THE NEWSLETTER OF ACTIVE MOTIF — November 2006 • volume 7 • number 4

New: Magnetic Beads Make Chromatin
Immunoprecipitation Faster and Easier
Active Motif’s new ChIP-IT™ Express Kits use protein G-coated magnetic beads
instead of traditional agarose beads to make chromatin immunoprecipitation(ChIP) simpler and faster than ever before. These innovative beads have madeit possible to simplify and streamline the protocol, optimize buffers andreduce or eliminate several time- and labor-intensive steps. The result is that
ChIP-IT Express enables you to perform ChIP in a single day.
New technique for speed and success
The new components and protocolof ChIP-IT Express make it faster andeasier than any other ChIP procedure.
The kits include all buffers, reagentsand components required to shear 5
samples of chromatin, using your choiceof sonication or enzymatic digestion,then perform 25 ChIP reactions. We’ve
even included a strong bar magnet. The
protein G-coated magnetic beads, provided ready-to-use, have a high binding
capacity for IgG. This enables them tobe used in small volumes, which reduces
non-speciﬁc binding. This improved
speciﬁcity enables the number andlength of washing steps to be reduced. It
is not even necessary to block the beads
or pre-clear the chromatin.
Optimized buffers eliminate steps
In addition to the magnetic beads andbar magnet, ChIP-IT Express Kits containnew buffers that eliminate the need for
DNA puriﬁcation, so you can simultane-

www.activemotif.com

ously elute your DNA from the beads
while reversing the cross-links. These
simple protocol changes give you results
quickly and easily while offering thefreedom to simultaneously perform
multiple ChIP assays: now you can evenperform ChIP in PCR tubes with a multichannel pipettor (Figure 1).
The power of magnetic beads
With ChIP-IT Express Kits, ChIP canbe performed in the included Eppendorf tubes or in 8-well PCR strips. PCR
strips make it convenient to process
larger numbers of samples. The protein
G-coated magnetic beads pellet inseconds, much faster than centrifuging
agarose beads. An added advantage isthat magnetic beads pellet on the tube
side instead of the bottom, even coming
completely out of the wash buffer. This
makes it easy to remove buffers without
disturbing the beads, which reduces
sample loss and helps ensure sample-tosample consistency.

INSIDE THIS ISSUE
for Quantiﬁcation of
2 ELISA
Activated Ras GTPase
34

Simplify Co-Immunoprecipitation

5

New: Study Your Choice ofTarget in Any Cell Signaling
Pathway

7

TransAM™: The Simple Way
to Assay Transcription Factor
Activation

8

Simpliﬁed Analysis of
DNA Methylation

Investigate SUMOylation with
Simple, Efﬁcient SUMOlink™

Figure 1: Multiple-sample ChIP using ChIP-IT Express.
Washing magnetic beads is fast and easy because thepellet forms against the side of the tube in seconds.
This makes it possible to ChIP multiple samples in
8-well PCR tubes using a multi-channel pipettor.
continued on page 61

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

2FormatCatalog 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. Please give us a call, return the enclosed reply card or visit our
website at www.activemotif.com/gtpase
for complete information, including
downloadable manuals. To get to thebest Ras activation assay available, try
the Ras GTPase Chemi ELISA Kit.

Toll Free — 1 877 222 9543

November 2006 • volume 7 • number 4

Co-Immunoprecipitation

Simplify Co-Immunoprecipitation
Active Motif’s Nuclear Complex Co-IP Kit simpliﬁes co-immunoprecipitation
studies of nuclear protein complexes by providing you with optimized reagentsfor both 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
binding beads

Add antibody directed against
one of the proteins of interest

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: Flow chart of the Co-Immunoprecipitation process.

Product

FormatCatalog No.

Nuclear Complex Co-IP Kit

50 rxns54001

www.activemotif.com

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
to performing traditional immunoprecipita-tions. To ﬁnd out more about the
Nuclear Complex Co-IP Kit, visit us on
the web at www.activemotif.com.

3

SUMOylation

Investigate SUMOylation with Simple, Efﬁcient SUMOlink™
Active Motif’s SUMOlink™ Kits provide a simple method for generating
SUMOylated proteins in vitro. These fast, efﬁcient and robust assays contain all
the necessary reagents for SUMOylation of target proteins, and include positive
and negative controls to ensure assay success.
Post-translational modiﬁcation by SUMO
(small ubiquitin-like modiﬁer) can alter
protein function in many different
cellular processes including disrupting
protein-protein interactions in signaling
pathways, inﬂuencing intracellular protein localization and playing a regulatoryrole in transcription factor activity.
SUMOlink Kits enable you to easily
perform and detect in vitro SUMO conjugation of your target protein alongsideprovided controls. In the kit, all required
reagents are provided along with positive control p53 protein and antibody.

“The positive control protein andantibody supplied in SUMOlink Kits
help ensure the accuracy of yourin vitro SUMOylation assays.”

SUMOlink applications• Investigate SUMOylation effects ontranscription factor activity• Study the role of SUMOylation incellular processes• Identify novel SUMOylation targets
SUMOlink advantages• Simple and efﬁcient method• Complete kit with validated reagentsand positive control protein• SUMOylate proteins or cell extracts• Antibodies cross-react with human,
mouse and rat samples

MW

1

SUMO-12
3

1SUMO-1
2
3

98

98

64

64

50

50

36

36

30

The SUMOlink method
In easy-to-use SUMOlink Kits, you
simply add the assay components to a
microcentrifuge tube with your protein
of interest. After a 3-hour incubation,
the reaction is stopped and results canbe analyzed by Western blot. With the
kit’s p53 antibody and either SUMO-1 or
SUMO-2/3 antibodies, you can easily determine the extent to which your target
protein has been SUMOylated (Figure 1).

4

MW

SUMO-1 and SUMO-2/3 kits available
In vertebrates, the SUMOylation enzyme
cascade can result in 3 distinct types of
SUMO modiﬁcation: SUMO-1, -2 or -3.
Active Motif provides kits to detect andinvestigate SUMOylation of your protein
of interest by each of these SUMO
proteins. The kits contain E1 activatingand E2 conjugating enzymes along withwild-type and mutant SUMO-1 protein(SUMO-1 Kit) or SUMO-2 and -3 proteins(SUMO-2/3 Kit). Antibodies to detect
SUMO-1 or SUMO-2/3 modiﬁcations aswell as control p53 protein and antibody
are included. For more information
please give us a call or visit our website
at www.activemotif.com.

1

SUMO-22
3

1SUMO-32
31

SUMO-22
3

1SUMO-3
2
3

30

16

16

A

p53 antibody

SUMO-1 antibody

B

p53 antibody

SUMO-2/3 antibody

Figure 1: Speciﬁc protein labeling using SUMOlink.
Western blot analysis of in vitro SUMOylation of p53 protein by wild-type and mutated isoforms of SUMO-1, SUMO-2 and
SUMO-3 proteins. A. SUMO-1 analysis: Western blots incubated with p53 antibody (1:5000 dilution) and SUMO-1 antibody(1 :4000 dilution); B. SUMO-2/3 analysis: Western blots incubated with p53 antibody (1:5000 dilution) and SUMO-2/3
antibody (1:4000 dilution).
Lane 1: Wild-type SUMO protein conjugation reaction.
Lane 2: Mutated SUMO protein conjugation reaction.
Lane 3: No p53 control protein used in the conjugation reaction.

Product

FormatCatalog No.

SUMOlink™ SUMO-1 Kit

20 rxns

40120

SUMOlink™ SUMO-2/3 Kit

20 rxns

40220

Toll Free — 1 877 222 9543

November 2006 • volume 7 • number 4

Phospho-Protein Detection

New: Study Your Choice of Target in Any Cell Signaling Pathway
Measure downstream effects of signal transduction events with FACE™
Maker. This new, adaptable version of Active Motif’s FACE (Fast Activated
Cell-based ELISA) enables you to study any phosphorylated protein you
choose in a simple, fast and sensitive cell-based assay. Using antibodies
speciﬁc to your desired target protein, modiﬁcation-state speciﬁc analysis
is performed directly within the cell without the need for lysates, gels andtime-consuming immunoblotting.
The FACE method
Cells are grown in a 96-well plate andstimulated to induce the pathway ofinterest. Following stimulation, the cells
are ﬁxed to preserve protein modiﬁcations, and each well is then incubatedwith a primary antibody speciﬁc to thetarget of interest. Original FACE Kits contain primary antibodies speciﬁc to particular targets, but FACE Maker Kits now
give you the option to use your own
choice of primary antibody. Subsequent
incubation with secondary HRP-conjugated antibody and developing solution

provides an easily quantiﬁed colorimetric or chemiluminescent readout(Figure 1). Results may be normalized forcell number using the included Crystal
Violet solution. With FACE, a duplicate
assay for the non-modiﬁed state of theprotein can easily be run for comparisonof constitutive levels versus the stimulation effect.
Prevent unwanted sample modiﬁcations
FACE eliminates the need to prepare celllysates, which greatly reduces the possibility of introducing undesired altera-

Add anti-IgG
HRP conjugate

Add primary
antibody
3 hrs.

tions of the protein of interest. FACE Kits
use a ﬁxation step that “freezes” the cellular state and prevents further protein
modiﬁcations. This enables detectionof the exact protein state in the cells
at your chosen timepoint(s), providing
more accurate and reproducible results.
Two types of detection
All FACE Kits are available in both colorimetric and chemiluminescent formats.
The colorimetric kits detect your protein
of interest using an HRP-colorimetricsignal at a wavelength of 450 nm andcan be read with a standard plate reader.
The more sensitive FACE Chemi Kits use
chemiluminescent detection that ismeasured by a luminometer, which
accurately monitors the slightestchanges in protein phosphorylation.
FACE advantages• Simple, quantiﬁable method• Fixing cells preserves protein
activation state• Cell-based assay eliminates lysate
preparation and immunoblotting• Fast – results after less than 3 hoursof hands on time

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.

Product

FormatCatalog No.

FACE™ Maker

1 x 96 rxns
5 x 96 rxns

48000
48500

FACE™ Maker Chemi

1 x 96 rxns
5 x 96 rxns

48050
48550

www.activemotif.com

What’s in the kit
Active Motif already offers FACE Kits
that have been optimized for 25 different targets. With new FACE Maker Kits,
the possibilities are now endless because
you decide which protein to target. FACE
Maker Kits include 96-well plates for cell
culture, optimized Reaction Buffers andCrystal Violet Cell Quantiﬁcation Solution. You supply only your own phospho-speciﬁc and total antibodies and an
appropriate HRP-conjugated secondary
antibody. For the simplest way to study
phospho-proteins, try FACE and the new
FACE Maker Kits.

5

Chromatin Immunoprecipitation
continued from page 1 — New: Magnetic Beads Make Chromatin Immunoprecipitation Faster and Easier

Figure 2: Use of a standard tip box for magnetic ChIP.
ChIP-IT Express Kits include a strong bar magnet thatcan be used to convert tip boxes into a magnetic standfor washing the protein G-coated magnetic beads.
Figure 3: Flow chart of chromatin immunoprecipitation with ChIP-IT Express.

A powerful magnet
ChIP-IT Express kits include a strong bar
magnet that can be used to turn pipette
tip boxes into magnetic stands for
immunoprecipitation and washing. P1000
tip boxes can be used with Eppendorf
tubes (Figure 2), while P200 boxes workwith PCR strips (Figure 1, page 1). Commercially available magnetic tube stands,
such as those from Ambion andPromega, can also be used.
ChIP-IT Express advantages• Dramatically reduced hands-on time
• Simpliﬁed wash steps improveconsistency• No pre-clearing, blocking or DNA
puriﬁcation• High-throughput compatible
Improvements of ChIP-IT Express
ChIP-IT Express improves on traditional
ChIP by reducing or eliminating several
time-consuming steps (Figure 3). The
magnetic beads have much lower background than traditional agarose beads,
so pre-clearing and blocking are no longer necessary. This dramatically reduces
the amount of hands-on time during
the assay. Washing is easier because thespin steps have been replaced by rapid
magnetic pull-down. Other optimizations include a specialized ChIP Elution

6

Buffer that eliminates the need for DNA
puriﬁcation after the ChIP is complete.
This saves time, minimizes manipulations
and eliminates the DNA loss that canoccur during puriﬁcation.
Secrets to successA feature that made the original ChIP-IT
Kits successful was the positive andnegative controls, as these help troubleshooting and results interpretation. But,
because many customers requested
species-speciﬁc controls, we’ve removed
these human-only controls from ChIP-IT
Express Kits and are pleased to release
new ChIP-IT Control Kits for human, rat
or mouse samples. These kits provide
positive and negative control antibodies and species-speciﬁc PCR primer sets,
PCR buffer and a convenient 10X DNA
loading dye that makes your PCR reactions gel-ready. The human control kit
is now available; check our website forrelease of the rat and mouse kits.

Are you Ready-to-ChIP?
For your convenience, Active Motif
now offers Ready-to-ChIP Chromatin.
Combined with our controls, this ensuresthat all components are conﬁrmed towork in ChIP, so you can more easily
validate your own antibodies and primer
sets. Ready-to-ChIP Chromatin has
been optimally sheared by sonicationand validated in ChIP. It can be used inconjunction with all of the ChIP-IT Kits,
so you can be certain the only variable
in testing an antibody for ChIP is theantibody itself.
Get started today
Active Motif’s ChIP-IT products provide you with everything you need tomake your ChIP experiments easier andmore reproducible. No ChIP is fasterthan ChIP-IT Express, and the validated
ChIP-IT controls and reagents help
ensure your success. For more complete
information, please give us a call or visit
us online at www.activemotif.com/chip.

Product

FormatCatalog No.

ChIP-IT™ Express

25 rxns

53008

ChIP-IT™ Express Enzymatic

25 rxns

53009

ChIP-IT™ Protein G Magnetic Beads

25 rxns

53014

ChIP-IT™ Control Kit - Human

5 rxns

53010

Ready-to-ChIP HeLa Chromatin

10 rxns

53015

Toll Free — 1 877 222 9543

November 2006 • volume 7 • number 4

Transcription Factor ELISAs

Simplify the study of activated transcription factors with the combined
convenience and increased sensitivity of Active Motif’s non-radioactive,
ELISA-based TransAM™ assays.

? What is TransAM?
Active Motif’s TransAM Kits are simple
DNA-binding ELISA-based assays thatfacilitate the study of transcription
factor activation in mammalian tissue
and cell culture extracts. The TransAM
method is fast, delivering results in lessthan 5 hours, and it is up to 100-foldmore sensitive than classic EMSA, or
gelshift, techniques.

? How does TransAM work?
Commonly, transcription factors areactivated by phosphorylation as aresult of a cell-signaling cascade. Upon
activation, transcription factors bindto their DNA targets to regulate gene
expression. TransAM Kits operate on
this principle by using a 96-well platein which multiple copies of a doublestranded oligonucleotide that contains
a speciﬁc target-binding sequence areimmobilized. When nuclear or wholecell extracts are added to the plate,the activated transcription factor ofinterest binds its consensus sequenceon the plate-bound oligonucleotide.
Next, a primary antibody speciﬁc tothe transcription factor is added. This isdetected by incubation with a secondary
HRP-conjugated antibody and developing reagent. Colorimetric changes aremeasured by a spectrophotometer; withthe sensitive chemiluminescent kits,
results are read with a luminometer. The
readout provides a sensitive measure of
the activated transcription factor.

www.activemotif.com

is TransAM an improvement? Howover other methods?

Two commonly used methods to study
transcription factor activation are
gelshift/EMSA and reporter gene assays.
TransAM has a number of advantages
over these procedures. With respect togelshift/EMSA, TransAM assays are 10-foldmore sensitive so you get more accurate
results while using less sample. TransAM
eliminates the use of radioactivity as
well as the need to run, blot, expose anddevelop gels. This saves you a considerable
amount of time; TransAM is complete inless than 5 hours, while gelshift takes days.
In addition, data from TransAM is easier
to interpret and compare than gelshift
results (Figure 1). TransAM also overcomesthe problems associated with the inconsistency of reporter gene assays. Because
transfection is a variable process and gene
expression is subject to many confounding
factors, reporter assays must be repeatedmany times to obtain statistically reliable
data. And, unlike reporter assays, TransAM
can also be used with tissue samples.
types of samples can be? Whatassayed with TransAM?

TransAM assays are typically used with
nuclear extracts. Most commonly,
extracts are made from cells in theirnormal state as well as after stimulationto activate the targeted transcription
factor; the results are compared todetermine the effect of the treatment
on transcription activation. For sometranscription factors, such as NFκB,
whole-cell extract can be used.

NFκB activation (OD450 nm)

TransAM™: The Simple Way to Assay Transcription Factor Activation
2.5

Unstimulated
IL-1α stimulated

2
1.5
1
0.5
0

0.5

1

2
5
10 25
Cell extract/well (µg)

IL-1α stimulated
0.5 1

5 10 25 50

50

Unstimulated
0.5 1

5 10 25 50µg of cell extract

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

transcription factors can be? Whichstudied with TransAM?

Active Motif has developed a TransAM
Kits to study over 30 individualtranscription factors, as well as kits
designed to study transcription factor
families. TransAM Family Kits containmultiple antibodies so that you cansimultaneously proﬁle the activationlevels of different family members.
TransAM Flexi Kits provide you with
optimized reagents, but give you the
ﬂexibility to use your own oligonucleotide, enabling you to study alternative binding sites. For more information complete about the TransAM Kits,
please give us a call or visit our website
at www.activemotif.com/transam.

7

DNA Methylation

Simple Analysis of DNA Methylation
Active Motif’s MethylDetector™ Bisulﬁte Modiﬁcation Kit provides an
efﬁcient method for DNA methylation analysis by combining optimized
reagents for performing DNA conversion with time-saving DNA puriﬁcation
columns, as well as positive control PCR primers to validate your results.
Proven controls ensure success
DNA methylation analysis frequently
begins by treating DNA with bisulﬁteto convert unmethylated cytosines touracils while leaving methylated cytosines
unchanged. The bisulﬁte-treated DNA isthen ampliﬁed by PCR and analyzed bysequencing or restriction digest. However,the conversion process can be technicallychallenging. To help, MethylDetector
provides optimized conversion reagentsthat enable a streamlined protocol. Positive control PCR primers that are speciﬁc
for bisulﬁte-converted DNA enable you
to conﬁrm that the conversion step was
successful before beginning costly down-

8

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

stream sample analysis, saving you time,effort and materials (Figure 1).
MethylDetector advantages• Flexible – works with high G/Ccontent sequences and uncut DNA• Consistent results – 99% conversion efﬁciency• Proven reagents – optimized
reagents, simple protocol andpositive controls• High yield – plenty of converted
DNA for further analysis

M

1

2

3

4

5

500 bp400 bp300 bp200 bp100 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 andreproducibility of the MethylDetector Kit.

Product

FormatCatalog No.

MethylDetector™

50 rxns

55001

THE NEWSLETTER OF ACTIVE MOTIF — November 2006 • volume 7 • number 4

PRSRT STD
US POSTAGEPAID
PERMIT #236

92054