuming, radioactive and lack sensitivity. Not only this, but they yield limited
data, which is prone to non-specific effects
that can complicate data interpretation.
Fortunately, science and consequently thetechnology around it, is constantly evolving and improving. TransAM Kits are one
such improvement. The highly sensitive
ELISA-based approach of TransAM makes
obtaining and interpreting results simple,
safe, fast and consistent. Plus, you can easily analyze entire families of transcription
factors in a single experiment, while using
a fraction of the sample needed to obtain
results using EMSA (Figure 1). Why not try
the new “gold standard” of transcription
factor measurement today?
The latest additions
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
can simultaneously profile the activationlevels of several different family members.
The new TransAM MAPK Kit will even enable you to profile the activity of multiple
families of transcription factors, which are
regulated by the MAPK kinase cascade.
For better transcription factor analysis, try
TransAM Kits.

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Cell-based ELISAs

continued from page 1 — Faster Phospho-specific Analysis using Cell-based ELISAs

FACE advantages• Cell-based format – no extracts, gels,blotting or radioactivity• Fast – requires less than 2 hours ofhands on time
• Flexible – high-throughput chemi andcolorimetric formats• Accurate – sensitive readout means
less well-to-well variation• Quantitative – provides moremeaningful results
Try FACE Kits today
FACE Kits offer you a fast, sensitive andquantitative alternative to traditional
assays. New kits are available for studying
the activation of ATF-2, c-Jun, EGFR, ErbB-2,
GSK3β and MEK1/2. To get to the most upto-date information available on FACE Kits,
visit our website at www.activemotif.com.

4

0

A

0.1

1

10

C

43 kDaOD450 nm

How does FACE work?
FACE Kits are easy to use and requireless than 2 hours of hands on time. In
FACE, cells are cultured in the provided
96-well plates and stimulated to induce
the pathway of interest. The cells arethen rapidly fixed with formaldehyde topreserve protein modifications, including phosphorylation. Each well is thenincubated with a primary antibody specificfor the activated protein of interest. Thisis followed by secondary HRP-conjugated antibody and developing reagent,
which provides a sensitive colorimetric or
chemiluminescent readout. FACE Kits aresupplied complete with everything needed
to perform one 96-well assay with thephosphorylated antibody and one 96-wellassay with the total antibody for theprotein of interest. Finally there’s a kit thatmakes it easy for you to compare activatedto native protein levels at the same time
(Figure 1, page 1 and Figure 2C).

0

B

0.1 1

10
43 kDa

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

Anisomycin induction (µg/ml)

Phospho-p38
Total-p38

0

0.1
1
10
Anisomycin induction (µg/ml)

Figure 2: Phospho- and total-p38 MAPK assays.
Macrophage 4/4 cells were grown in 10 cm dishes to 80% confluency, serum-starved for 16 hours and stimulated with anisomycin for 15 minutes. Cell lysates
were made and Western blots performed using phospho- (A) and total-p38 (B) antibodies. For FACE, 4/4 cells were grown in 96-well plates, stimulated asabove, fixed and then assayed in triplicate using the FACE p38 Kit (C). Data were corrected for cell number through use of the kit’s Crystal Violet Dye.

Product

FormatColorimetric Kit
Catalog No.

Chemiluminescent Kit
Catalog No.

FACE™ AKT

1 x 96 rxns
5 x 96 rxns

48120
48620

48220
48720

FACE™ ATF-2

1 x 96 rxns
5 x 96 rxns

48115
48615

48215
48715

FACE™ c-Jun (S63)

1 x 96 rxns
5 x 96 rxns

48125
48625

48225
48725

FACE™ c-Jun (S73)

1 x 96 rxns
5 x 96 rxns

48135
48635

48235
48735

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™ MEK1/2

1 x 96 rxns
5 x 96 rxns

48180
48680

48280
48780

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

Toll Free — 1 877 222 9543

MAY 2004 • VOLUME 5 • NUMBER 2

Transcription Factor Antibodies

High-Quality 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 directedagainst 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 andchromatin 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.

www.activemotif.com

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

Supershift

BandshiftFree 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.

Antibodies for ChIP
Chromatin immunoprecipitation is one ofproteomics’ powerful new tools for analyzing genome regulation (see page 2). However, one of the drawbacks of performing
ChIP is finding antibodies that are proven towork in this technique. Although not all of
our antibodies have been tested for use in
ChIP, a high proportion have been validated
in EMSA using our Nushift Kits. This is a good
indication that the antibody will also function in ChIP, which increases your chances ofgetting the antibody you need for ChIP thefirst time. Currently our antibodies against
AP-2, ATF-6, E2F-1, E2F-6, GATA-1, HDAC3,
HDAC4, IRF-3, Pax-5 and Sp1 are ChIP positive. As this list is growing, be sure to checkour website for up-to-date information.

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 a positive
control with a particular antibody in Western blotting or gelshift.

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.

5Protein Transfection

continued from page 1 — Simple, Efficient Delivery of Proteins into Cells
How does Chariot work?
Chariot delivers antibodies, peptides andproteins into cells through a 4-step process(Figure 1):
1. Association of Chariot to your macromolecule to form a stable complex;
2. Membrane uptake of the complex at
the external cell surface;
3. Insertion into the cell membrane,
conformational change and pore
formation;
4. Release of the complex into the cytoplasm, followed by complex
dissociation.1
Non-covalent delivery
Many protein delivery systems require thatyou fuse a carrier protein to your macromolecule. However, this can change thefolding characteristics of your protein and,ultimately, its function. Because Chariot
uses non-covalent, charged interactions to“cage” proteins, it does not affect protein
folding or function. The result is the delivery of highly active, native proteins.
Independent of the endosomal pathway
Experiments at 4°C have shown that
Chariot delivery is independent of theendosomal pathway.2 This is importantbecause endocytosis can cause protein
modifications, and may also prevent releaseof the protein into the cytoplasm, bothof which will effect the success of yourexperiment. In contrast, the Chariot complex enters the cell through an interaction
with the cell membrane. When the Chariot
complex crosses the cell membrane, thecomplex is destabilized, leaving unmodified antibody, protein or peptide free toperform its function.
Don’t just take our word for it
There is a lot of exciting research taking
place with Chariot; much of it would havenot been possible without the help of
this innovative technology. The ability todelivery functionally active protein directly
into mammalian cells makes it easy to
perform studies that are not possible using
conventional methods. Our website offers
an extensive list of publications describing

Cell line or model

Cell type

Macromolecule delivered

3T3-L1

Mouse fibroblast

Antibody

80%

A549

Human lung carcinoma

Antibody

95%

Arabidopsis

Primary plant protoplasts

ProteinNot provided

COS-7

Monkey kidney

Protein, oligopeptide

80%

CV-1

Monkey kidney

Antibody

55%

HCA2

Human fibroblasts

Protein90%

HeLa

Human cervix carcinoma

Protein, peptide, antibody

95%

HMSC

Primary human mesenchymal stem cells

Peptide

80%

HS-68

Human foreskin fibroblastProtein, peptide, antibody

95%

IMR90

Human fibroblastProtein, peptide

80%

JurkatHuman T-cell leukemia

Protein, peptide, antibody

75%

Mono Mac 6

Primary human monocyte

Protein50%

Mouse hepatocytes

Primary liver

Protein95%

Mouse model (in vivo)

Alveolar wall tissue

Protein88%

Neural retina cells

Primary chicken

Protein80%

NIH/3T3

Mouse embryoProtein, peptide

80%

NRK

Normal rat kidney

Antibody

30-60%

PC-12

Rat pheochromocytoma

Protein, peptide

80%

Sensory neurons (DRG)

Primary chick

Antibody, peptide

80%

Sensory neurons (DRG)

Primary rat

Protein80%

Thyrocytes

Primary humanProtein

90%

WI-38

Human lung fibroblastProtein, peptide, antibody

95%

WISH

Human placenta carcinoma

Protein30%

such novel research, as well as data that has
been furnished to us by respondents to ourChariot survey (see table above). Detailsof two recent, innovative publications areshown below:“Direct Activation of Bax by p53 Mediates
Mitochondrial Membrane Permeabilization
and Apoptosis” was published in Sciencein February 2004. It describes delivery ofwheat germ agglutinin by Chariot, thereby
blocking p53 accumulation in the nucleus.3
The American Journal of Respiratory Cell &Molecular Biology published “Alveolar Wall
Apoptosis Causes Lung Destruction andEmphysematous Changes” in 2003. Here, anin vivo model was used to study apoptosis
through delivery of Caspase-7 protein into
living mouse lung tissue by Chariot.4

Delivery efficiency (%)

Association

Cell targeting

Membrane translocation

Decaging

Figure 1: Schematic model for translocation of the Chariot complex.
Figure generously provided by Dr. Gilles Divita, Biophysics Dept.,
CNRS, Montpellier, France.1

To get ideas on what you might dowith Chariot, please visit our website atwww.activemotif.com.
1. Deshayes et al. (2004) Biochemistry 43:1449-1457.
2. Morris et al. (2001) Nature Biotech. 19:1173-1176.
3. Chipuk et al. (2004) Science 303:1010-1014.
4. Aoshiba et al. (2003) Am. J. Respiratory Cell & Mol. Bio. 28:555-562.

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.

6Toll Free — 1 877 222 9543

MAY 2004 • VOLUME 5 • NUMBER 2

Gene Silencing

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

www.activemotif.com

Pep-2/gripNA

Pep-2/PNA

A

B
1

2

3

4

5

6

1

7

2

3

4

5

6

7

Cyclin B1
CdK2

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

Hit your target only
A recurring problem of many gene silencing
reagents is a lack of specificity. This cancause unintended phenotypes because
these reagents may bind and silence
expression of more than just the intended
target. In vivo mismatch discrimination experiments have shown that gripNA binding
is reduced or eliminated by the presence ofa single base-pair mismatch. Recent experiments performed in Zebrafish embryos
indicate that this specificity can improveyour results. While wild-type gripNA andmorpholino probes were comparable inpotency at silencing the chordin, uroDand no tail genes, the intentional inclusionof 2 and 4 base-pair mismatches in themorpholino probes caused non-specificeffects that were not triggered by comparable gripNA probes.1

Order anytime
Active Motif’s custom gripNA synthesis
service can provide you with better materials for your gene silencing experiments. All
the information needed to design, orderand use gripNA probes can be accessed atwww.activemotif.com/gripna. Our simple,
online ordering system makes it easy toplace your gripNA order 24 hours a day, 7days a week. gripNA probes can be orderedwith or without Chariot II. In addition, you
can add a 3´ modification (Biotin, FITC or
primary amine) to your probe. Each gripNA
is verified by mass spectrophotometry andsupplied with a fluorescently labeled positive control for human CREB. Log on now
and order your custom gripNA today.
1. Urtishak et al. (2003) Developmental Dynamics 228(3).
2. Morris et al. (2004) Gene Therapy (Feb 12, e-pub ahead of print).

Product

FormatCatalog No.

Custom gripNA™ Probe

200 nmol

24001

Custom gripNA™ Probe w/Chariot™ II

200 nmol

24002

Primary Amine Modification

24004

Biotin Modification

24005

Fluorescein Modification

24006

Chariot™ II

96 rxns

24008

gripNA™ Human CREB Positive Control

25 nmol

24007

gripNA™ Chordin Positive Control

5 nmol24009

7

DNA Repair

Fast, Quantitative Measurement of DNA Repair Protein Activity
Active Motif’s DNA Repair Kits provide
a fast, user-friendly format for studying
DNA damage and repair protein activity.
This is because our DNA Repair Kits utilize
the DNA-binding properties of DNA repair
proteins to selectively capture activatedprotein, which is then detected and quantified using the kit’s highly characterized
antibodies. Assaying protein activity using
the DNA Repair Kits is fast and simple,
which saves you both time and money.
In recent years, a clear link has been
established between cancer and genome
instability. Defects within the cellular
recombination and repair machinery canhave disastrous effects to the cell and leadto cancer. This makes DNA repair proteins
ideal drug targets and creates a need for
new sensitive, accurate and convenient
assays for monitoring DNA repair protein
activity. To meet this need, Active Motif
has developed its innovative DNA Repair
Protein Kits.
Advantages• Non-radioactive, colorimetric method• 10X more sensitive than EMSA
• Results in under 5 hours• Works with cells and tissues• High-throughput compatible
GTBP

Absorbance (OD450 nm)

A

B

1.25

1
0.75

0.5

0

Raji

PANC-1

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

8

Oligonucleotide
coated plate

1 hr.

Add developing
and stop solution

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

Figure 1: Flowchart of the DNA Repair Kit procedure.

The DNA Repair Kit method
The DNA Repair Kit method is simple andefficient and can be used with both tissue
and cell culture extracts. Each kit includes a
96-well plate in which multiple copies of a
specific oligonucleotide have been immobilized. When cellular extract is added, therepair protein of interest binds to the oligonucleotide. Each well is then incubatedwith a primary antibody that is specific forthe repair protein being studied. Additionof HRP-conjugated secondary antibody
results in an easily quantified colorimetricreadout (Figure 1).

illustrate, GTBP activity was measured andcompared using Active Motif’s GTBP DNA
Repair Kit and by Western blot. The GTBPDNA Repair Kit results are clearly morequantitative than Western blot (Figure 2).

Easily quantitate your data
Being able to measure small changes in
DNA repair protein activity is important.
While traditional methods such as Western
blot are sensitive, they typically provide
qualitative, rather than quantitative data.
Active Motif’s DNA Repair Kits, however,provide a colorimetric readout that is
easily quantified, so you can detect eventhe slightest change in protein activity. To

What’s available
Active Motif’s DNA Repair Kit product line
is continually growing. Currently available
kits include those for studying Ku70/86,
GT Binding Protein (GTBP) and Replication
Protein A (RPA). Please give us a call or visit
our website for complete kit configurations, downloadable manuals and to learn
about new additions to the DNA Repair Kit
product line.

Product

0.25

Add anti-DNA
repair antibody

Add cell extract
Cell extractcontaining DNA
repair protein

All you need
DNA Repair Kits come complete with optimized reagents including positive control
nuclear extract to simplify the way you
study interactions between damaged DNA
and repair proteins. Don’t waste time withinefficient Western blots or EMSAs; use
DNA Repair Kits 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

MAY 2004 • VOLUME 5 • NUMBER 2

Nuclear Receptor Binding Assays

Quickly Quantitate Estrogen Receptor Activity

Estrogen Receptor regulation
ERα activation is dependent upon ligand
binding to the receptor; this causes thereceptor to dimerize and bind to estrogen
response elements (EREs) located within
the promoters of estrogen-responsive
genes. While both agonist and antagonist
compounds can bind to ERα, they have opposite effects on ERα conformation. Whenan agonist is bound, a hydrophobic grooveis exposed that allows the binding of coactivator proteins such as SRC1 and RIP140.
In contrast, when an antagonist is boundthe co-activators needed for transcriptionof target genes are unable to bind. Because
inappropriate activation of ERα has been
associated with numerous diseases, there
is an urgent need to identify ligands thatmodulate its activity.
The Nuclear Receptor ELISA method
The Nuclear Receptor ERα ELISAs aredesigned to measure the effects thatvarious agonist and antagonist ligands haveon the activation of ERα. Each kit contains
a 96-stripwell plate that is coated with asequence-optimized peptide that includes
the consensus-binding motif of the ERα
co-activators. When sample containing
ERα and an agonist or antagonist ligand is
added to a well, the ligand-activated ERα
binds to the Capture Peptide. Incubation
with primary antibody specific for ERα
followed by HRP-conjugated secondary
antibody and developing solution provides an easily quantified colorimetric or
chemiluminescent readout. The result is aquantitative measurement of the effect of
the agonist or antagonist on ERα activation(Figure 1).

www.activemotif.com

ERα activation (OD450 nm)

Active Motif’s Nuclear Receptor ERαELISAs are a simple solution for monitoring the effects of agonists and antagonists
on the activity of Estrogen Receptor alpha(ERα). The assays utilize a unique peptidecapture system that provides quantitative
results in only three hours, while enabling
you to study both cellular samples andrecombinant proteins.

0.8

estradiol
DES
genisteintamoxifen
clomiphene
ICI 182,780

0.6
0.4
0.2
0

estradiol

DES

genistein tamoxifen clomiphene ICI 182,780

Drug (25 µM)
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 detected with an ERα antibody. Secondary antibody and detection solution are then used to quantitate ligand-activated ERα. Note that in MCF-7 cells
estradiol, DES and genistein are ERα agonists while tamoxifen, clomiphene and ICI 182,780 are ERα antagonists. (DES, genistein, clomiphene and ICI 182,780
results are provided for demonstration purposes only; these compounds are not included in the Nuclear Receptor ERα ELISA Kits.)

Faster, more reproducible 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 thecloning and transfection of reporter
plasmids or the construction of stable celllines. Nuclear Receptor ERα ELISAs arecomplete in hours, rather than days, haveincreased specificity and provide quantitative results. Moreover, the method is far
more reproducible, which is crucial whencomparing results from a large number ofdifferent compounds. With the Nuclear
Receptor ERα ELISAs, studying ERα has
never been easier.
Nuclear Receptor ELISA advantages• Quantitative analysis of agonist/
antagonists effects• Results in ~3 hours• Specific, antibody-based
measurement• High-throughput compatible• Complete kit with proven controls
Product

Choice of sensitivity
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 theuse of a microplate luminometer. The
flexible measurement parameters ofchemiluminescence mean that you canadjust the detection limits to ensure thatthe sensitivity is appropriate for the sample
type that you are assaying.
Get it today
Nuclear Receptor ELISAs make studying theeffects of agonist and antagonist ligands on
receptor activation both faster and more
reproducible. In addition to the ERα ELISAs,
we will be releasing new Nuclear Receptor
ELISAs for studying other nuclear receptors
throughout 2004. Please give us a call orvisit our website for complete kit configurations, downloadable manuals and to learn
about new additions to this innovative
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

9

BLAST Enhancement

TimeLogic® DeCypher® Helps Unlock Key Bioinformatics Questions
The DeCypher® Solutions combine innovative software and reconfigurable hardware forhigh-performance sequence analysis:• DeCypherBLAST™ – rapid genomic and protein sequence comparisons• DeCypherHMM™ – powerful Hidden Markov Model analysis for rapid protein classification• DeCypherSW™ – rigorous Smith-Waterman methods find optimal alignments across frameshifts
and introns for homology modeling, microarray probe design and siRNA studies• GeneDetective™ – align cDNAs or amino acids to genomic DNA for fast, accurate identificationof gene models and splice variants
What is DeCypher?
DeCypher allows you to extend the process
of experimental design from your bench
to the computer. DeCypher employs acomprehensive set of ultra-fast analysis
tools that let you experiment with varied
parameter settings—or completely different algorithms—to define the best possible
sequence annotations.
The turnkey DeCypher solutions arecapable of the large-scale analysis routinely
required for genome comparisons. Installed
within a single computer, they allow you
to complete sequence comparisons at the
speed of hundreds to thousands of CPUs.
DeCypher features both a web interface &a command line client, enabling all users to
execute powerful bioinformatics analyses.
How would I use DeCypher to predict thefunction of a gene? DeCypher enables you
to quickly process comprehensive BLASTX
searches of your gene sequences against
a well-curated protein database of known
proteins. For high-scoring matches, you
can assume a similar protein function. To
reinforce your conclusions, it’s possible tocompare your protein sequence with anannotated Hidden Markov Model databasesuch as PFAM. A match to a specific protein
family can imply similar function. DeCypher’s HMM-Framesearch method is alsoavailable for automatic translation of yournucleic sequences and frameshift tolerantalignments while assigning protein families.

10

How can DeCypher be used to identify
distantly-related sequences?
DeCypherHMM enables you to build yourown Hidden Markov Model database froma collection of similar proteins from different organisms. This model can be used tosearch for remote homologs through comparison with a large sequence database.
DeCypherHMM’s performance enables you
to begin exploring results from extremelylarge HMM comparisons in minutes.

How can I use DeCypher to rapidly identify exons and splice variants? TimeLogic’s
new GeneDetective™ solution allows you
to map ESTs or amino acid sequences togenomic data to build sophisticated genemodels. The system uses multiple sequencealignment methods to accurately describe
exons and introns, then delivers a zoomable, graphical view of your gene model for
exploration of splice variants.
Can DeCypher be used for protein structure modeling? For homology modeling,
DeCypher’s BLAST and robust SmithWaterman methods can be used to findsequences with known protein structure bycomparison with Protein Data Bank (PDB)
sequences. If no significant homology to
PDB is found, DeCypherHMM can efficiently identify protein structural families
reported in databases such as SCOP.
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DeCypherBLAST and DeCypherSW can also
be used for comparing distantly related
genomes by processing protein-spacecomparisons of nucleic data.
To learn more about these methods, orabout the DeCypher solutions’ speed andcapability, please contact TimeLogic today
at 877-222-9543 or at www.timelogic.com.

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Toll Free — 1 877 222 9543

MAY 2004 • VOLUME 5 • NUMBER 2

Gene Enrichment

Rapid Gene Isolation using RecAThe RecActive™ Kit for gene enrichmentoffers a fast, flexible and efficient method
for isolating target DNA from complementary DNA (cDNA) or genomic DNA (gDNA)
libraries. With up to 10,000-fold enrichmentof your target gene(s) in only two rounds ofpurification, RecActive is the most effective
gene isolation technology available.
Isolate genes without hybridization or PCR
Conventional methods for isolating clones
from cDNA and gDNA libraries require thepreparation and hybridization of a large
number of filters from library master plates.
This is time-consuming, labor-intensive and,depending on the method, often requires
the use of radioactivity. PCR amplification
is faster, but can introduce errors into theamplified DNA. Furthermore, PCR is noteffective for isolation of full-length genes
or splice variants. In contrast, RecActive
utilizes the natural properties of RecA
recombinase from E. coli to form stable,
multi-stranded hybrids that can be easily
purified, removing the need for membrane
hybridization or PCR amplification.
Save time by enriching for your DNA
With the RecActive technique, a short sequence (200-600 bp) corresponding to yourtarget DNA is prepared as a biotinylated
DNA probe and coated with RecA. When
added to the pooled library DNA, the RecA
catalyzes formation of stable multi-stranded complexes consisting of probe DNA andhomologous target sequences. Streptavidin-coated magnetic beads are then used
to enrich for clones that are bound by thebiotinylated probe (Figure 1). After enrichment, the purified DNA is transformed into
bacteria and plated. As only enriched DNA
is transformed, you save time and materials
by working with only the DNA you need.

www.activemotif.com

Denature the doublestranded, biotinylated probe
and coat with RecA

RecA recombinase

Double-stranded librarySingle-stranded, RecAcoated, biotinylated probes

Formation of stable, multistranded hybrid ismediated by RecA

Streptavidin-coatedmagnetic beads

Clone enrichment
by magnet= RecA= Biotin= Streptavidin-coatedmagnetic beads

Figure 1: Flowchart of the RecActive process.
A short (200-600 bp) double-stranded, biotinylated probe is producedby PCR using the Biotin-dNTP Mix. The probe is denatured and coatedwith RecA. Each strand of the probe then finds and hybridizes to its
complementary sequence in the DNA library. After removal of RecA,
streptavidin-coated magnetic beads are used with a magnet to enrich
for the homologous clone(s).

Ensure you isolate all the splice variants
The RecActive method is very effectiveat isolating splice variants. Traditional PCR
methods will not amplify unknown splicevariants unless the splice sites are internal
to the PCR primers. However, RecActiveenriches for all clones that share homology to the 200-600 bp piece of probe
DNA. That means that you will enrich for
all splice variants contained within yourlibrary, irrespective of the splice site.
Proven performance
To demonstrate the powerful results possible with the RecActive Kit, a “library” wasconstructed by combining white and blue
colony-forming plasmids at a ratio of
1:10,000. Following a single enrichment
using RecActive, 65% of transformants werewhite colony-forming clones (Figure 2).
Gene enrichment from model species
Model systems are widely used for drug
discovery and other modern research
projects due to their ease of handling.
However, functionally related genes fromother species must then be isolated for
efficient drug discovery and basic research
to be performed. Often the DNA homology between these related genes is too low
to enable effective use of hybridizationor PCR-based approaches. By altering thebinding conditions, RecA can catalyze formation of probe:target complexes with up
to 30% heterology. This makes RecActive
the system of choice for isolating interspecies and/or interfamily genes.

AA

B
B
Preselection: 0.01%

Postselection: 65%

Figure 2: Gene specific enrichment.
RecActive enrichment of White Plasmid from a library containing aratio of 1 White:10,000 Blue plasmids.

Find your gene of interest today
For the simplest, most versatile gene enrichment tool available, try the RecActive
Kit today. For complete details, please give
us a call or visit our website.

Product

FormatCatalog No.

RecActive

10 rxns*
5 x 10 rxns

52010
52050

11

Recombinant Proteins

Recombinant Proteins for Your Research
Active Motif now offers a growing line ofrecombinant proteins that are ideal for use
in many different biological applications.
The NFκB p50, NFκB p65, p53, c-Myc and
1.5

Absorbance (OD450 nm)

1

0.5

0
0.5

1

1.5

2

2.5

p50 Recombinant Protein (ng)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.

12

technical data sheets that specify protein
length, the species it was produced in,method of purification, etc., can be foundat www.activemotif.com.

Recombinant Protein Liney = 0.468x - 0.055 r2 = 0.999

0CREB proteins are currently optimized
for use in our TransAM ELISAs (Figure 1,
see page 3). Complete information on therecombinant proteins, including detailed

3

AKT1

ER

p53

RARγ

ATF-2

FXR

p53 R273H

RXRα

BRCA1

GR

p300

RXRβ

BRCA2

IκBα

PPARα

RXR-LBDc-Fos

JNK2α1PPARβ(δ)

Sp1

c-Jun

JNK2α2

PPARγ

STAT1

c-Myc

LXRα

pRB

TRα1

CREB

LXRβ

Rad51

TRβ1

CTF1 (NF-1)

NFκB p50

RARα

eIF2α

NFκB p65

RARβ

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 314
92101