THE NEWSLETTER OF ACTIVE MOTIF — February 2006 • volume 7 • number 1

New Products
for Kinase Biology

IN
THIS
ISSUE

2 Fast, Efﬁcient Analysis of Protein Phosphorylation

8 NEW: Fluorescent Detection Products

3 NEW: Highly Active Recombinant Kinases

9 Sensitive Protein Quantiﬁcation Using
Fluorescence

4 NEW: Colorimetric Quantitation of Activated Kinases
5 Sensitive, Speciﬁc Transcription Factor Analysis
6 Use ChIP-IT™ for More Successful Chromatin
Immunoprecipitation
7 NEW: Rapid DNA Methylation Detection

10 NEW: Transcription Factor Protein Arrays
11 NEW: Learn More Faster with AlmaKnowledgeServer
12 Improved Co-IP of Nuclear Protein Complexes

Kinases

Fast, Efﬁcient Analysis of Protein Phosphorylation
0.6
0.5
OD450 nm

As interest in kinase activation and their effect on cellular regulation increases,
so does the need for fast, efﬁcient and robust assays to measure protein
phosphorylation. That is why Active Motif developed its Fast Activated
Cell-based ELISA (FACE™) Kits. FACE Kits provide a simple, sensitive, cell-based
method for monitoring protein phosphorylation.

0.4
0.3
0.2
0.1
0

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-conjugatedantibody and developing solution provides
a colorimetric or chemiluminescent readout that is quantitative and reproducible(Figure 1). FACE Kits also contain a primary
antibody for the native non-modiﬁedprotein, so you can monitor both native
and activated protein levels in the same
experiment (Figure 2).

Speciﬁcity you can count on
A concern for many researchers studying
phosphorylated proteins is the lack of
speciﬁcity of phospho-speciﬁc antibodies. To be certain that you detect only
the protein you are interested in, allantibodies in FACE Kits are stringently
tested for cross-reactivity using Western
blot analysis. All phospho-speciﬁc antibodies are veriﬁed to detect only theactivated form of the target protein. In
addition, the phospho-speciﬁc and total
antibodies are used in tandem to ensure
the phospho-speciﬁc antibody doesn’tinteract with other phosphorylated
proteins. This ensures that FACE Kits arehighly speciﬁc and detect only the protein you are interested in, at the speciﬁc
phosphorylated site.

Add anti-IgG
HRP conjugate

Add primary
antibody

Uninduced
InducedTotal-AKT

Phospho-AKT

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

FACE advantages• Cell-based ELISA – no extracts, gels,blotting or radioactivity• Fast – requires less than 2 hours ofhands on time
• Flexible – high-throughput chemi
and colorimetric formats• 2 Antibodies – compare both
phosphorylated and native protein
levels in the same kit• Quantitative results – ELISAs
provide more meaningful data thanWesterns“FACE Kits enable detection ofphosphorylated proteins within thecell, eliminating the need for cell
extractions, gels & Western blotting.”

3 hrs.
1 hr.

Cells arefixed following
stimulation

P

P

P

Add developing
and stop solution

Stimulatecells
Cells cultured in
96-well plate
Figure 1: Flow chart of the FACE process.
Cells are grown, stimulated and fixed in the same 96-well plate. Addition of primary and secondary antibodies detects
phosphorylated protein.

2

High-throughput analysis
Many cellular kinase cascades containcritical control points, where multiple downstream components canbe phosphorylated by a single kinase.
The phosphorylation of one or othersubstrates can cause signiﬁcant shifts
in the cellular response, which meansthat monitoring phosphorylation events
across multiple proteins can become

Toll Free — 1 877 222 9543

February 2006 • volume 7 • number 1

commonplace. However, trying to monitor this type of “signaling switch” using
Western blotting or classical radioactive
kinase assays is a labor intensive and expensive undertaking, especially if there
are multiple treatments to be measured.
To overcome this problem using FACE
can be as simple as an extra pipetting
step. This is because in the FACE method,
cells are cultured and analyzed within
the same well and therefore do not require the use of multiple culture plates,
extract preparations, gels, membranes,
etc. And, because you can easily increasesample throughput, your results will bestatistically more relevant.

Kinases & Proteins

Eliminate unwanted sample modiﬁcations
When analyzing protein phosphorylation
via Western blotting or ELISA, it isnecessary to prepare cellular extracts.
However, additional protein modiﬁcations can occur during the extraction
process, which may alter your results. To
eliminate this problem, FACE Kits use aﬁxation step that “freezes” the cellular state and prevents further protein
modiﬁcations. This enables detection of
the exact protein state in the cell at achosen time point, which provides
you with more accurate results. For abetter phospho-speciﬁc assay, try FACE.

FACE™ Product Line
FACE™ AKT (S473)

FACE™ ATF-2 (T71)

FACE™ Bad (S112)

FACE™ c-Jun (S63)

FACE™ c-Jun (S73)

FACE™ c-Src (Y418)

FACE™ EGFR (Y992)

FACE™ EGFR (Y1173)

FACE™ ErbB-2 (Y877)

FACE™ ErbB-2 (Y1248)

FACE™ ERK1/2 (T202/
Y204 & T185/Y187)

FACE™ FAK (Y397)

FACE™ FKHR (T24)

FACE™ GSK3β (S9)

FACE™ JAK1 (Y1022/
Y1023)

FACE™ JNK (T183/Y185)

FACE™ MEK1/2 (S217/
S221)

FACE™ NFκB Proﬁler(S468 & S536)

FACE™ p38 (T180/Y182)

FACE™ PI3 Kinase p85

FACE™ STAT2 (Y689)

FACE™ STAT4 (Y693)

FACE™ STAT6 (Y641)

NEW: Highly Active Recombinant Kinases
Active Motif provides an extensive lineof recombinant transcription factor andcell signaling-related proteins. These
recombinant proteins are ideal for all
your research needs and can be used
in a variety of applications including asprobes in the new TransArray™ transcription factor protein array (see page 10) or
as protein standards in ELISAs. The c-Fos,c-Jun, c-Myc, CREB, NFκB p50, NFκB
p65, p53 and Sp1 proteins have beenvalidated for use in making standard
curves in our TransAM™ Transcription
Factor ELISAs (see page 5). Complete information on the recombinant proteins,
including detailed technical data sheetsthat specify the protein length, themethod used for puriﬁcation, etc. canbe found at www.activemotif.com.

www.activemotif.com

Recombinant Kinase & Transcription Factor Product Line
ABL

AKT

ATF

B-RAF

BRCA

BRK

AP-1

c-Myc

CHK

CK2

CREB

CSK

CTF1

EGFR

eIF2

ER

ErbB

ERK

FAK

FGF

FGR

FXR

GR

GSK3

HCK

IGF1-R

IκBα

IKK

INS-R

IRAK

ITK

JAK

JNK

LCK

LXR

Lyn

MAPKAPK

MEK

MKK

NFκB

p38

p53

p300

PAK

PDGFR

PDK

PKA

PKC

PPAR

PXR

Rad

RAR

pRb

RXR

S6K

SNK

Sp1

Src

STAT

SYK

TR

VEGF-R

VRK

WEE

Yes

ZAP70

3

Kinase ELISAs

NEW: Colorimetric Quantitation of Activated Kinases
Active Motif’s KineActive™ ELISAs improve the study of highly conserved
members of kinase families in cell and tissue extracts by providing unmatchedsensitivity and speciﬁcity in a non-radioactive, 96-well format.

“KineActive provides rapid,
speciﬁc detection of activatedkinases in a 96-well format.”
The KineActive method
In the KineActive ELISA method, acellular extract or recombinant kinase
is added to the wells of the provided
96-well, antibody-capture plate. This isfollowed by addition of a kinase-speciﬁc antibody, which binds to the plateand captures the kinase of interest.
After the unbound proteins are washedaway, a biotinylated, unphosphorylated
peptide substrate is added in the presence of ATP. The immobilized kinase
phosphorylates this substrate peptide,which is then captured by the additionof a second antibody that is speciﬁc forthe phosphorylated form of the peptide.
Addition of streptavidin-HRP and subsequent developing solution provides aquantitative colorimetric readout that is
directly proportional to the activity of
the kinase (Figure 1).

4Advantages• Quantitative• Unmatched speciﬁcity• Non-radioactive detection• High throughput compatible• Improved sensitivity• Assay cell and tissue extracts or
puriﬁed protein

2.52OD450 nmSpeciﬁc kinase detection
A major limitation when using traditional
kinase assays is that many kinase families, such as Src, contain members that
phosphorylate a common target. Therefore, to accurately measure the activityof a speciﬁc kinase, it is necessary toisolate it from others that can act upon
the same peptide substrate. The highspeciﬁcity of the KineActive Kits enable
the study of conserved kinase families
whose activity is otherwise difﬁcult tomeasure accurately.

based radioactive kinase assays may
require several hundred micrograms ofwhole-cell extract in order to detect Src
activity, KineActive Src can measure Src
activity using less than 20 µg of extract(Figure 2). Try KineActive today to simplify your kinase biology research.

Desiredkinase

ATP

1
0.5
0

Improved sensitivity
KineActive offers improved sensitivity over other methods, which enables
you to choose from a variety of sample
sources including puriﬁed protein orcell and tissue extracts. For example,
while traditional immunoprecipitation-

Non-phospho
peptide substrate

1.5

50

12.5
6
3
25
HeLa whole-cell extract (µg)

50

(no 1º ab)

Figure 2: Sensitive detection of activated Src in HeLa
cells using KineActive.
HeLa whole-cell extract was diluted down to 3 µg per
well and assayed using the KineActive Src ELISA Kit.

BiotinPhosphorylated
peptidesubstrate

Streptavidin-HRP
conjugate

P

Kinaseantibody

Phospho
Peptide antibody

Figure 1: Flow chart of KineActive ELISA process.
In KineActive, sample containing the desired kinase is added to a 96-well antibody capture plate. This is followed by additionof kinase antibody, which binds to the plate and specifically captures the kinase of interest. After washing, a biotinylated,
non-phosphorylated peptide substrate is added in the presence of ATP. The immobilized kinase phosphorylates this peptide,which is captured by addition of an antibody against the phosphorylated peptide. Addition of Streptavidin-HRP and developing solution provides a quantitative colorimetric readout.

Product

FormatCatalog No.

KineActive™ Lyn

1 x 96 rxns

56096

KineActive™ Src

1 x 96 rxns

56196

KineActive™ Yes

1 x 96 rxns

56296

Toll Free — 1 877 222 9543

February 2006 • volume 7 • number 1

Transcription Factor ELISAs

Sensitive, Speciﬁc Transcription Factor ELISAs
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.”A

2.5

IL-1α stimulated

Unstimulated
IL-1α stimulated

2

0.5

1

5

10

Unstimulated

25

50

0.5

1

5

10

25

50

1.5
1

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.
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

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

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 youneed in a single kit, including positive control antibodies and primers, 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 makeyour ChIP 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: Flow chart 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

February 2006 • volume 7 • number 1

DNA Methylation

NEW: Rapid DNA Methylation Detection
Active Motif’s MethylDetector™ Bisulﬁte Modiﬁcation Kit simpliﬁes analysis
of DNA methylation. The kit provides optimized reagents for performing
DNA conversion with bisulﬁte, plus time-saving DNA puriﬁcation columns
and positive control PCR primers to validate your results.
Methylation is important
DNA methylation is a naturally occurring event that affects cell function byaltering gene expression. As aberrant
methylation is prevalent in many humancancers, much research depends on accurately quantifying DNA methylation.
Proven controls ensure success
Many DNA methylation analysis methods begin by using bisulﬁte to convert
unmethylated cytosines to uracils.
During conversion, methylated cytosines
remain unchanged. The DNA is thenampliﬁed by PCR and analyzed by sequencing or restriction digest. However,bisulﬁte conversion can be technicallychallenging, and it is desirable to conﬁrm
that the process was successful before
spending time and money on sample
analysis. To help ensure your success, theMethylDetector Kit provides optimized
conversion reagents, an easy-to-use protocol and positive control PCR primersthat are speciﬁc for bisulﬁte-converted
DNA. Because these primers produce
PCR product only if conversion has occurred, you can conﬁrm the procedure
worked before starting sequencing orother analysis methods (Figure 1).

“MethylDetector improves DNA
methylation analysis byproviding optimized reagentsand proven controls.”

www.activemotif.com

M

1

2

3

4

5

500 bp400 bp300 bp200 bpThe MethylDetector method
In the MethylDetector method, DNAof interest is rapidly heat denatured
in a thermocycler in the presence ofthe bisulﬁte conversion reagent. The
temperature is then lowered and theconversion reaction is performed. Unlike
other methods, MethylDetector does
not require an initial acid denaturation
step as the conversion reagent includes
a DNA denaturant, which saves you
time and effort. After DNA conversion,
the sample is added to the included
DNA puriﬁcation columns, and a simple,
on-column desulfonation is performed.
Ready-to-use DNA is then eluted fromthe columns (Figure 2).

100 bpFigure 1: Agarose gel analysis of PCR products generated
with MethylDetector.
Three different DNA conversions were performed(Lanes: 1-3) and compared to an unconverted DNA
control (Lane 5) and to a no DNA control (Lane 4). The
presence of PCR product in only the converted samples
demonstrates the conversion efficiency and reproducibility of the MethylDetector Kit.

Advantages• Works efﬁciently with high G/Ccontent sequences and uncut DNA• Reproducible assay consistently
provides 99% conversion efﬁciency• Optimized reagents and protocol
with proven controls• Combined thermal denaturationand conversion reaction streamlines
procedure• DNA puriﬁcation columns eliminate precipitation and a separate
desulfonation step
• High yield of converted DNA is idealfor downstream analysis

Denature DNA withheat followed by
Conversion Reagent

Desulfonate,
Clean-up and PCR

Figure 2: Flow chart of the MethylDetector process.

Order one today
The MethylDetector Kit will improveyour bisulﬁte conversions. Find more
information about this new innovative
kit from Active Motif by giving us a callor visiting www.activemotif.com.

Product

FormatCatalog No.

MethylDetector™

50 rxns

55001

7

Fluorescent Detection

NEW: Fluorescent Detection Products
Active Motif is pleased to announce the launch of the new Active Motif
Chromeon ﬂuorescent detection product line. This new product range
offers innovative ﬂuorescent tools using the proprietary ﬂuorescent dyes
and substrates developed at Chromeon GmbH.

Albumin Quantitation Kit
Serum albumin is a reliable prognostic indicator for morbidity and mortality, liverdisease, kidney disease and malnutrition.
The new Albumin Blue Fluorescent Assay
Kit from Active Motif Chromeon is aquantitative assay designed to measure
albumin levels in biological samples suchas serum and urine. The kit combines unmatched sensitivity, high speciﬁcity anda quick and easy protocol. Learn more atwww.activemotif.com.

Chromeo Dyes
Fluorescent labels for bioanalysis
The Chromeo Dyes – Chromeo 494,
Chromeo 546 and Chromeo 642 –provide unmatched photostability,
which enables multiple exposures andincreased exposure time. They arecompatible with most excitation sources
including diode lasers, LEDs, tungsten
lamps and xenon arc lamps (Table 1). In
addition, Chromeo Dyes display a large
tolerance to pH. Their luminescent properties together with their low toxic effects make Chromeo Dyes ideal for use

absorb (494 nm)

emit (628 nm)

1.0

Fluorescence excitation

The Chromeon advantage
The dyes developed at Active Motif
Chromeon exhibit superior luminescence properties, including a broad
range of ﬂuorescence excitation andemission spectrums, large Stokes
shifts, limited photobleaching and pH
stability. These features enable thedevelopment of a versatile platform
for novel ﬂuorescent assays such as the
new ProStain™ Protein Quantiﬁcation
Kit (see page 9), CE Dyes for capillaryelectrophoresis and the Albumin Blue
Fluorescent Assay Kit. These products
and the Chromeo™ Dyes represent onlya small fraction of dyes available from
Active Motif Chromeon. To ﬁnd more
information about other ﬂuorescentdyes, visit www.chromeon.com.

as labels of biomolecules in cell culture
staining experiments. Chromeo Dyes areavailable as amine reactive NHS-Esters,
used for conjugation with amino-groups
in peptides, proteins and amino-modiﬁed DNA, or as carboxylic acids.

0.8

0.6

0.4

0.2
0
300 350 400 450 500 550 600 650 700 750 800

Wavelength (nm)Figure 1: Chromeo 494 enables ﬂuorescent multiplexing.
Chromeo 494 is excited at 488 nm. With its large Stokes
shift of 124 nm, Chromeo 494 is the ideal partner for multiplexing with other, short-shifted 488-excitable dyes.

Absorption

Emissione L/(molcm)

Quantum
Yield (%)

Stokes
Shift

Chromeo™ 494

494

628

20,00025

124 nmChromeo™ 546

545

561

110,000

8

16 nm

Chromeo™ 642

642

660

170,000

17

18 nm

Dye

Table 1: Chromeo Dye properties.

Product

FormatCatalog No.

Chromeo™ 494 Carboxylic Acid

1 mg
5 mg

15110
16110

Chromeo™ 494 NHS-Ester

1 mg
5 mg

15111
16111

Chromeo™ 546 Carboxylic Acid

1 mg
5 mg

15210
16210

Chromeo™ 546 NHS-Ester

1 mg
5 mg

15211
16211

Chromeo™ 642 Carboxylic Acid

1 mg
5 mg

15310
16310

Chromeo™ 642 NHS-Ester

1 mg
5 mg

15311
16311

Albumin Blue Fluorescent Assay Kit

1 kit

15002

You can ﬁnd additional Chromeon Dyes at www.chromeon.com

8

Toll Free — 1 877 222 9543

February 2006 • volume 7 • number 1

Fluorescent Protein Quantification

Sensitive Protein Quantiﬁcation Using Fluorescence
Active Motif’s ProStain™ Protein Quantiﬁcation Kit is a simple, sensitive alternative to traditional methods for determining protein levels. Offering high signal
strength, unique spectral properties and robust conjugation, the kit offers limitsof detection that are 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 ProStain
Protein Quantiﬁcation Kit makemeasurement fast and accurate.”
Fast, simple conjugation
Using ProStain is fast and easy; the kit
provides ﬂuorescent dye, dilution buffer
and a protein standard. Simply resuspend the dye and add it to the wells ofa microplate, then add a serial dilutionof the standard protein to produce aStandard Curve, along with your sample.
After a 30-minute, room-temperatureincubation, simply read the ﬂuorescence
to quantify your samples.

www.activemotif.com

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.

Contaminating substances
Unlike many protein determination
methods, the ProStain Protein Quantiﬁcation Kit has been shown to be resistant to the effects of many contaminating agents, such as detergents and salts(Figure 2). However, to achieve the best
results the Standard Curve should be
80

Relative fluorescence

Broad spectral shift for better results
A disadvantage of many protein quantiﬁcation methods, such as Bradford assays,
is that the absorbance spectra of thefree and conjugated forms of the dyepartially overlap. This causes non-linear
protein measurement because free dye
is excited by the same wavelength oflight used to excite the bound dye. In
contrast, the free versus conjugatedabsorbance maxima of the ﬂuorescentdye provided in ProStain are separated
by 108 nm. This means that when conjugated sample is excited at ~500 nm (forexample, at 488 nm), only conjugated
dye is excited. In addition, the emission
intensity of free dye is 50-fold lower
than conjugated dye; taken together,
these features effectively eliminate
background (Figure 1).

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

1

2

3

4

5

BSA (µg)
Figure 2: Standard Curves of BSA.
Increasing amounts of BSA protein were quantified
using the Fluorescent Protein Quantification Kit in thepresence of a variety of contaminants.

created using the same conditions
as your samples. For example, if yoursamples are prepared in an unusual buffer, then your Standard Curve should be
prepared using the same buffer.
Consistent complex quantiﬁcation
To be effective, a protein quantiﬁcation
method must be consistent when used on
different samples. To show the advantagesof ProStain, it and the Bradford assay wereused to produce BSA Standard Curves
and then to quantify known amounts ofthree different nuclear extracts. Results
obtained with ProStain showed a greater
degree of accuracy than the Bradford
assay, which displayed inconsistent
results between different sample types
and concentrations (Table 1). This typeof inconsistency will impair downstream
analyses performed with your samples as
it will cause signiﬁcant loading errors inyour Westerns, ELISAs and other assays. To
improve the results of your downstream
assays, quantify using ProStain.
HeLa (µg/ml)

ProStain

Bradford

0.5

0.57 (13.8%)

1.39 (177.0%)

1.0

1.18 (17.9%)

0.82 (18.3%)

5.0

5.31 (6.2%)

5.21 (4.2%)

Jurkat (µg/ml)

ProStain

Bradford

0.5

0.46 (7.7%)

0.88 (75.8%)

1.0

0.98 (1.9%)

1.63 (63.2%)

5.0

5.15 (2.9%)

4.84 (3.2%)

MCF-7 (µg/ml)

ProStain

Bradford

0.5

0.51 (1.1%)

0.48 (3.2%)

1.0

1.02 (1.6%)

0.79 (20.7%)

5.0

5.63 (12.7%)

3.52 (29.6%)

Table 1: ProStain and Bradford quantiﬁcation of extracts.
Known amounts of 3 nuclear extracts were assayed intriplicate. The average concentration calculated, and its
error from the known amount quantified, are shown.

Product

FormatCatalog No.

ProStain™ Protein Quantiﬁcation Kit

1000 rxns

15001

Nuclear Extract Kit

100 rxns

40010

9Transcription Factor Arrays

NEW: Transcription Factor Protein Arrays
Active Motif’s new line of TransArray™ TF Protein Arrays make itpossible to simultaneously study 38 activated transcription factors ina fast and easy-to-use format.
Multiplexed detection
Post-transcriptional regulation andtranslational modiﬁcations can produce
dramatic changes in protein levels andactivity that are not detectable in DNA
arrays. This makes identifying protein:protein and protein:DNA interactions ina high-throughput format an enormous
challenge as current assays enable onlya single protein to be characterized at atime. In contrast, the new TransArray Kits
enable high-throughput characterizationof multiple transcription factor proteins.

Why use TransArray?
• Simultaneously measure 38 activated transcription factors (see Table 1)• Highly speciﬁc• Quantitative detection• Simple alternative to pull-down
assays, yeast two-hybrids and Co-IPs• Study protein:protein or protein:
DNA interactions
The TransArray method
Each array consists of highly active
puriﬁed proteins that are spotted intriplicate on a nitrocellulose membrane.
A protein, DNA or ligand of interest isthen used as a probe to search for interactions with the immobilized proteins.
These interactions are assessed either bytagging the probe with a small molecule,by radioactivity or through using an antibody to the probe (Figure 1). Currently,
the signals are visualized by radioactive
detection using photographic ﬁlm or bya phosphor imager.

orProtein

RNA, DNA
Incubate withTransArray™

“TransArray enables multiplexed analysis of transcription
factor interactions with both DNA and proteins ”

DetectInteractions

A

B

C

D

E

1

STAT3

Rap74

hRPB5

c-Fos

BRG-1 Mutated

2

STAT1

Rap30

Negative

c-Jun

PC4

3

BRCA1

TFIID

Sp1

AP-1

PC4 F77P

4

TFIIA-p55

TBP

CTF-1

DR1

p52

5

TFIIA-p12

TFIIH

E2F-1

HMG-1

p75

6

TFIIB

TFIIH-p62

pRB

TOPO WT

p75-CTD

7

TFIIEα-p56

RNA pol II

p53

TOPO Y723F

p300

8

TFIIEβ-p34

RNA pol II CTD

c-Myc

BRG-1 WT

Negative

Table 1: Activated transcription targets included in the TransArray™ TF Protein Arrays

Product

FormatCatalog No.

TransArray™ TF membrane

2 arrays

31400

10

Wash Away
Unbound Probe

Strength of signal corresponds
to amount of bound probe
Figure 1: Flow chart of the TransArray process.

Try TransArray today
The innovative TransArray format enables multiplexed detection and interaction assessment of transcription factor
proteins with DNA, RNA and/or ligands
of interest. For your convenience, Active
Motif also offers a full line of highly
active recombinant proteins (see page 3)that are ideal for use as protein probes
in TransArray. To ﬁnd more information
about these new protein arrays, visit
www.activemotif.com.

Toll Free — 1 877 222 9543

February 2006 • volume 7 • number 1

Knowledge Mining

NEW: Learn More Faster with AlmaKnowledgeServer
AlmaKnowledgeServer (AKS) is a powerful text mining system that makes it
possible for you to uncover relationships that exist in the scientiﬁc literature
between genes, proteins, chemical compounds and diseases. AKS can evendiscover links that exist, but that have not yet been published.
A new way to keep pace
Researchers read journal articles to learn
about new developments in their ﬁeld.
But, with 1000’s of journals and morethan 15 million abstracts on Medline(plus over 2,000 more added daily), it isphysically impossible to keep current,
let alone to explore all of the areas thatmay provide new and useful insights.
The answer is AlmaKnowledgeServer(AKS), a powerful text mining systemthat, unlike you, reads every paper everyday. AKS examines all abstracts added
to PubMed, then applies statistics andrules-based analysis to uncover relationships that exist in the scientiﬁc literature
between genes, proteins, chemical compounds and diseases. This enables you tomake better informed decisions about
your research in far less time.
Bioentity and synonym recognition
AKS understands and retrieves biologicalconcepts (bioentities) instead of simple
words. The bioentities recognized by
AKS include genes and proteins, diseases
and chemical compounds. AKS has beentaught the many synonyms, acronyms
and other scientiﬁc naming ambiguities
that plague traditional searching. For
example, searching PubMed for IL-2, IL2,
IL 2 or interleukin 2 return different
results, though all are the same gene.
So, an exhaustive search for a single
gene may actually require 5-10 individual
searches. With AKS, one search returns
all documents that are relevant to IL-2.

Search ﬁrst, then analyze
After a search, AKS ranks the relevant
concepts and bioentities. Documents
can be explored by viewing sentencesin which two or more bioentities occur(Figure 1). By reading the top ranking
sentences, it’s possible to build a signiﬁcant understanding of the interrelations
between bioentities. You can also read
abstracts with co-occurring bioentities
highlighted, and apply ﬁlters to ﬁne tune
your results. AKS also provides links to
Medline, so you can quickly access themost essential papers.

Visualize biological relationships
Because new insights often come from
exploring data from different perspectives, AKS also provides a graphical viewof the relationships between bioentities(Figure 2). The links between terms represent documents or sentences wheremultiple entities co-occur. Though thisvisual representation may contain thecontent of thousands of documents, it
can be interpreted quickly and easily,
helping you to obtain new insights.
Achieve more with better information
AKS can help answer questions that areimportant to your research, like if a given
protein is involved in a disease, or who
are the thought leaders in an emerging
ﬁeld? To learn more, give us a call or visit
Active Motif’s biocomputing division,
TimeLogic, at www.timelogic.com.

Figure 1: Sentences displaying co-occurrences of “leptin” and “obesity” bioentities.

Figure 2: AKS includes a powerful visual environment to map relationships between bioentities.

www.activemotif.com

11

Co-Immunoprecipitation

Improved Co-IP of Nuclear Protein Complexes
Using the Nuclear Complex Co-IP Kit from Active Motif will improve yourco-immunoprecipitation studies of nuclear protein complexes because
it provides optimized reagents for both nuclear extract preparation andimmunoprecipitation.
Optimized method and reagents
Co-immunoprecipitation (Co-IP) isoften used to analyze protein/protein
interactions. However, traditional Co-IP
methods are not optimal for studying
DNA-binding protein complexes as thesecomplexes are unstable. The Nuclear
Complex Co-IP Kit helps maintain thesecomplexes by providing both high andlow stringency buffers, as well as additional salt and detergent. Addition of salt
and detergent is ideal for robust protein
interactions as it reduces background.
However, many complexes may not withstand high stringencies, thus stringency
can be modiﬁed as required.

Advantages• Simple and efﬁcient• Optimized extraction procedure
preserves nuclear protein complexes• Easily alter IP stringency to detect
interactions of varying strengths

12

U.S.

EUROPE

Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351
Email: sales@activemotif.com

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

JAPAN
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
Email: japantech@activemotif.com

Try the new Co-IP Kit today
The Nuclear Complex Co-IP Kit enables
you to study tightly bound or weakprotein complexes with equal ease,which improves your results. To get more
information on this innovative productplease give us a call or visit our website
at www.activemotif.com.

250
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

PRSRT STD
US POSTAGEPAID
PERMIT #236

92054