THE NEWSLETTER OF ACTIVE MOTIF — September 2005 • volume 6 • number 3

Drop In & Learn About New
Immunoprecipitation& Fluorescent
Detection
Products

IN
THIS
ISSUE

2 New: Simplify Co-Immunoprecipitation

7 Sensitive, Speciﬁc Transcription Factor Analysis

3 New: Effortless Quantiﬁcation of Your
Protein Samples

8 Fast, Efﬁcient Analysis of Protein Phosphorylation

4 New: Capillary Electrophoresis Made Easy

9 Deliver Functional Proteins Directly into Living Cells

5 ELISA for Quantiﬁcation of Activated Ras GTPase

10 Accurate Detection of Signal Pathway andNuclear Receptor Proteins

6 Use ChIP-IT™ for More Successful
Chromatin Immunoprecipitation

11 Positive Control Recombinant Proteins andCell Extracts for All Your Needs

Co-Immunoprecipitation

New: Simplify Co-Immunoprecipitation
Active Motif’s new Nuclear Complex Co-IP Kit simpliﬁes co-immunoprecipitation
studies of nuclear protein complexes by providing you with optimized reagents forboth nuclear extract preparation and immunoprecipitation.
The Nuclear Complex Co-IP’s extraction
process provides a simple and effective
method to obtain and maintain protein
complexes contained in nuclear compartments of the cell, speciﬁcally those previously bound to DNA, while the versatile
Co-IP reagents offers you the ﬂexibility
to vary the stringency of the Co-IP buffer
compositions. This improves your results
and enables you to study tightly boundor weak protein complexes with ease.
Co-Immunoprecipitation (Co-IP) is often
used to ﬁnd and study protein/protein
interactions. In Co-IP, a ﬁrst antibody
is used to immunoprecipitate a target
antigen, which also co-precipitates any

bound, interacting proteins. The complex
is then detected by Western blot using asecond antibody targeted against one of
the bound, interacting proteins. However,traditional methods for performing Co-IP
are not optimal for studying complexes of
DNA-binding proteins as these complexes,
being fragile, are frequently disrupted
during the extraction process. In addition,
many protein complexes are altered by
the salt and detergent composition of thebuffers used in the immunoprecipitation
process, which can complicate theiranalysis. To overcome these problems,
the Nuclear Complex Co-IP Kit extraction
reagents were designed to help maintain
nuclear protein complexes.

“The Nuclear Complex Co-IP Kit simpliﬁes co-immunoprecipitation of DNAbinding proteins from cell and tissue samples by providing both extraction andimmunoprecipitation components that maintain nuclear protein complexes.”
Cell nucleus containing the twointeracting proteins of interest

1

2

3

Nuclear extraction

Add antibody directed against
one of the proteins of interest

Add antibody
binding beads

Lane 1. Mol. Weight Marker
Lane 2. Extract
Lane 3. negative IP (no ab)
Lane 4. Co-IP

Wash & collect theimmunoprecipitated proteins

Immunoprecipitate theproteins of interest

Figure 1: Flowchart of the Co-Immunoprecipitation process.

Product

FormatCatalog No.

Nuclear Complex Co-IP Kit

50 rxns

54001

2

1

2

3

Figure 1: Western blot analysis of the IP’d p33 subunitof the RNA pol II complex.
HeLa cells were grown to confluence on 100 mm plates
and nuclear extracts were prepared using the kit’s
extraction reagents. For IP experiments, the stringencyof the IP High Buffer was increased by supplementing
with NaCl and Detergent. 100 μg of nuclear extract wasused per IP reaction and incubated with either 2 μg
p33 antibody or no antibody. Following the IP, Western
blot analysis was performed using RNA pol II mouse
mAb at 0.1 μg/ml followed by anti-mouse HRP at 1:1000.
Detection of the p33/RNA pol II complex by the RNA
pol II antibody (lane 3) demonstrates that the Co-IP
was successful in maintaining the protein complex. The
input HeLa extract (lane 1) was run as a control for theWestern blot using 0.1 μg/ml RNA pol II.
Lane 1 Western blot control
Lane 2 Negative Control (no antibody used in IP)
Lane 3 Co-IP: IP using p33/WB using RNA pol II

The Co-IP Kit also contains high and low
stringency IP buffers, as well as salt anddetergent. Addition of salt and detergent
is ideal for robust protein/protein interactions because it reduces background.
However, as unstable protein complexes
may not withstand high stringencies, thisconvenient kit format enables stringency
to be modiﬁed as required for eachparticular protein complex.
Advantages• Simple and efﬁcient• Optimized extraction procedure
preserves nuclear protein complexes• Flexible IP reagents to detect
protein/protein interactions ofvarying strength4

Western blot analysis of the immunoprecipitated proteinsusing an antibody directed against the second protein of interest

250

Order one today
The Nuclear Complex Co-IP Kit offers
you a simple and ﬂexible alternative toperforming traditional immunoprecipitations. To ﬁnd out more about the new
Nuclear Complex Co-IP Kit, visit us on
the web at www.activemotif.com.

Toll Free — 1 877 222 9543

September 2005 • volume 6 • number 3

Fluorescent Protein Quantification

New: Effortless Quantiﬁcation of Your Protein Samples
Active Motif’s Fluorescent Protein Quantiﬁcation Kit is a simple, sensitive
alternative to traditional methods for determining protein levels. Offeringhigh signal strength, unique spectral properties and robust conjugation, thekit offers limits of detection (60 ng/ml) superior to other ﬂuorescent-based
systems, as well as traditionally used methods, such as the Bradford assay.

“The spectral properties and robust
conjugation of the FluorescentProtein Quantiﬁcation Kit makemeasurement fast and accurate.”absorb (503 nm)

emit (600 nm)

absorb (612 nm)

Fluorescence excitation

1.0

Unbound
Conjugated

0.8
0.6
0.4
0.2

emit (665 nm)

0
400 450 500 550 600 650 700 750 800

Wavelength (nm)Figure 1: Absorption/emission spectra of free/bound dye.
Normalized absorption and emission spectra of free(solid lines) and conjugated dye (dotted lines) in
phosphate buffer of pH 7.2.

www.activemotif.com

Fast, simple conjugation
Using the Fluorescent Protein Quantiﬁcation Kit is fast and easy. The kit provides
ﬂuorescent dye, dilution buffer anda protein standard. Simply resuspend
the dye and add it to the wells of a
microplate, then add a serial dilution of
the standard protein to produce a Standard Curve, along with your sample. On
conjugation, the dye changes from blue
to red (Figure 2). After a 30 minute, room
temperature incubation, simply read theﬂuorescence to quantify your samples.

Consistent performance
To ensure accurate determination ofprotein levels, it is important that theprotein detection reagent performconsistently between samples. To demonstrate this, the Fluorescent Protein
Quantiﬁcation Kit was used to prepareStandard Curves using several different
proteins. The ﬂuorescence intensity
obtained with the kit showed little variation between samples across a range ofconcentrations (download a kit manualfor data). This means that you can haveconﬁdence that your results will not be
inﬂuenced by protein-speciﬁc effects.
80

Relative fluorescence

Broad spectral shift for better results
A disadvantage of many methods used
for protein quantiﬁcation, such as theBradford assay, is that the absorbance
spectra of the free and conjugated formsof the dye partially overlap. This causes
non-linear protein measurement because
free dye is excited by the light used toexcite bound dye. In contrast, the freeversus conjugated absorbance maxima of
the dye provided in the Fluorescent Protein Quantiﬁcation Kit are separated by
108 nm. This means that when conjugatedsample is excited at ~500 nm (for example, at 488 nm), free dye is not excited. In
addition, the ﬂuorescent intensity of free
dye is 50-fold lower than conjugated dye;taken together, these features effectively
eliminate background (Figure 1).

your samples. For example, if your samples
are prepared in an unusual buffer, thenthe standards used for the Standard Curve
should be prepared in the same buffer.

70
60
50

BSA alone
BSA + 20 mM NaCl
BSA + 2 mM MgCl2
BSA + 10 mM Tris
BSA + 5 mM (NH4)2SO4
BSA + 0.02% SDS

40
30
20
10
0

Figure 2: Color shift caused by dye conjugation.
The dye is blue in its free state. After a 30 minute, room
temperature conjugation, the dye’s color changes to red.

Contaminating substances
Unlike many protein determination methods, which are inﬂuenced by the presenceof contaminants such as detergents andsalts, the Fluorescent Protein Quantiﬁcation Kit has been shown to be resistantto the effects of many contaminating
agents (Figure 3). However, to achieve thebest results the protein standards should
be assayed under the same conditions as

1

2

3

4

5

BSA (μg)
Figure 3: Standard Curves of BSA.
Increasing amounts of BSA protein were quantified
using the Fluorescent Protein Quantification Kit in thepresence of a variety of contaminants.

A simple way to improve your results
The large difference in the absorption
maxima of the free dye compared to the
conjugated dye and the ease of conjugation make the new Fluorescent Protein
Quantiﬁcation Kit an ideal tool for
protein quantiﬁcation. To improve theresults of your downstream assays, use
the Fluorescent Protein Quantiﬁcation Kit.

Product

FormatCatalog No.

Fluorescent Protein Quantiﬁcation Kit

1000 rxns

15001

3

Capillary Electrophoresis

New: Capillary Electrophoresis Made Easy
Active Motif is pleased to offer a new selection of novel dyes for capillaryelectrophoresis (CE). CE Dyes are a signiﬁcant improvement over existing
methods for analyzing proteins using CE. Their ease of use, spectral shift
upon conjugation, high quantum yield and ability to maintain the natural
ionic character of labeled protein make them ideal for use in CE.
Derivatization of proteins with ﬂuorescent labels prior to CE is commonly used
to overcome the lack of sensitivity whenanalyzing proteins via UV absorbanceor natural ﬂuorescence. In general, theproteins are labeled by attaching dye
molecules to the primary amines oflysine residues. Lysine is used primarily
because it is a relatively abundant aminoacid, and also because lysine residues
are often located at the surface of theprotein. However, as dye labeling is nonuniform, and because proteins usuallycontain many lysine residues, inconsistent labeling of multiple lysine residues
within a single protein is common. If
the dye used affects the overall protein
charge, then a single protein will give rise
to a series of products, each with a different charge. This can cause problems
during separation and other steps.
One charge – for clear separation
Like other dyes, Active Motif’s CE Dyes
utilize highly abundant and accessible
lysine residues for their attachment
chemistry. However, CE Dyes overcome
the limitation of other dye systems by
maintaining the positive charge of theamine group following dye conjugation(Figure 1). This means that proteins labeled with CE Dyes will not display bandbroadening or require adjusted ioniccharacter calculations to be performed.
The result is that CE Dyes provide ahighly sensitive labeling system thatmaintains the natural charge propertiesof every protein within your sample.

4

added, followed by incubation at either
room temperature or 50ºC, dependingon the CE Dye used. Completion of thereaction can be observed by eye due toa change in sample color from blue tored. It’s as simple as that!
Large Stokes Shift minimizes backgroundIn addition to their non-toxic and charge
properties, CE Dyes also undergo a significant change in spectral properties upon
conjugation to amine groups. In their
free state, CE Dyes have very weak ﬂuorescence (quantum yield < 1%), with anabsorbance maximum of 612 nm and anemission maximum of 665 nm. However,upon reaction with a primary amine theabsorbance maximum shortwave-shifts
by more than 100 nm to 503 nm andemits strongly with a quantum yield of
more than 50% at 600 nm. This change
in spectra and quantum yield meansthat the presence of unconjugated label
does not affect downstream analysis andsigniﬁcantly reduces background effects.

Non-toxic – for user-friendly labeling
A further disadvantage of commonlyused dyes, such as o-pthaldialdehyde,
naphthalene-2,3-dicarboxaldehyde and3-(2-furoyl)quinoline-2-carboxaldehyde,
is that derivatization must be performedin the presence of cyanide. Use ofsuch a highly toxic compound is not
only undesirable, but creates signiﬁcant disposal problems for the unusedmaterial. In contrast, the CE Dye labelingprocess is simple to perform, non-toxic
and requires only a 60 minute incubation at either room temperature or 50ºC,
depending on the CE Dye used.
The CE Dye method
Labeling samples with CE Dyes is fastand convenient. The protein sample ofinterest is dissolved in bicarbonate solution (pH unadjusted) and stock CE Dye is

Get better CE results starting now
To improve your CE results, give us a callto order CE Dyes today.

“Labeling proteins for capillary electrophoresis is easy with CE Dyes, while
the large Stokes Shift and charge maintenance help improve your results.”
CH3
Protein(CH2)4

NH3

O

CH3
R

Protein(CH2)4

CH3

R

N
CH3

Figure 1: Labeling with CE Dyes maintains each protein’s natural charge.
Chemical equation depicting the chemistry of CE Dye labeling, which demonstrates the preservation of the positive chargeof the lysine residue of a protein. R stands for the respective chromogenic/fluorogenic group.

Product

FormatCatalog No.

CE Dye 503
CE Dye 540

1 kit
1 kit

15101
15102

Toll Free — 1 877 222 9543

September 2005 • volume 6 • number 3

Small GTPase Activation

ELISA for Quantiﬁcation of Activated Ras GTPase

In contrast, the Ras GTPase Chemi ELISA
uses Raf-RBD protein and antibodies ina 96-well format to capture and quantify the activated Ras in your sample.
This faster, more sensitive alternative
enables you to use less of your precious
sample, yet still detect low-level events.
In addition, because ELISAs provide morequantitative results than Westerns, thedata generated is more meaningful.
The Ras GTPase Chemi ELISA method
Because activated Ras binds speciﬁcallyto the Ras-binding domain (RBD) of the
Raf effector protein, Raf-RBD is used

as a probe to isolate activated Ras. The
Ras ELISA Kit contains a Raf-RBD protein
fused to GST and a 96-well, glutathionecoated assay plate. GST-Raf-RBD is ﬁrst
incubated on the plate for one hour toimmobilize this capture protein. Addition of sample to the plate results in thebinding of activated Ras to the Raf-RBD.
A primary antibody speciﬁc for Ras isthen added, followed by an HRP-conjugated secondary antibody and developing reagent (Figure 1). The plate is thenread on a luminometer, which provides asensitive, quantitative chemiluminescentreadout of activated Ras (Figure 2).

“ELISAs are faster and more sensitive than other methods used to
study GTPase activation, and eliminate the need for IP, gels and blots.”
Add anti-IgG
HRP conjugate

Add primary
antibody
45 min45 min45 minAdd developing
solution, thenquantify

Add cell
extract toplate

Cell extractcontaining activatedRas GTPase

GST-Raf-RBD
coated plate

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

Product
Ras GTPase Chemi ELISA Kit

www.activemotif.com

FormatCatalog No.

1 x 96-well plate

52097

Ras ELISA advantages• More sensitive – assay uses only
25 μg of extract, or 20-fold lessthan pull-down/Western methods• Better results – quantitative data
makes it easier to compare results• Less effort – no need to perform IP,
run gels or develop Western blots• Save time – results in < 5 hours• Versatile – assay activated extracts
from cells or tissue samples, or
study recombinant Ras protein
Activated Ras Signal (RLU)

Active Motif’s Ras GTPase Chemi ELISA Kit is the ﬁrst ELISA-based kit
designed to detect and quantify activated Ras GTPase. The method offers
several advantages over other commercially available kits, which require youto perform immunoprecipitation of Ras, followed by Western blotting.

800000

Unstimulated
EGF stimulated

600000400000200000
0

6.25

12.5
25
Cell extract (μg/well)50

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

Try the quantitative, sensitive assay
The Ras GTPase Chemi ELISA Kit makes
it fast and easy to detect and quantify
activated Ras GTPase. The kit is ideal
for the study of novel signaling pathways that activate Ras, as well as fordetermining if a particular malignancy
is related to inappropriately activated,oncogenic Ras. Additional assays todetect other GTPase proteins, such as
RhoA, are currently in development.
Please give us a call, return the enclosed
reply card or log on to www.activemotif.
com/gtpase for complete information,
downloadable manuals and to learn
about new additions to this innovative
product line. To get to the best Ras activation assay available, try the Ras GTPase
Chemi ELISA Kit.

5

Chromatin Immunoprecipitation

Use ChIP-IT™ for More Successful Chromatin Immunoprecipitation
Chromatin immunoprecipitation (ChIP) is a powerful, yet technicallychallenging tool used to study protein/DNA interactions. Active Motif’s
ChIP-IT™ Kits make your ChIP more successful by combining nearly everything you need in a single kit, including positive control antibodies andprimers, DNA puriﬁcation columns and a comprehensive protocol.
The ChIP method
In ChIP, intact cells are ﬁxed using
formaldehyde, which cross-links andpreserves protein/DNA interactions. The
DNA is then sheared into small, uniform
fragments. The protein/DNA complexes
are then immunoprecipitated using
antibodies directed against the DNAbinding protein(s) of interest. Following
immunoprecipitation, the cross-links arereversed and DNA fragments are puriﬁed
and screened to determine which gene,or group of genes, was bound by theprotein of interest (Figure 1).
ChIP-IT advantages• Easy to use – all critical buffers,
Protein G beads and DNA puriﬁcation columns are provided• No need to optimize reagents andprotocol• Your choice of enzymatic orsonication shearing• Direct measurement of transcription
factor/DNA interactions or histone
modiﬁcations
Choose enzymatic or sonication shearing
The key to a successful ChIP experiment
begins with correctly shearing the DNA
into 200-1000 bp fragments. This can beachieved by using one of two methods: enzymatic digestion or sonication.
Sonication shearing is an effective
method for shearing DNA but can be

6

into fragments suitable for ChIP. And, as
digestion is dependent only on time andtemperature, very little optimization isrequired. For your convenience, ChIP-IT
Kits are available with your choice ofeither sonication or enzymatic shearing.
Start your ChIP today
Active Motif’s ChIP-IT Kits provide youwith everything you need to make yourChIP experiments easier and more reproducible. For more information on ChIP-IT
visit www.activemotif.com/chip.

difﬁcult to optimize due to complications from overheating, emulsiﬁcationand dependence on sonicator type. In
contrast, enzymatic digestion can beused to quickly and easily digest DNA“ChIP-IT makes performing ChIP with transcription factors more successful
by combining all of the critical components you need in a single kit.”
Cross-link protein to DNA inliving cells with formaldehyde

Break open cells
and shear DNA

Add primary
antibody of interest

Analyze purified DNA
using PCR or
hybridization methods

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

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

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

Product

FormatCatalog No.

ChIP-IT™

25 rxns

53001

ChIP-IT™ w/o controls

25 rxns

53004

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

10 rxns

53002

ChIP-IT™ Enzymatic

25 rxns

53006

ChIP-IT™ Enzymatic w/o controls

25 rxns

53007

Enzymatic Shearing Kit (included in 53006 & 53007)

10 rxns

53005

Toll Free — 1 877 222 9543

September 2005 • volume 6 • number 3

Transcription Factor ELISAs

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

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

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

2.5

0.5

1

5

10

Unstimulated

25

50

0.5

1

5

10

25

50

1.5
1

TransAM advantages• Non-radioactive, colorimetric method provides quantiﬁable results• Results in less than 5 hours• 10-fold greater sensitivity thangelshift (100-fold with Chemi Kits)• Simultaneous proﬁling of multiple
family members• Assay both cell and tissue samples

0.5
0

A

IL-1α stimulated

Unstimulated
IL-1α stimulated

2

Flexi or Original format
The Original TransAM Kits offer a fast,
non-radioactive alternative to gelshiftby providing a 96-well plate that isprecoated with oligonucleotide that
contains a consensus-binding site for thefactor of interest. Activated transcription factor binds the oligo and is quantiﬁed on a spectrophotometer using the
supplied antibodies and developing
reagent. This format is convenient formeasuring binding at the consensus
site, but does not enable you to study
alternative sites. For this reason, we developed TransAM Flexi Kits; they containall of the optimized reagents provided
in the Original kits, including antibodies,but give you the ﬂexibility to immobilize
any oligo in the 96-well plate.

0.5

1

2
5
10 25
Cell extract/well (μg)

50B

μg of cell extract

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

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

TransAM™ Product Line
TransAM™ AP-1 Family

TransAM™ AML-1/Runx1

TransAM™ C/EBP α/βTransAM™ HNF-1

TransAM™ Flexi NFκB p65

TransAM™ GATA Family

TransAM™ AML-3/Runx2

TransAM™ CREB & pCREB

TransAM™ IRF-3

TransAM™ NFκB p65*

TransAM™ HNF Family

TransAM™ AP-1 c-Fos

TransAM™ Elk-1

TransAM™ MEF2

TransAM™ Oct-4

TransAM™ IRF Family

TransAM™ AP-1 c-Jun

TransAM™ ER

TransAM™ MyoD

TransAM™ p53

TransAM™ MAPK Family

TransAM™ AP-1 FosB

TransAM™ FKHR (FOXO1)TransAM™ NF-YA

TransAM™ PPARγTransAM™ Flexi NFκB Family

TransAM™ AP-1 JunD

TransAM™ GATA-4

TransAM™ NFATc1

TransAM™ Sp1 & Sp1/Sp3

TransAM™ NFκB Family

TransAM™ ATF-2

TransAM™ GR

TransAM™ Flexi NFκB p50

TransAM™ STAT3

TransAM™ STAT Family

TransAM™ c-Myc

TransAM™ HIF-1

TransAM™ NFκB p50*

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

www.activemotif.com

7Cell-based ELISAs

Fast, Efﬁcient Analysis of Protein Phosphorylation
As interest in kinase activation and its effect on cellular regulation increases,
so does the need for fast, efﬁcient and robust assays to measure protein
phosphorylation. To ﬁll this need, Active Motif developed its Fast Activated
Cell-based ELISA (FACE™) Kits.
FACE Kits provide a simple, sensitive,
cell-based method for monitoring protein phosphorylation. They enable youto perform modiﬁcation-speciﬁc analysis
directly within the cell, without the needfor time-consuming cell extractions, gelelectrophoresis or membrane blotting,all of which saves you time and money.
The FACE method
In the FACE method, cells are cultured in
96-well plates and stimulated to induce
the pathway of interest. Following stimulation, the cells are rapidly ﬁxed, which
preserves activation-speciﬁc protein modiﬁcations. Each well is then incubated witha primary antibody speciﬁc for the protein
modiﬁcation of interest. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides acolorimetric or chemiluminescent readoutthat is quantitative and reproducible(Figure 1). FACE Kits also contain a primary
antibody for the native non-modiﬁedprotein, so you can monitor both native
and activated protein levels in the same
experiment (Figure 2).
Uninduced A-431
EGF-Induced A-431

OD450 nm0.4
0.3

Speciﬁcity counts
To be certain that you detect only yourprotein of interest, all FACE antibodies
are stringently tested for cross-reactivity. All phospho-speciﬁc antibodies are
veriﬁed to detect only the activated formof the target protein. In addition, thephospho-speciﬁc and total antibodies areused in tandem to ensure the phosphoantibody doesn’t interact with otherphosphorylated proteins. This ensuresthat FACE Kits are highly speciﬁc, so you’ll
get the results you need.

Choose from a variety of kits
FACE Kits are available for a variety ofdifferent factors, listed below, and eachassay is available in both colorimetric andchemiluminescent formats. To simplify themeasurement of your phospho proteins,try a FACE Kit today.
Add anti-IgG
HRP conjugate

Add primary
antibody
3 hrs.

1 hr.

Cells arefixed following
stimulation

P

P

P

Add developing
and stop solution

Stimulatecells

Cells cultured in
96-well plate
Figure 1: Flowchart of the FACE process.
Cells are grown, stimulated, fixed and assayed in a single 96-well plate. Addition of primary and secondary antibodies
and developing solution enables quantitation of phosphorylated and total protein levels on a spectrophotometer orluminometer.

“FACE Kits make it possible to detect phosphorylated proteins in the cell,
eliminating the need for cell extractions, gels and Western blotting.”

0.2

FACE™ Product Line

0.1
0FACE™ AKT
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 the levels of total and phosphorylated ErbB-2
contained within uninduced or EGF-induced A-431 cells.

8

FACE advantages• Simple, quantitative method• Cell-based assay eliminates extractions and gel electrophoresis• Fixing cells preserves protein
activation state• Fast – requires less than 3 hours ofhands on time
• Primary antibodies to compare
phospho and native protein levels

FACE™ ATF-2

FACE™ Bad

FACE™ c-Jun (S63)

FACE™ c-Jun (S73)

FACE™ c-Src

FACE™ EGFR (Y992)

FACE™ EGFR (Y1173)

FACE™ ErbB-2 (Y877)

FACE™ ErbB-2 (Y1248)

FACE™ ERK1/2

FACE™ FAKFACE™ FKHR (FOXO1)FACE™ GSK3β

FACE™ JAK1FACE™ JNKFACE™ MEK1/2

FACE™ NFκB Proﬁler

FACE™ p38

FACE™ PI3 Kinase p85

FACE™ STAT2

FACE™ STAT4

FACE™ STAT6

Toll Free — 1 877 222 9543

September 2005 • volume 6 • number 3

Protein Transfection

Deliver Functional Proteins Directly into Living Cells
Chariot™ is Active Motif’s patented* protein delivery reagent that efﬁciently
transports biologically active proteins, peptides and antibodies directly into
cultured mammalian cells. Delivery is complete in less than two hours andprovides efﬁciencies of 65-95%. After delivery, living cells can be assayed
immediately to determine the effects of the introduced material. Thesefeatures make Chariot an ideal tool for a variety of functional studies.
Targeted delivery
Chariot is a peptide that forms a noncovalent complex when incubatedwith your puriﬁed protein, peptide
or antibody for 30 minutes at room
temperature. Addition of the complex
to cells results in its rapid internalization. Once inside the cell, the complex
dissociates and Chariot is transported to
the nucleus, while the delivered protein
is biologically active and free to proceed
to its cellular target (Figure 1).

Deliver biologically active proteins
The ability of Chariot to deliver biologically active protein is shown using
a 119 kDa subunit of β-galactosidase.
β-galactosidase is composed of four
subunits that must assemble to form
functional protein. HeLa cells turn blue
when X-gal is added after delivery of theChariot-galactosidase complex, demonstrating successful delivery of functional
β-galactosidase (Figure 2).

Advantages• Delivers active protein directly into
living cells• Up to 95% efﬁciency in < 2 hours• Works in a variety of cell lines, aswell as in vivo
• Study living cells – no ﬁxing needed
Why use protein delivery?
Direct delivery of active protein makes
it easy to perform studies not evenpossible using DNA transfection andexpression. Successful Chariot deliveryof proteins, peptides and antibodies has
been shown in a wide range of cell lines,
including hard-to-transfect neuronal, primary and plant cells. For a list of papers
that cite the use of Chariot, simply returnthe enclosed reply card or download thelist at www.activemotif.com/chariot.

“The ability to deliver active proteins directly into living cells and then assay
their effect makes feasible studies not even possible with other methods.”
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 into hard-to-transfect and nondividing cells. To learn what your protein isreally doing, study it using Chariot.

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

Non-covalent delivery of native protein
Many protein delivery systems requirethat you begin by fusing a carrier protein
to your macromolecule. In addition to
being time-consuming, this can alter thefolding characteristics of your protein
and, ultimately, its function. Because
Chariot forms a non-covalent bond withyour protein, it does not affect the delivered protein’s folding or function.

www.activemotif.com

Figure 2: Chariot delivery of β-galactosidase.
One μg of a 119 kDa subunit of β-galactosidase wascomplexed with Chariot for 30 minutes and delivered
into HeLa cells. Cells were fixed and stained with X-gal
2 hours post-delivery.

Product

FormatCatalog No.

Chariot™

25 rxns**
100 rxns

30025
30100

β-Galactosidase Staining Kit

75 rxns

35001* Chariot is covered under U.S. Patent No. 6,841,535. Purchase includes the right to use for basic
research purposes only. Other-use licenses are available; please contact Active Motif Technical
Services for additional information.
** A rxn is deﬁned as sufﬁcient Chariot reagent to deliver protein to cells in a 35 mm plate.

9Sandwich ELISAs

Accurate Detection of Signal Pathway and Nuclear Receptor Proteins
FunctionELISA™ & NR Sandwich Kits offer a simple, rapid method to monitor changes in protein levels. They utilize the Sandwich ELISA technique tocapture and quantify the amount of a speciﬁc protein in your sample.
y = 0.018x - 0.018 r2 = 0.998
0.75

0.25

0
0

Convenient format saves time
FunctionELISA & NR Sandwich Kits
are faster and simpler than Western
blots because your samples are readon a spectrophotometer (or on aluminometer for IκBα). This means thatyou don’t need to run, blot and develop
gels. In just hours, you’ll have accurate,quantitative results that are easier tointerpret than blots (Figure 2). Plus, theconvenient 96-well format makes it possible to process multiple samples quickly,
at your level of throughput.

10

10

20

30

40

50

60

Cytochrome c (ng/ml)
Figure 1: Cytochrome c Standard Curve.
A Standard Curve was generated by assaying increasing
amounts of recombinant human cytochrome c with the
FunctionELISA Cytochrome c Kit.

0

“Sandwich ELISA Kits make it
simple to capture and quantiﬁably
measure the amount of a speciﬁc
protein in your sample.”

Complete kits ensure your successOur Sandwich ELISA Kits contain all of
the reagents required to rapidly quantify
your protein. FunctionELISA Kits even include recombinant protein for producing
a standard curve. For your convenience,
the Capture Antibody is supplied immobilized in a 96-well assay plate, so you
won’t waste time with overnight incubations. For complete details, please visit
our website at www.activemotif.com.

0.5

5
10
30
time (minutes)

60

A

chemiluminescence

Speciﬁc, quantitative assay
Sandwich ELISAs utilize two antibodies
that recognize different epitopes on
the protein to be measured. The ﬁrst
antibody, termed the Capture Antibody,binds the protein of interest from thecell lysate. A Detecting Antibody is thenused to bind to the captured protein. In
FunctionELISA Kits, accurate quantiﬁcation can be made by comparing thesample to a standard curve made using
recombinant protein included in each kit(Figure 1).

1

OD450 nmThe Sandwich ELISA method is fasterand simpler to perform than othertechniques used to study proteins, like
Western blot. And, it enables you tomake quantitative rather than justqualitative protein measurements.

Sandwich ELISA advantages• Quantitative results in just hours• ELISA format eliminates the need torun, blot and develop gels
• Easily analyze multiple samples• Ready-to-use format with Capture
Antibody precoated on the plate• Assay both cell and tissue samples

200000160000120000
80000
40000

B

0

0

10

20

30
40
time (minutes)

50

60

70

Figure 2: FunctionELISA provides a more quantitative measurement of IκBα phosphorylation than Western blot.
Jurkat cells were induced with 1 nM TNF-α and harvested at the indicated time points. Whole-cell extracts were assayedin Western blot analysis (A) using Phosphorylated-IκBα mAb (Cat. No. 40904), and with the FunctionELISA IκBα Kit (B).

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

NR Sandwich AR

1 x 96 rxns
5 x 96 rxns

49196
49696

NR Sandwich ERα

1 x 96 rxns
5 x 96 rxns

49296
49796

NR Sandwich PR

1 x 96 rxns
5 x 96 rxns

49396
49896

Toll Free — 1 877 222 9543

September 2005 • volume 6 • number 3

Recombinant Proteins & Cellular Extracts

Positive Control Recombinant Proteins and Cell Extracts for All Your Needs
Active Motif offers you an extensive lineof recombinant proteins against a varietyof transcription factors and cell-signaling
related proteins that are sure to suit yourresearch needs. These proteins are great
for use as positive controls in Western
blot, EMSA, kinase assays and as standards in ELISAs such as our TransAM Kits.
Finding a good positive control is essential for any experiment. With Active
Motif’s line of recombinant proteins, you
can feel conﬁdent of your results as eachprotein has undergone extensive testing
to ensure its functionality.
Cellular extract controls
In addition to our recombinant proteins,
Active Motif offers a broad line of readyto-use nuclear, cytoplasmic and whole-cell
extracts that are prepared using extraction protocols that have been optimized
to produce high yields of cell signaling
proteins. In addition, many of our extracts
are prepared from cells that have beenstimulated or treated to speciﬁcally induce
activation of hard-to-detect transcription
factors. Be sure to check out our online
search tool to ﬁnd your extract of choiceeither by keyword or tied to the antibody
for which you need a positive control.

Figure 1: Active Motif’s cell extract search engine.
Active Motif’s online cell extract search engine enables you
to find cell extracts by cell type, application, catalog number,keyword, stimulation condition or antibody of choice.

www.activemotif.com

Product
Recombinant AKT1 protein
Recombinant ATF-2 protein
Recombinant BRCA1 protein
Recombinant c-Fos protein
Recombinant c-Jun protein
Recombinant c-Myc protein
Recombinant CREB protein
Recombinant CTF1 (NF-1) protein
Recombinant eIF2α protein
Recombinant ER protein
Recombinant FXR protein
Recombinant GR protein
Recombinant IκBα protein
Recombinant JNK2α1 protein
Recombinant JNK2α2 protein
Recombinant LXRα protein
Recombinant LXRβ protein
Recombinant NFκB p50 protein
Recombinant NFκB p65 protein
Recombinant p53 protein
Recombinant p300 protein
Recombinant PPARα protein
NEW: Recombinant PPARα-LBD protein
Recombinant PPARβ(δ) protein
NEW: Recombinant PPARβ(δ)-LBD protein
Recombinant PPARγ protein
NEW: Recombinant PPARγ-LBD protein
Recombinant pRB protein
NEW: Recombinant PXR protein
Recombinant Rad51 protein
Recombinant RAR-α protein
Recombinant RAR-β protein
Recombinant RAR-γ protein
Recombinant RXR-α protein
Recombinant RXR-β protein
Recombinant RXR-LBD protein
Recombinant Sp1 protein
Puriﬁed Sp1 protein
Recombinant STAT1 protein
NEW: Recombinant STAT3 protein
Recombinant TRα1 protein
Recombinant TRβ1 protein

Format5,000 units
25,000 units
2,000 units
5,000 units
5,000 units
5,000 units
25,000 units
5,000 units
25,000 units
4,000 units
10,000 units
5,000 units
25,000 units
5,000 units
5,000 units
10,000 units
10,000 units
5,000 units
5,000 units
5,000 units
4,000 units
10,000 units
10,000 units
10,000 units
10,000 units
10,000 units
10,000 units
5,000 units
10,000 units
10,000 units
5,000 units
5,000 units
5,000 units
10,000 units
5,000 units
10,000 units
5,000 units
2,000 units
25,000 units
10,000 units
10,000 units
10,000 units

Catalog No.
31105
31106
31113
31115
31116
31117
31107
31118
31108
31119
31120
31121
31109
31110
31111
31122
31123
31101
31102
31103
31124
31125
31141
31126
31142
31127
31143
31128
31144
31129
31130
31131
31132
31133
31134
31135
31136
31137
31112
31140
31138
31139

11

Active Motif Ofﬁces
U.S.

EUROPE

JAPAN

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

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

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2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
Email: japantech@activemotif.com

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

12

Australia

Austria

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Denmark

Finland

Hungary

India

Israel

Italy

Japan

Korea

Spain

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