THE NEWSLETTER OF ACTIVE MOTIF — SEPTEMBER 2003 • VOLUME 4 • NUMBER 3

Quantitative Measurement of the GATA, HNF and IRF
Transcription Factor Families
Traditionally, transcription factor activity has
been studied using either Electrophoretic
Mobility Shift Assays (EMSA), immunoblottingor reporter gene assays. These methods
are time-consuming and at best provide
only semi-quantitative results. Moreover,determination of transcription factor subunit
activity requires the investment of significant
amounts of both time and money, or may noteven be possible. Now, TransAM™ Family Kits
make it straightforward to measure the activity of families of transcription factors in one
simple experiment and in less than 5 hours*.
Plus, the TransAM Family Kits can be used on
all sample types, including cell lines and tissues, giving you unsurpassed flexibility.

The TransAM method
Each TransAM Family Kit includes two
96-well plates in which multiple copies of adouble-stranded oligonucleotide have been
immobilized. When nuclear or whole-cell
extract is added, the various members of
the transcription factor family of interest bind to the consensus-binding site on
the oligonucleotide. Primary antibodies
specific for the activated forms of eachfamily member are then added to individual wells, followed by incubation withsecondary HRP-conjugated antibody anddeveloping reagent. Reading the plate on aspectrophotometer provides a quantitative,
colorimetric readout (Figure 1, page 4).
continued on page 4

IN THIS ISSUE
1Quantitative Measurement ofTranscription Factor Families

1

An Improved Method to StudyDNA Damage and Repair

Normalization for
2 Reproducible
More Meaningful Results
Chemiluminescent
3 Ultra-sensitive
Assay for MAPK Phospho-proteinscient Delivery of Biologically
6 Effi
Active Proteinsand Simple Mitochondrial
7 FastFractionation
Yields of Specifically
7 HighSegregated Extracts
c Antibodies & Quality
8 Specifi
Positive Control Extracts

An Improved Method to Study DNA Damage and Repair

DNA repair proteins act to maintaingenome integrity by recognizing, binding toand repairing damaged DNA. Damage recognition proteins such as G/T binding protein (GTBP, also called MSH6) and Ku70/86
bind as heterodimers to DNA with specificlesions. GTBP recognizes mismatched G/Tbase pairs in double-stranded DNA and triggers excision and repair. In contrast, Ku70/
86 binds directly to DNA ends and repairs
double-stranded DNA breaks. Deficiencies
in the activity of these proteins is linked
to the development of many pathologicaldiseases, including cancer. Yet, despite thegrowing interest in studying the relationshipbetween DNA damage and repair, there isa lack of convenient, sensitive assays thatare suitable for processing large numbers ofsamples. To overcome this, we are introducing a new line of high-throughput assays
to quantify DNA repair proteins, beginningwith kits for GTBP and Ku70/86 (Figure 1).

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

Active Motif’s new DNA Repair Kits offer
an improved method for studying DNA
repair protein activity. Current techniques
used to study DNA damage and repair include time-consuming EMSAs and Western
blots. Our new DNA Repair Kits are DNAbinding ELISAs that eliminate radioactivity
and provide quantitative results in less than
five hours. Studying DNA repair has neverbeen easier.

0.6

0.4

0.2

0

0

1

2

3

4

5

6

Raji nuclear extract (µg)

Figure 1: Monitoring Ku70 with the Ku70/86 DNA Repair Kit.
Different amounts of nuclear extract from Raji cells were tested for
activity using the Ku70/86 DNA Repair Kit.

continued on page 5

www.activemotif.com

Protein Normalization

Fast, Reproducible Normalization Method for More Meaningful Results

HRP

E

E

Secondary
Antibody

Substrate

Detecting
Antibody
Ag

Ag

A

CaptureAntibody

GAPDH Assay“Sandwich”

0.8B
Lamin B1 Assay“Sandwich”

Figure 1: Sandwich ELISA schematics.

Why be normal?
Before performing many cell biology
assays, the protein concentration of eachsample must be quantified, commonly
using a Bradford or Lowry protein assay.
“Equal” amounts of each sample are thenassayed. However, these methods can yield
variable results depending on the protein
composition of the extract and its buffer
type. This can introduce significant errors in
subsequent assays because, in fact, unequalamounts of the different samples are being
assayed. Unlike these methods, Active
Motif’s normalization kits use an antibodybased detection method, so your results
are not altered by the protein makeup of
your sample or its buffer composition.

2

Absorbance (OD450 nm)

HRP

Simple, specific method
Active Motif’s new normalization kits provide a simple, specific method for protein
normalization. They utilize the “Sandwich”
ELISA method, which uses two antibodies,
each recognizing a distinct epitope on theprotein. Each kit includes an ELISA plate
coated with the first antibody, called the
Capture Antibody. This is used to capture
either GAPDH or Lamin B1 from the sample.
A second antibody, called the Detecting
Antibody, is used to detect the protein
bound by the Capture Antibody. Horseradish peroxidase (HRP) is then used forcolorimetric detection. In the GAPDH Kit,
HRP is directly conjugated to the Detecting Antibody, while the Lamin Kit utilizes
an HRP-conjugated secondary antibody(Figures 1A & 1B). Each kit provides quantifiable and reproducible results through thegeneration of standard curves using theincluded GAPDH (Figure 2) or Lamin B1 protein standard. Using the curve, the amountof GAPDH or Lamin B1 per sample can thenbe used to normalize your assay results.
y = 0.015x + 0.032 r=0.999

0.6

0.4

0.2

0

0

10

20

30

40

50

60

Verify the integrity of your samples
The Lamin Nuclear Normalization Kit canalso be used to verify the integrity of yoursamples. Because Lamin B1 is found onlyin the nucleus, its presence in cytoplasmic
extracts would indicate contamination of
the extract by nuclear proteins. To demonstrate, nuclear and cytoplasmic extracts
were made from NIH/3T3 cells using our
Nuclear Extract Kit (page 7). The absenceof Lamin B1 in the cytoplasmic extract confirms the integrity of the extracts (Figure 3).

Absorbance (OD450 nm)

Active Motif’s new GAPDH Whole-cell and
Lamin Nuclear Normalization Kits make itfast and easy to normalize your samples
prior to downstream applications, suchas PAGE, Western blot and ELISA analysis.
This reduces inaccuracies by ensuring thatyou are comparing equivalent amounts ofprotein in each sample. The Whole-cell andNuclear Normalization Kits quantify thehousekeeping proteins GAPDH and Lamin,
which are widely used for normalization as
they are present in high levels in the cytoplasm and nucleus, respectively. The kitsutilize an ELISA-based assay that is more
reproducible than other methods. They
also feature a convenient 96-stripwell format, so you can process multiple samples
quickly, at any level of throughput.

0.5

0.25

0

20

10

5

Nuclear

2.5

20

10

5

Cytoplasmic

2.5

NIH/3T3 extracts (µg)
Figure 3: Assaying cytoplasmic and nuclear extracts for Lamin B1.
The Lamin Nuclear Normalization Kit was used to assay cytoplasmic
and nuclear extracts that were isolated using the Nuclear Extract Kit.

Get better results every experiment
Active Motif’s new normalization kits area simple way to improve the accuracy of
your cell biology assays. They eliminate thevariability introduced into your research byinaccurate protein determination methods.
In many instances, the normalization kits
make it possible for you to perform yourassays without even determining the protein concentration of your samples. Withnormalization this easy, there’s no longer areason to not improve all of your results.

GAPDH Control protein (ng)Figure 2: Production of a GAPDH Standard Curve.
The GAPDH Whole-cell Normalization Kit was used with known
amounts of the GAPDH Control protein to generate a standard curve.

Product

FormatCatalog No.

GAPDH Whole-cell Normalization Kit

1 x 96 rxns
5 x 96 rxns

48007
48507

Lamin Nuclear Normalization Kit

1 x 96 rxns
5 x 96 rxns

48008
48508

Toll Free — 1 877 222 9543

SEPTEMBER 2003 • VOLUME 4 • NUMBER 3

Cell-based ELISAs

Ultra-sensitive Chemiluminescent Assay for MAPK Phospho-proteins

Regulation of cellular protein activity
via phosphorylation has been shown tobe critical for normal functioning of the
majority of cellular pathways. Indeed,
many disease states such as cancer
and autoimmune responses have been
attributed to the aberrant control ofprotein phosphorylation. Yet despitewidespread interest, measurement ofprotein phosphorylation has remained lowthroughput and labor intensive. FACE Kits
are designed to overcome the limitationsof classical assays and at the same time
improve the quality of your results.
The FACE method
In the FACE method, cells are cultured in
96-well plates and stimulated to induce thepathway of interest. Following stimulation,
the cells are fixed, which preserves protein
modifications, including phosphorylation.
Each well is then incubated with a primary antibody specific for the activatedprotein of interest. Subsequent incubation with secondary HRP-conjugatedantibody and developing solution provides an easily quantified, colorimetric or
chemiluminescent readout. The signals arethen normalized for cell number using theprovided Crystal Violet solution and plotted. FACE Kits are easy to use and requireless than two hours hands on time, giving
you convenience not available with otherphosphorylation assays.

www.activemotif.com

Flexibility with chemiluminescent detection
Colorimetric ELISA measurement of protein
phosphorylation offers a more convenient
alternative to Western blotting and in-gel
kinase assays. However, use of colorimetricdetection can make it difficult to maintain linearity within the assay readout,
meaning that small changes in protein
phosphorylation can be lost due to suboptimal detection. The new FACE Chemi
Kits, however, use chemiluminescence, so
the assay detection limits can be adjusted
to maintain linearity and ensure that yourdetection sensitivity is appropriate to yoursample type. This means that FACE Chemi
Kits can accurately monitor even the smallest changes in protein phosphorylation. For
experiments that require maximum sensitivity, try the new FACE Chemi Kits.
Reproducible - for more meaningful results
FACE Kits are highly reproducible, which
is vital for getting the most meaningful
results possible when measuring smallchanges in the amounts of phosphorylated
protein. To demonstrate, FACE Chemi assays
were performed on three different samplesof macrophage 4/4 cells that had beentreated in an identical manner with PMA to

induce ERK phosphorylation (Figure 1). The
combination of the high reproducibility andincreased throughput of FACE Chemi Kits
means that you’ll get the most meaningfulphospho-specific results available.

ERK Chemi signal (RLU)

Fast Activated Cell-based ELISA (FACE™)
Kits are an innovative alternative toclassical methods for monitoring protein
phosphorylation. FACE Kits enable modification-specific analysis directly within thecell, without the need for time-consuming cell extractions, gel electrophoresisor membrane blotting. In addition to thehighly popular colorimetric version, FACE
Kits are now available in a new ultra-sensitive, chemiluminescent format. Even thesmallest change in protein phosphorylation
can be detected simply and quickly with
the new FACE Chemi Kits.

Experiment 1
Experiment 2
Experiment 3

35000
30000
25000
20000
15000
10000
5000
0

0

10
100
PMA induction (ng/ml)

1000

Figure 1: Reproducible assay of phosphorylated ERK.
Murine macrophage 4/4 cells were cultured in 96-well plates andserum starved for 16 hours. Cells were then stimulated with Phorbol
12-myristate 13-acetate (PMA) for 10 minutes and fixed. Levels ofphospho-ERK were assayed in triplicate using the FACE ERK1/2 Chemi
Kit. Data was plotted after normalization for cell number (performed
through use of Crystal Violet).

FACE Kits are available for monitoring thecellular levels of phosphorylated p38,
JNK, AKT, ERK1/2 and EGFR, with manyother kits to be released throughout
2003. For a better way to monitor protein
phosphorylation, use FACE.

Product

FormatCatalog No.

FACE™AKT Chemi

1 x 96 rxns
5 x 96 rxns

48220
48720

FACE™ EGFR Chemi

1 x 96 rxns
5 x 96 rxns

48250
48750

FACE™ ERK1/2 Chemi

1 x 96 rxns
5 x 96 rxns

48240
48740

FACE™ JNK Chemi

1 x 96 rxns
5 x 96 rxns

48210
48710

FACE™ p38 Chemi

1 x 96 rxns
5 x 96 rxns

48200
48700

3

DNA-binding ELISAs

continued from page 1 — Quantitative Measurement of the GATA, HNF and IRF Transcription Factor Families

The complete solution for all your needs
Reporter gene assays expressed eithertransiently, or from a stable cell line, arecommonly used to monitor transcription

factor activation. However, it can be difficult to relate this data to actual in vivo
experiments, which can limit more relevant,downstream research. TransAM Family
Kits provide a complete solution that iswell suited for both model systems and invivo studies. Don’t waste time with DNA
transfections or stable cell line construction, which can’t be used in vivo; TransAM
Kits are designed to meet all your needs.

IRF activation (OD450 nm)

Proven specificity – for improved accuracy
To accurately study transcription factor
activation, you must be able to determine
which of the complex mixture of related
heterodimers is involved in pathway regulation. To ensure that this is possible, allantibodies supplied in TransAM Family Kits
are tested for cross-reactivity with otherfamily members and shown to recognize
only the isoform of interest. In addition,
TransAM Family Kits are tested for specificity by assaying in the presence of an excessof oligonucleotide containing a wild-type
or mutated consensus-binding site (Figure2). This competitive assay proves that thetranscription factor isoform detected isbinding specifically to the probe that has
been immobilized to the TransAM plate.
With the guaranteed specificity of theTransAM Family Kit antibodies, you’ll get
the accuracy you want for all your experiments.

2

TransAM IRF FamilyInterferon Regulatory Factors (IRFs) area family of transcription factors thatplay a critical role in host defense. The
function and activity of the individual
IRFs can depend both on their activation state as well as that of other IRF
family members. The ability of theTransAM IRF Family Kit to simultaneously profile the activation states of
IRF-1, -2, -3 and -7 makes it easy tounderstand the critical role of eachisoform in the regulation of immune
function and oncogenesis.

Extract alone
Extract + wild-type oligo
Extract + mutated oligo

1.5

TransAM HNF Family
Hepatocyte Nuclear Factors (HNFs) areprimarily required for cellular differentiation and metabolism. Unlike mosttranscription factor families, this groupof evolutionary conserved proteins does
not have a common consensus-bindingsite. This makes assaying HNF family
activity using classical EMSA or reporter
assays cumbersome and time consuming. The new TransAM HNF Family Kit
contains multiple binding sites in eachwell, making it quick and easy to monitor HNF-1, -3α, -3β and -4α activity ina single assay. In addition, a TransAM
HNF-1 Kit is available for researchers
who want to monitor this important
regulator of liver function separately.

1

0.5
0

IRF-1

IRF-2

IRF-3

IRF-7

Figure 2: Specificity of TransAM Kits.
TransAM IRF assays are performed in the absence or presence of competitor oligonucleotides that contain either a wild-type or mutated
IRF consensus-binding site. IRF-1, IRF-2 and IRF-3 activity was assayedusing 5 µg/well nuclear extract from U-937 cells stimulated with TPA +IFNγ, while IRF-7 activity was assayed using 5 µg/well nuclear extractfrom Jurkat cells stimulated with PHA.

TransAM GATA Family
The GATA family of transcription factors contains six zinc-finger binding
proteins that regulate differentiationand cell proliferation. GATA proteinsare grouped into two subfamilies. The
new TransAM GATA Family Kit measures
GATA-1, -2, and -3, the hematopoietic
subfamily. As hematopoietic cells are
difficult to transfect, they are often
studied as primary isolates, making reporter gene assays and EMSA impractical. The TransAM method, however, canbe used easily on primary isolates, so it
is ideal for GATA research.

Figure 1: Flowchart of the TransAM procedure.

Product

FormatCatalog No.

TransAM™ AP-1 Family

2 x 96-well plates

44296

TransAM™ GATA Family

2 x 96-well plates

48296

TransAM™ HNF Family
TransAM™ HNF-1

2 x 96-well plates
1 x 96-well plate5 x 96-well plates

46296
46196
46696

TransAM™ IRF Family

2 x 96-well plates

45296

TransAM™ NFκB Family

2 x 96-well plates

43296

TransAM™ STAT Family

2 x 96-well plates

42296

4

*

Technology covered under AAT-filed patents and licensed exclusively
to Active Motif.

Toll Free — 1 877 222 9543

SEPTEMBER 2003 • VOLUME 4 • NUMBER 3

DNA Repair

continued from page 1 — An Improved Method to Study DNA Damage and Repair

Advantages• Non-radioactive, colorimetric method• Quantitative results in less than 5 hours• 10X more sensitive than gelshift• High-throughput compatible• Ability to assay both cells and tissues
Precise, quantitative results
Being able to precisely measure small changes in DNA repair protein activity within any
sample is important. Although traditional
methods such as Western blot are sensitive, they are typically more of a qualitative rather than quantitative tool. Active
Motif’s DNA Repair Kits, however, provide acolorimetric readout that is easily quantified, so you can detect even the slightestchange in protein activity. To illustrate, GTBP
activity was measured using Active Motif’s
GTBP DNA Repair Kit and compared to aWestern blot of the same sample. Assays
were performed on Raji, PANC-1 and Jurkat
nuclear extracts. The GTBP DNA Repair Kit
results are clearly more quantitative thanWestern blot (Figure 3).

Figure 2: Flowchart of the DNA Repair Kit procedure.

Improved accuracy
The DNA Repair Kits are accurate over a
wider range of sample concentrations thanother methods because they use colorimetric detection, which is linear over abroader sample range (Figure 1, page 1). This
saves time and money, as fewer samples
will need to be diluted and reassayed toobtain accurate results. Get the most outof your experiments by using the new DNA
Repair Kits.
GTBP

A

Absorbance (OD450 nm)

How it works
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 ofa specific double-stranded oligonucleotide
have been immobilized. The oligonucleotide
in the GTBP Kit contains a G/T mismatch,while that in the Ku70/86 Kit simply has ablunt end. When cellular extract is added,
the repair protein of interest binds to theoligonucleotide. Each well is then incubatedwith a primary antibody that is specific forthe repair protein being studied. Additionof a secondary HRP-conjugated antibody
and developing solution provides an easily
quantified colorimetric readout (Figure 2). This
allows you to easily monitor the regulation of
DNA repair proteins in a particular cell line,
animal model or tumor biopsy.

1.25

1
0.75

0.5

Try it today
Our DNA Repair Kits will simplify the way
you study interactions between damaged
DNA and repair proteins. These easy-to-use
kits are flexible enough to use in a variety
of functional studies, such as examining thespecificity of repair proteins for a particular
DNA damage. And, the increased sensitivity means you can accurately detect smallchanges in activity levels. If you’re tired ofusing radioactive probes, try one of our
DNA Repair Kits today.

0.25
0

Raji

PANC-1

JurkatB

Figure 3: Measurement of GTBP activity in different cell lines using
Western blot and the new GTBP DNA Repair Kit
Jurkat, PANC-1 and Raji nuclear extracts were assessed for GTBP activity using Western blot (A) and the new GTBP DNA Repair Kit (B).

Productwww.activemotif.com

What’s available
DNA Repair Kits are currently available forstudying GTBP and Ku70/86. The GTBP Kit
contains an antibody specific for GTBP,
while the Ku70/86 Kit contains two antibodies, specific for either Ku70 or Ku86. Be
sure to check our website for complete kit
configurations, downloadable manuals andto learn about new additions to the DNA
damage and repair product line.

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

5

Protein Transfection

Efficient Delivery of Biologically Active Proteins
Chariot™ is a revolutionary delivery reagent
that efficiently transports biologically
active proteins, peptides and antibodies
directly into cultured cells in less than
two hours. Typical delivery efficiencies
are 65-95%. After delivery, living cells canbe assayed immediately to determine theeffects of the introduced material, without
the need for fixing. This makes Chariot
ideal for functional studies.
Simple delivery method
Chariot is a peptide that forms a noncovalent complex when combined withpurified protein, peptide or antibody. This
protects the protein from degradation
during internalization. Upon addition tocells, the complex is rapidly internalized,
where it dissociates. The delivered protein
is biologically active and free to proceed toits cellular target.

A

0

G1

10

15

20
(h)

G2

B

0
G1

10

15

20
(h)

G2

Figure 1: Chariot delivery of functional protein.
HS-68 cells arrested in G0 phase by serum deprivation for 48 hours
were released by addition of serum for 3 hours. Flow cytometry
performed 0, 10, 15 and 20 hours after addition of Chariot alone and aChariot-p27kip1 complex indicate that cells receiving Chariot alone progressed into G2 phase (A), while over 90% of cells receiving the Chariotp27kip1 complex remained in G1 phase (B). Data generously provided by
Dr. Gilles Divita, Biophysics Dept., CNRS, Montepellier, France.

6

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

Protein

30%

Deliver biologically active proteins
The ability of Chariot to deliver biologically
active compound was demonstrated using
p27kip1, a 27 kDa cyclin-dependent kinase
inhibitor that causes cell-cycle arrest in G1
phase. Over 90% of cells receiving Chariotp27kip1 complex were unable to progressbeyond G1 phase (Figures 1A & 1B), demonstrating efficient delivery of active p27kip1.
What can Chariot do for you?
Many papers have been published in scientific journals citing the use of Chariot. Successful delivery has been shown in a varietyof cell types, including hard-to-transfectTransfection efficiency (%)

cells like neuronal and plant cells. Recently,
the first paper showing usage of Chariot invivo was published. The table above wascompiled from data furnished to us byrespondents to our Chariot survey. For thecomplete survey, or to download a list of
papers that cite Chariot, visit our website.
Chariot delivers results
Chariot speeds and simplifies a variety offunctional studies because it efficiently delivers biologically active proteins, peptides andantibodies directly into cells, even hard-totransfect and non-dividing cells. For a new
way to study your protein, try Chariot.

Product

FormatCatalog No.

Chariot™

25 rxns
100 rxns

30025
30100

Toll Free — 1 877 222 9543

SEPTEMBER 2003 • VOLUME 4 • NUMBER 3

Cell Extraction & Fractionation

Fast and Simple Mitochondrial Fractionation
The Mitochondrial Fractionation Kit isolates
highly enriched mitochondrial and cytosolic
fractions from mammalian cell lines. This
simplifies the study of protein translocation

Absorbance (OD450 nm)

0.8
0.6

Cytosolic
Mitochondrial

0.4
0.2
0

Uninduced

16 hr apoptosis

Cellular fractions (2 µg/well)Figure 1: Monitoring cytosolic and mitochondrial cytochrome c levels.
HeLa cells were grown to 90-95% confluence and treated with 10 nM
Actinomycin D to induce apoptosis. Cell samples were harvested prior
to treatment as well as 16 hours post-treatment. The Mitochondrial
Fractionation Kit was used to isolate cytosolic and mitochondrial
fractions from each sample. Two µg of each fraction was assayedusing the FunctionELISA™ Cytochrome c Kit to monitor changes in the
mitochondrial and cytosolic cytochrome c levels.

events that occur during apoptosis and in
signal transduction pathways. The kit’s highquality reagents and optimized protocol eliminate cross-contamination and produce highyields of properly segregated mitochondrialand cytosolic fractions.
Precise fractionation
To demonstrate its utility, Active Motif’s
Mitochondrial Fractionation Kit was used
to isolate mitochondrial and cytosolic fractions from both uninduced and apoptotic
HeLa cells. The FunctionELISA™ Cytochromec Kit was used to determine the levels ofcytochrome c in the mitochondrial andcytosolic fractions because the release ofcytochrome c from mitochondria is a key
indicator of apoptosis. As expected, nearly
all cytochrome c in uninduced cells wasfound in the mitochondrial fraction, while
nearly all the cytochrome c in the apoptotic
sample was in the cytosol (Figure 1).
Upstream quality for downstream successUsing the Mitochondrial Fractionation Kit,
with its optimized protocol and uniqueformulation of buffers, you’ll be certain to
get the specifically segregated fractions you
need. For more consistent results, try the
Mitochondrial Fractionation Kit.

Product

FormatCatalog No.

Mitochondrial Fractionation Kit

100 rxns

40015

FunctionELISA™ Cytochrome c

1 x 96-well plate5 x 96-well plates

48006
48506

High Yields of Specifically Segregated Extracts

Nuclear Extract Kit advantages• Complete kit with all required reagents• No need to optimize your procedure• QC’d reagents ensure reproducibility
Results you can rely on
Accurate research often depends on
precisely isolating proteins from a specificcellular compartment. If nuclear extractcontains cytoplasmic proteins or vice-versa,
your results may not be valid. The Nuclear
Extract Kit helps ensure that proteins remain segregated in their particular fraction.

www.activemotif.com

Specific segregation for accurate results
To demonstrate the importance of using aquality extraction procedure, nuclear andcytoplasmic extracts were prepared fromunstimulated and TNF-α-stimulated HeLa
cells using our Nuclear Extract Kit and assayed for activated NFκB p50 using the
2.5

NFκB activation (OD450 nm)

Active Motif’s Nuclear Extract Kit enables
you to isolate high yields of active nuclear,whole-cell and cytoplasmic extracts frommammalian cell and tissue samples. Its use
eliminates the need to optimize your own
reagents and protocols, so you get errorfree results every time.

Unstimulated
TNF-α stimulated

2.0
1.5
1.0
0.5
0.0

0.625

1.25

2.5

5

10

0.625

1.25

2.5

5

10

Cytoplasmic

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

TransAM™ NFκB p50 DNA-binding assay(Figure 1). As activated NFκB is not present
in the cytoplasm, only nuclear extract fromthe stimulated cells should test positive.
This is precisely what was seen, demonstrating the specificity of the kit.
Quality in means quality out
Extracts prepared using the Nuclear Extract
Kit are ideal for use in any experiment that
requires nuclear, whole-cell or cytoplasmic
extract. In addition, Active Motif offers
over 100 different ready-to-use extracts
from a variety of cell lines and tissue types(page 8). Whether you choose to makethem using our kit or purchase them fromus, the use of high-quality extracts will
improve the accuracy of your downstream
experiments. To get reproducible results,
try the Nuclear Extract Kit.

Product

FormatCatalog No.

Nuclear Extract Kit

100 rxns
400 rxns

40010
40410

7

Antibodies & Extracts

Specific Antibodies & Quality Positive Control Extracts

Whether you are researching AP-1, C/EBP,
NFκB/Rel, Rb, Sp1 or STAT, we can help.
Don’t waste your time and money on
custom antibody services when we may
already have the antibodies you need.

8A-431 (EGF)

A-431

Hep G2

JurkatNIH/3T3 SR

NIH/3T3

HeLa SR

With over 200 antibodies to choose
from, evaluation of cell cycle regulatorsand transcription factors is easier thanever before. Our antibodies are highly
characterized using appropriate cellular
extracts, so you can be sure of their quality.
And, to make using our antibodies as simple
as possible, each antibody is accompanied
by a detailed, lot-specific protocol.

Ready-to-use positive control extracts
Many types of cell biology research require
high-quality cell and tissue extracts. However,there’s no longer a need to isolate these
extracts yourself. Active Motif offers quality
extracts available from a variety of cell types
and tissue sources. Many of our extracts havebeen stimulated or treated to specificallyinduce transcription factor activation. All of
our extracts are tested for activity and crosscontamination by Western blot (Figure 1) orwith our TransAM™ transcription factor ELISAs(page 1). So, why spend time developing yourown positive control when we have alreadydone it for you?

HeLa

Highly characterized transcription factor
antibodies
Active Motif offers one of the broadestlines of mono- and polyclonal antibodies
for cell signaling and transcription factorrelated research. These antibodies are ideal
for performing Western blots, ELISAs andsupershift/electrophoretic mobility shift
assays (EMSA).

Find the right positive control & antibody
To make your life easier, we have tested
our extracts to determine which antibodies
test positive in each extract. This allows
you to quickly pick the right positive control for your antibody of interest.
Active Motif’s website has an easy-to-use
search engine; you can search for antibodies and extracts, download their data
sheets and determine which extracts areappropriate for use as a positive control.
To use this valuable tool and save time
while finding the antibodies and positive
controls you need, logon to our website atwww.activemotif.com.

A
B

Figure 1: Western blot analysis using Active Motif antibodies & extracts.
HeLa, HeLa SR (I hour serum response), NIH/3T3, NIH/3T3 SR (1 hour serum
response), Jurkat, Hep G2, A-431 and A-431 (EGF) nuclear extracts were loaded at
9 µg per well and separated by SDS-PAGE. The membranes were then analyzed
by Western blot using NF-YB pAb (Panel A) and C/EBPβ pAb (Panel B).

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