THE NEWSLETTER OF ACTIVE MOTIF — June 2009 • volume 10 • number 2

look inside forthe latest assays
for DNA methylation,transcriptional regulation,
histone purification& sequential ChIP

IN
THIS
ISSUE

2 Aberrant DNA Methylation andPediatric Syndromes

9 Recombinant and Puriﬁed Proteins forYour Biochemical Assay Needs

4 NEW: MethylCollector™ Ultra for
Enhanced Methylation Enrichment

9 ChIP-validated Antibodies for Your
Chromatin Experiments

5 NEW: UnMethylCollector™ Positively
Identiﬁes Unmethylated CpG Regions

10 Faster, More Accurate Measurement of
Nitric Oxide

6 Identify Protein Co-localization In Vivo
Using Sequential Chromatin IP

10 Efﬁcient, Extremely Affordable CompetentE. coli in a More Convenient Format

7 Histone Puriﬁcation Made Simple in Your
Choice of Assay Format

11 ELISA for Quantiﬁcation of
Activated Ras GTPase

8 TransAM™ Transcription Factor
DNA-binding ELISAs

11 Investigate the Effects of SUMOylation
with SUMOlink™

8 Improve Luciferase Assay Responsewith RapidReporter®

Review Article

Aberrant DNA Methylation and Pediatric Syndromes
Methylation of mammalian DNA has long been recognized to play a major role in a variety of different cellularfunctions. Dysregulation(s) in DNA methylation or in the DNA methylation machinery has been shown to lead tothe development of severe pediatric syndromes.
Methylation of mammalian DNA haslong been recognized to play a majorrole in different cellular functions, suchas development or control of gene
expression, and is often associated withtranscriptional repression. The DNA
methyltransferases (DNMTs) catalyze
the transfer of a methyl group from
S-adenosyl methionine to the 5´ position of cytosines, mostly within CpG
dinucleotide motifs. Three families of
DNMTs have been identiﬁed: DNMT1,
DNMT2 and DNMT3. The DNMT3 family,
containing two active methyltransferases, DNMT3A and DNMT3B, and one
DNMT3-Like protein (DNMT3L), establishes the initial CpG methylation pattern de novo and shows the same propensity for methylating unmethylated
duplex DNA as it does for hemi-methylated DNA. DNMT1 is considered amaintenance DNMT. Indeed, this enzyme
shows a speciﬁcity for hemi-methylated
DNA and is responsible for the establishment and regulation of tissue-speciﬁc
patterns of DNA methylation in regulatory sequences (reviewed in 1).
The complex series of events leading
to a repressive chromatin state involvethe coordinated regulation of DNA
methyltransferases, other proteins called
Methyl-CpG binding proteins (MBD
proteins) and the Kaiso family proteins.
The MBD family proteins include MeCP2,
MBD1, MBD2, MBD3 and MBD4.2, 3 In
mammalian cells, DNA methylation is
generally associated with gene silencing,either directly by inhibiting binding oftranscription factors to their recognition
sequences4, or indirectly by preventing
transcription factors from accessing
their target sites through attachment

2of MBD proteins that “read” DNA
methylation patterns.1 These MBDs canrecruit histone deacetylases and histone
methyltransferases, thereby resulting in formation of a closed repressive
chromatin structure. DNA methylation
is involved in many cellular functions
such as embryonic development, genetic
imprinting, X chromosome inactivation

Aberrant methylation patterns areassociated with certain human tumorsand developmental abnormalities.
and control of gene expression. Aberrant
methylation patterns are associated withcertain human tumors and developmental abnormalities. In this review, we
will focus on the role of aberrant DNA
methylation patterns in the development of pediatric syndromes.
Dysregulations of genomic imprinting
Diploid organisms possess two copies of each autosomal gene, one fromeach parent. Most autosomal genes are
expressed simultaneously from bothalleles. However, for a subset of genes,
one allele is silenced in a parent-oforigin-dependent manner. This process iscalled imprinting. DNA methylation andhistone modiﬁcations have been shown
to play a major role in imprinting mechanisms.5 Typical disorders associated withimprinted genes include Prader-Willi andAngelman syndromes, Beckwith-Wiedemann syndrome and multiple forms ofneoplasia.6-8
The Prader-Willi syndrome (PWS) andthe Angelman syndrome (AS) are two
clinically distinct disorders, each causedby several genetic and epigenetic dys-

regulations within chromosome 15q11-q13,
containing a number of imprinted genes.
Prader-Willi syndrome (PWS)
Individuals with Prader-Willi syndrome
show neonatal hypotonia, failure to
thrive, hyperphagia in early childhood
leading to obesity, hypogonadotropism,short stature, behavior problems andmoderate mental retardation.9 One
percent of PWS patients show a paternal
copy of chromosome 15 that has amaternal pattern of imprinted gene
expression and epigenetic modiﬁcationsof 15q11-q13. In 85% of PWS patients whoshow imprinting defects, both copies
of several genes (such as SNRPN, NDN
or MKRN3) within chromosome 15 aremethylated.7
Angelman syndrome (AS)Angelman syndrome is a neurogenetic
disorder characterized by severe mental
retardation, seizures, abnormal gait, frequent smiling and laughter, microcephaly
and ataxia.10 Three to ﬁve percent of AS
patients show an imprinting defect, witha maternally inherited chromosome 15that has a paternal imprint and epigenetic modiﬁcations on the UBE3A gene,considered as the AS gene.7, 11
Beckwith-Wiedemann syndrome (BWS)
Beckwith-Wiedemann syndrome isa congenital overgrowth syndrome
characterized by a number of developmental abnormalities as macroglossia,
macrosomia or neonatal hypoglycemia.7
Moreover, children with Beckwith-Wiedemann syndrome have an increased risk
to develop embryonal tumors (Wilm’stumor, hepatoblastoma, etc.).12 This syndrome is a multigenic disorder, implying

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June 2009 • volume 10 • number 2
dysregulation of one or more imprinted
genes of a major imprinted gene cluster
on chromosome 11p15, involved inregulation of growth and the cell cycle.
These genes include the paternallyexpressed genes IGF2 and KCNQ10T1
and the maternally expressed genes
H19, CDKN1C and KCNQ1B.13 It seems
that there are several genotypes for the
Beckwith-Wiedemann syndrome. In the
majority of sporadic BWS cases, several
groups have reported abnormal, biallelicexpression of IGF2. This phenomenon isdesignated as loss of imprinting. Anothersubgroup of sporadic BWS cases shows
biallelic expression of IGF2 and silencingof H19. These events are accompanied bya paternal methylation pattern on both
chromosomes (reviewed in 6).
Dysregulations of tumor suppressor
function and retinoblastoma
Some genes such as p53 or Rb1 have atumor suppressor function. Dysregulation of these genes causes developmentof many cancers. Retinoblastoma is achildhood malignant tumor of the retina
that can be caused by genetic and epigenetic alterations. Genetic alterations
include mutation of one allele of thetumor suppressor gene Rb1, combinedwith chromosomal deletion or loss ofthe other allele.14 Epigenetic alterations
include the hypermethylation of the Rb1
gene leading to the inactivation of its
tumor suppressor function15 and to thedevelopment of retinoblastoma.
Dysregulations of DNA methylation
machinery: DNMT and MBD proteinsRett syndrome
Rett syndrome is an X-linked neurodevelopmental disorder, affecting female
children of 17-18 months. Rett syndrome
is characterized by an apparently normal
development of the child prior to theonset of the symptoms. This period isfollowed by a skill regression in earlychildhood (speech, hand use) and pa-

www.activemotif.com

tients develop severe dementia, autism
and many other hallmark characteristics.16, 17 Rett syndrome causative mutations occur mainly (in 85% of the cases)in the gene coding for the CpG-methyl
binding protein, MeCP2.18 Rett patientswith no MeCP2 mutation may show
mutation in MeCP2-related genes, suchas CDKL5, whose product interacts with
MeCP2. MeCP2 recruits Histone deacetylases (HDACs) 1 and 2.19 Mutations in the
MeCP2 gene result in hyperacetylationof histone H3 in mice and of histone H4

Review Article
Some studies established that the lossof methyltransferase activity, and notother functions of DNMT3B protein,is responsible for the disease. Indeed,mutations in ICF patients are frequently
found in the C-terminal portion of theprotein, which contains the catalytic
domain.24 All patients, including those
without mutations in DNMT3B, exhibit
hypomethylation of the DNA, targetedto speciﬁc sequences, such as satellite
DNA of the juxtacentromeric heterochromatin at chromosome 1, 9 or 16.

Typical disorders associated with imprinted genes include Prader-Willi,Angelman and Beckwith-Wiedemann syndromes and multiple neoplasias.
in cell cultures derived from Rett patients.20, 21 Several studies have provided
insights into the relationship between
the genotype (type of mutation(s) andposition of the mutation(s) in MeCP2)
and the severity of Rett syndrome
phenotypes.22
The immunodeﬁciency, centromericregion instability and facial anomalies
syndrome (ICF)ICF is an immunodeﬁciency, autosomal
and recessive disease. This syndrome ischaracterized by an instability of the
pericentrometric regions of chromosomes 1, 9 and 16, and by a severe immunodeﬁciency. The patients present with
an immunoglobulin deﬁciency in thepresence of normal B- and T-cell counts.
Other variable clinical signs are chronic
respiratory and gastrointestinal infections, and facial anomalies (macroglossia,
ﬂat nasal bridge) (reviewed in 23). Mutations in the gene coding for the enzyme
DNMT3B were found to be responsible
for clinical presentations of ICF. Sixty
percent of the patients have mutations in both alleles of DNMT3B within
the coding sequence.24 ICF patients
who don’t show mutations in DNMT3B
display an additional hypomethylationin centromeric satellite alpha DNA.25, 26

These are sites of whole-arm deletions,decondensation, multiradial chromosome junctions and chromosome breaks,
leading to aberrant rearrangements in
mitogen-stimulated lymphocytes.27
All these studies show the importanceof epigenetics in general, and of DNA
methylation in the development of anorganism and in various cell functions.
References

1. T. Latham, N. Gilbert, B. Ramsahoye, (2008) Cell Tissue
Res 331:31.
2. B. Hendrich, A. Bird, (1998) Mol Cell Biol 18:6538.
3. R. J. Klose, A. P. Bird, (2006) Trends Biochem Sci 31:89.
4. F. Watt, P. L. Molloy, (1988) Genes Dev 2:1136.
5. F. Y. Ideraabdullah, S. Vigneau, M. S. Bartolomei, (2008)
Mutat Res 647:77.
6. E. R. Maher, W. Reik, (2000) J Clin Invest 105:247.
7. B. Horsthemke, J. Wagstaff, (2008) Am J Med Genet A
146A:2041.
8. S. Manipalviratn, A. DeCherney, J. Segars, (2009) Fertil
Steril 91:305.
9. A. P. Goldstone, (2004) Trends Endocrinol Metab 15:12.
10. C. A. Williams et al., (2006) Am J Med Genet A 140:413.
11. T. Kishino, M. Lalande, J. Wagstaff, (1997) Nat Genet 15:70.
12. W. Reik, E. R. Maher, (1997) Trends Genet 13:330.
13. R. Weksberg, A. C. Smith, J. Squire, P. Sadowski, (2003)
Hum Mol Genet 12 Spec No 1, R61.
14. J. R. Thompson, C. J. Williams, (2005) Semin Reprod Med
23:285.
15. N. Ohtani-Fujita et al., (1997) Cancer Genet Cytogenet
98:43.
16. R. H. Mount, R. P. Hastings, S. Reilly, H. Cass, T. Charman,(2003) Am J Ment Retard 108:1.
17. A. M. Kerr et al., (2001) Brain Dev 23:208.
18. M. D’Esposito et al., (1996) Mamm Genome 7:533.
19. F. Mari et al., (2005) Hum Mol Genet 14:1935.
20. M. Shahbazian et al., (2002) Neuron 35:243.
21. M. Wan, et al., (2001) Hum Mol Genet 10:1085.
22. S. Kudo et al., (2001) Brain Dev 23 Suppl 1, S165.
23. M. Ehrlich et al., (2008) Autoimmunity 41:253.
24. C. Wijmenga et al., (2000) Hum Mutat 16:509.
25. Y. L. Jiang et al., (2005) Hum Mutat 25:56.
26. T. Kubota et al., (2004) Am J Med Genet A 129A:290.
27. D. Gisselsson et al., (2005) Chromosoma 114:118.

3

DNA Methylation

NEW: MethylCollector™ Ultra for Enhanced Methylation Enrichment
The new MethylCollector™ Ultra Kit* is an enhanced version of our MethylCollector™ Kit*. It improves the enrichmentof CpG-methylated DNA by incorporating the Methylated CpG Island Recovery Assay (MIRA), which uses a combination of methyl-binding proteins (MBD2b and MBD3L1) to increase the afﬁnity for methylated DNA fragments.1 This
unique protein complex provides greater speciﬁcity for methylated CpG dinucleotides than alternative methyl-binding protein or antibody immunoprecipitation (MeDIP) methods, and in less than half the time. This makes MethylCollector Ultra suitable for use with smaller samples, as the new format requires only 1 ng of fragmented DNA. The kit
also includes positive control human, male genomic DNA and methylation-speciﬁc PCR primers to ensure your success.
His-MBD2b/MBD3L1 Capture(MethylCollector™ Ultra)
888

SNRPN (methylated)

SNRPN (methylated)

6

∆Rxn

FOXD2 (unmethylated)

4

SNRPN (methylated)
6

FOXD2 (unmethylated)

∆Rxn

6

∆Rxn

Antibody Immunoprecipitation
Anti-5-methyl cytidine (MeDIP)

MBD-Biotin Capture(Competitor MM)

4

2

2

12

16

20

24

28

32

36

0

12

40

16

20

24

REFERENCE

28

32

36

40

12

16

20

24

Cycle

Cycle

MethylCollector Ultra advantages• Improved efficiency – high-afﬁnity
binding provides greater enrichment
than other MBD capture or
antibody immunoprecipitation(MeDIP) methods• Faster procedure – the convenient
magnetic protocol can becompleted in less than 3 hours• Uses minimal sample material –requires as little as 1 ng of DNA(~200 cells), fragmented bysonication or enzymatic digestion• Controls ensure success – includes
positive control DNA andmethylation speciﬁc PCR primers• Versatility – the eluted DNA issuitable for use in a variety ofdownstream applications such as
endpoint or real time PCR, bisulﬁteconversion, or ampliﬁcation andlabeling for microarray analysis

4

2

0

0

FOXD2 (unmethylated)

28

32

36

40

Cycle

Figure 1: Real time PCR analysis reveals the speciﬁcity of MethylCollector Ultra versus competing technologies.
100 ng of human, male genomic DNA was digested with Mse I and tested in MethylCollector Ultra, competitor MM’s kit
and using an anti-5-methyl cytidine antibody immunoprecipitation method. Eluted DNA was analyzed using PCR primers
for both methylated, SNRPN (red), and unmethylated, FOXD2 (blue) promoters. Only MethylCollector Ultra showed specificenrichment for CpG-methylated DNA as demonstrated by from the clear separation in amplification cycles. A comparisonof 500 ng and 1 µg of starting material for each method revealed similar amplification profiles (data not shown).

MethylCollector™ Ultra method

MeDIP method

Input material: 1 ng - 1 µg

Input material: 1 µg–

Denature DNA for 3 minutes at 95°C–

Pre-block beads

Combine DNA with MBD2b/MBD3L1 protein mix
and nickel-coated magnetic beads for 1 hour at 4°C

Combine DNA, antibody and magnetic beads,
incubate 5 hours to overnight

Wash beads 4 times using the included magnet

Wash beads 4 times

Elute methylated DNA at 42°C for 30 minutes

Elute methylated DNA at 55°C for 15 minutes

Add Proteinase K Stop Solution

Incubate at 100°C for 15 minutes

DNA clean up

DNA clean up

Total time required: < 3 hours

Total time required: > 7 hours

Table 1: Comparison of MethylCollector Ultra process with MeDIP.

Product

FormatCatalog No.

MethylCollector™ Ultra

30 rxns

55005

MethylCollector™

25 rxns

55002

Fully Methylated Jurkat DNA

10 µg

55003

* MethylCollector™ Ultra and MethylCollector™ are covered under U.S. Patent No. 7,425,415.

1. Rauch, T. and Pfeifer, G. (2005) Lab. Investigation 85: 1172-1180.

4

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June 2009 • volume 10 • number 2

DNA Methylation

NEW: UnMethylCollector™ Positively Identiﬁes Unmethylated CpG Regions
Active Motif is pleased to announce the launch of UnMethylCollector™*, the ﬁrst commercially available kit for the
speciﬁc isolation and enrichment of unmethylated CpG dinucleotides. UnMethylCollector utilizes the speciﬁcityof the CXXC binding domain towards unmethylated CpGs to capture and enrich for DNA fragments that lack
methylation. This enables researchers to identify hypomethylated promoters and to study the effects of compounds
that inhibit methylation. Instead of relying on negative data from methyl-speciﬁc binding techniques to identify
unmethylated promoters, UnMethylCollector offers a speciﬁc, reliable technique that provides positive identiﬁcationof unmethylated CpG regions.

UnMethylCollector™

ng DNA recovered

100

UnboundEluted

80
60
40

100

60
40
20

0

0

APC

p48

UnMethylCollector advantages• Sensitivity – detects unmethylated
CpGs from 10 ng - 1 µg of DNA
fragmented by sonication or
enzymatic digestion• Faster procedure – magnetic
protocol can be completed inless than 3 hours• Specificity – optimized reagentsensure successful capture from as
few as one unmethylated CpG• Controls ensure success – includes
positive control DNA and PCRprimers suitable for use in either
endpoint or real time PCR analysis• Versatility – eluted DNA is suitablefor use in various downstream applications such as PCR, sequencing,or ampliﬁcation and labeling for
microarray analysis

UnboundEluted

80

20

Xist

MethylCollector™ Ultra

120

ng DNA recovered

120

Xist

APC

NY-ESO

Figure 1: Direct comparison of UnMethylCollector andMethylCollector™ Ultra illustrates speciﬁcity.
Both UnMethylCollector and MethylCollector™ Ultra
were run according to the protocols using either 200 ng
or 100 ng respectively of the provided Mse I digested
human, male genomic DNA. Real time PCR analysis
was run across multiple loci on both the unbound andeluted fractions. UnMethylCollector clearly captures theunmethylated loci (APC and p48), while MethylCollector
Ultra enriches for the methylated loci (Xist and NY-ESO).
APC – contains 29 CpGs and is normally unmethylated
p48 – contains 22 CpGs and is normally unmethylated
Xist – contains 8 CpGs and is methylated in males
NY-ESO – contains 6 CpGs and is normally methylated

Bisulfite sequence results for APC (19 CpGs)
1
2
3
4
5
6
7
8

Methylated CpG
Unmethylated CpG

Figure 2: MethylDetector™ conﬁrms the speciﬁcity of UnMethylCollector for unmethylated CpG dinucleotides.
UnMethylCollector was used to enrich for unmethylated DNA fragments. This DNA was bisulfite treated using Active
Motif’s MethylDetector™ Kit. Bisulfite conversion changes unmethylated cytosines into uracils, while methylated cytosines
remain unchanged. The converted DNA was then amplified by PCR and the gel extracted PCR product was cloned, witheight colonies being selected for sequencing of the unmethylated APC promoter region. The presence of a CpG site in
the sequencing data indicates that the original CpG was methylated, thereby not converted. A C:T mismatch indicatesthat the original CpG was unmethylated, and therefore converted by the bisulfite treatment. Only one clone contained
a single methylated CpG of the 19 CpG dinucleotides within the sequenced APC promoter region. This validates that
UnMethylCollector specifically binds and enriches for unmethylated DNA fragments.

Product

FormatCatalog No.

UnMethylCollector™

30 rxns

55004

MethylDetector™

50 rxns

55001* Patent pending.

www.activemotif.com

5Sequential Chromatin IP

Identify Protein Co-localization In Vivo Using Sequential Chromatin IP
Performing sequential chromatin IP (also called Re-ChIP) was technically challenging and difﬁcult, until now.
Active Motif’s Re-ChIP-IT™ Kit makes it easy to perform sequential ChIP, so you can localize two different
proteins or histone modiﬁcations to the same genomic locus.
Extend the utility of ChIP
When performing ChIP experiments, it isoften useful to prove that two different
proteins or histone modiﬁcations arepresent at the same site in the genome.
Or, you may want to determine if aprotein coincides with a speciﬁc histone
modiﬁcation at the same regulatory
element. Re-ChIP is a relatively newtechnique in which sequential chromatin
immunoprecipitations are performed
using two different antibodies, enabling
you to assay for the simultaneouspresence of two proteins or distinct
histone modiﬁcations at the same
genomic region of interest (Figure 1).
How does Re-ChIP-IT work?
The Re-ChIP-IT Kit takes advantage ofthe same advances introduced in Active
Motif’s popular ChIP-IT Express Kit. Eachmethod uses protein G-coated magnetic
beads that have less background thanstandard agarose beads, so pre-clearing
and blocking steps are not needed.
Magnetic pull-down occurs in justseconds, and the low background has
eliminated the need for DNA puriﬁcation. With Re-ChIP-IT, the 1st ChIP is
performed as in regular ChIP. Chromatinthat has been immunoprecipitated isremoved from the magnetic beads witha special buffer that prevents the ﬁrst
antibody from participating in the 2nd
IP reaction. After desalting, a 2nd ChIP
is performed using an antibody different from that used in the ﬁrst ChIP.
The cross-links of these sequentiallyimmunoprecipitated protein-DNA complexes are then reversed and the DNA isanalyzed by PCR, similar to conventional
ChIP samples (Figure 2).

6

Figure 1: Sequential chromatin immunoprecipitation using Re-ChIP-IT.
The lane numbers are the same in each panel to indicate that the DNA is from the same chromatin sample. The left panel
shows the results of PCR performed on an aliquot of DNA removed from the experiment after the first ChIP step; theright panel represents PCR results on DNA from chromatin samples after both ChIP steps. For example, chromatin samples
subjected to first ChIP using Mouse IgG as a negative control (lanes 1 and 2 in the left panel) were then subjected to a second ChIP with an RNA Pol II antibody (lanes 1 and 2 in the right panel). Chromatin samples in which Mouse IgG was used aseither the first antibody (lanes 1 and 2) or second antibody (lanes 5 and 6) show little amplification of GAPDH DNA in either
the left (first ChIP) or right panel (first and second ChIP). Chromatin samples in which the first antibody used was anti-RNA
Pol II and the second antibody was anti-TFIIB (lanes 3 and 4) show good amplification of GAPDH DNA after the second ChIP
(right panel) indicating co-localization of RNA Pol II and TFIIB at the same region of the GAPDH promoter.

Figure 2: Schematic representation of the Re-ChIP-IT procedure.

Product

FormatCatalog No.

Re-ChIP-IT™

25 rxns

53016

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June 2009 • volume 10 • number 2

Histone Purification

Histone Puriﬁcation Made Simple in Your Choice of Assay Format
Now you can easily purify histones and further separate the fractions of core histones from any cell culture or tissue
sample while maintaining post-translational modiﬁcations like acetylation, methylation and phosphorylation. Active
Motif has two versions of our Histone Puriﬁcation Kit to meet your experimental needs.
Histone proteins are important regulators of genome function, comprising thescaffold around which genomic DNA
is organized. Working with histones
previously required long and tedious
puriﬁcation methods. Active Motif has
simpliﬁed the process with two complete histone puriﬁcation systems inyour choice of kit format: the original
Histone Puriﬁcation Kit, which can beused in either gravity ﬂow or spin column format, and the Histone Puriﬁcation
Mini Kit, with a convenient mini spincolumn for histone puriﬁcation (Table 1).
Both kits utilize proprietary puriﬁcation
columns and buffers to isolate very pure
fractions of histones. Using the Histone
Puriﬁcation Mini Kit, core histones canbe puriﬁed from cultured cells and tissue
samples as a single population containing H2A, H2B, H3 and H4 (Figure 1) fromas few as 8x105 cells. While you can not
purify the same amount of histones aswith the original Histone Puriﬁcation
Kit, mini spin columns make it easier toisolate histones from multiple samples
simultaneously. The original Histone
Puriﬁcation Kit, which has a higher
capacity than the Mini Kit, can purify all
four core histones using the spin-column
format, or purify the histones as separate fractions of H2A/H2B dimers andH3/H4 tetramers using the same column
in a gravity ﬂow format (Figure 2). Bothkits result in the preparation of extremely pure fractions of histones while
keeping important post-translational

Figure 2: Histone Puriﬁcation Kit.
The Histone Purification Kit was used in its gravity flow
format to purify separate H2A/H2B (Lane 1) and H3/H4(Lane 2) fractions from HeLa cells.

Kit
Original Histone
Puriﬁcation Kit
Histone Puriﬁcation
Mini Kit

modiﬁcations intact (e.g. acetylation,
phosphorylation and methylation).
Better substrates make for better assays
Core histones isolated using the original
Histone Puriﬁcation Kit are highly pureand suitable substrates for downstream
assays. Puriﬁed histones can be usedwith the Chromatin Assembly Kit togenerate chromatin that very closely
resembles native chromatin, ideal foruse in functional assays (Figure 3).

Figure 3: Chromatin assembled with puriﬁed histones.
Histones purified from HeLa cells were used with
the Chromatin Assembly Kit. The ordered spacing of
nucleosomes was confirmed by micrococcal nuclease
digestion and agarose gel electrophoresis.

FormatElutionCapacity

Gravity Flow

Separate H2A/H2B &H3/H4 fractions

0.5-2.5 mg

Spin Column

H2A, H2B, H3 & H4 ina single fraction

0.5-2.5 mg

Mini Spin Column

H2A, H2B, H3 & H4 ina single fraction

0.1-0.5 mg

Table 1: Comparison of the original Histone Puriﬁcation Kit and the Histone Puriﬁcation Mini Kit

Figure 1: Histone Puriﬁcation Mini Kit.
HeLa cell core histones (indicated by the bracket) were
purified with the Histone Purification Mini Kit and run
on an SDS-PAGE gel, which shows the first (1st) andsecond (2nd) elution fractions.

www.activemotif.com

Product

FormatCatalog No.

Histone Puriﬁcation Mini Kit

20 rxns

40026

Histone Puriﬁcation Kit

10 rxns

40025

Chromatin Assembly Kit

10 rxns

53500

7

Transcription

TransAM™ Transcription Factor DNA-binding ELISAs
TransAM™ Kits offer a great alternative to traditional methods to study transcription factor activity. TransAM Kits arehighly sensitive ELISA-based assays capable of detecting small changes in transcription factor activity without theneed for gels or radioactivity. The standard TransAM Kits are 10-fold more sensitive than conventional gelshift assays,
while TransAM Chemi Kits utilize chemiluminescent detection to improve sensitivity down to only 40 ng of extract.
NFκB activation (OD450 nm)

Flexible ordering options
Active Motif offers a large selection ofTransAM™ Kits for over 40 different transcription factor targets including NFκB,
AP-1, STAT, HIF and our newest kits for
Nrf2 and T-bet. For a complete productlisting and additional details, please visit
us at www.activemotif.com/transam.

TransAM advantages• Faster procedure – ELISA-based
assay can be completed in 5 hours,
yet provide more quantitative
results (Figure 1)• Sensitive – Up to 100-fold greater
sensitivity than gelshift assays• Less effort required – Our kits
are compatible with multi-channel
pipettors to streamline wash steps• Non-radioactive – colorimetric andchemiluminescent formats available

2.5

Unstimulated
IL-1α stimulated

2
1.5
1
0.5
0

A

0.5

1

2
5
10 25
Cell extract/well (µg)

IL-1α stimulated
0.5

Figure 1: TransAM Kits are more sensitive than gelshift.
Human fibroblast WI-38 cells were 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). The TransAM method is 10-fold moresensitive and provides quantitative results.

B

1

5

10

25

50

Unstimulated
50

0.5

1

5

10

25

50

µg of cell extract

Improve Luciferase Assay Response with RapidReporter®
Active Motif’s patented RapidReporter®* system includes a variety of vectors and assay reagents to study signaltransduction pathways with greater sensitivity than standard reporter assays. The RapidReporter vectors contain adouble destabilizing element that causes the luciferase mRNA and protein to degrade more quickly, enabling better
detection of transient or minor effects on transcriptional activity.
160
140

100
80
60
40
20
0

In addition to the “empty” vectors that
contain a multiple cloning site for insertion of promoters/enhancers of interest,
Active Motif also sells several pre-madeconstructs for widely studied promoterssuch as NFκB, CREB (CRE) or STAT.

pRR-High-NFκB
pGL3-NFκB

120
Fold induction

RapidReporter vectors
RapidReporter® vectors are based on
the extremely bright Gaussia luciferase
gene. The vectors are offered in two
different stringencies, pRR-High and pRRLow, to allow even weak transcriptional
effects to be detected.

0

1

2

3

4

Hours after addition of TNF-α

5

6

Optimized buffers for brighter signal
Active Motif’s Gaussia Luciferase Assay
Kits contain proprietary Lysis and Assay
Buffers and a Gaussia Substrate, which
are optimized for use with the RapidReporter vectors. For your convenience
the RapidReporter Gaussia Luciferase
Assay Kits are available in both 100 and1000 reaction formats.

Figure 1: RapidReporter “unmasks” hidden effects.
HeLa cells were transiently transfected with pRR-High-NFκB or pGL3 vector containing NFκB, then plated in
96-well plates. Twenty-four hours post-transfection, the cells were stimulated with 10 ng/ml TNF-α and measured
for Gaussia Luciferase (pRR) and firefly luciferase (pGL3) at the indicated time points. Because RapidReporter has
double destabilizing elements that reduce background, relatively small and transient events, like the natural
oscillation of NFκB from the cytosol to the nucleus during its activation, can be observed.

* RapidReporter is covered under U.S. Patent No. 7,157,272 and various other patents worldwide. It is sold under license granted by, and is a registered trademark of, GeneStream Pty Ltd.

8Toll Free — 1 877 222 9543

June 2009 • volume 10 • number 2

ChIP-validated Antibodies & Recombinant Proteins

Recombinant and Puriﬁed Proteins for Your Biochemical Assay Needs
Recombinant proteins are of particular interest in many aspects of research,from usage as substrates in biochemical assays to the reconstitution ofactive protein complexes. Active Motif offers a complete line of recombinant proteins to meet your research needs. These proteins are ideal for use
in many biological applications with NFκB p50, NFκB p65, p53, c-Fos, c-Jun,c-Myc, CREB and Sp1 proteins validated for use as standards in TransAM™
DNA-binding ELISAs. We also offer a variety of active serine/threonine andtyrosine kinases, as well as Myelin Basic Protein (MBP), which is useful asa substrate for in vitro kinase assays, including those for MAPK, PKA, PKC
cyclin-dependent and calmodulin-dependent protein kinases (Figure 1).
Active Motif Protein Categories• Kinase and kinase substrates• Transcription factors• Histone modifying enzymes• Nuclear receptors
A complete list of puriﬁed and recombinant proteins, as well as complete
details on each protein, can be found at www.activemotif.com/proteins.

Figure 1: Use of MBP as a kinase assay substrate.
Myelin Basic Protein was tested in an in vitro kinase
assay by incubation with (Lane 1) or without MAPK2(Lane 2), followed by Western blot detection using
an MBP phospho Thr98 antibody. To the right of theWestern blot is a lane from a Coomassie-stained
SDS-PAGE gel containing 4 µg of purified MBP.

ChIP-validated Antibodies for Your Chromatin Experiments
Chromatin immunoprecipitation (chromatin IP or ChIP) is anextremely important technique, allowing the in vivo detection ofsequences associated with chromatin proteins, and identiﬁcationof regions of the genome associated with speciﬁc histone modiﬁcations. ChIP is also a very challenging technique, so only antibodies of the highest quality will do. For an antibody to work in ChIP,
it must be of high titer and it must be very speciﬁc, with no detection of non-target proteins. Most importantly, it must be able torecognize the target protein in its native, chromatin-associatedcontext after it has been ﬁxed with formaldehyde. Active Motif
specializes in the manufacture and of antibodies for use in ChIP.

Figure 1: ChIP of methylated histone H3.
ChIP was performed with Histone H3 dimethyl Lys4 pAb (Catalog No. 39141)
using chromatin prepared from mouse embryonic stem cells (left lane) or
mouse erythroleukemia cells (right lane). PCR primers specific for the betaglobin gene were used to amplify a 210 base pair region of the promoter
following isolation of DNA.

To see our complete line of antibodies validated for use in chromatin IP, please visit www.activemotif.com/chipabs.

www.activemotif.com

9

Nitric Oxide Quantitation & Competent E. coli

Faster, More Accurate Measurement of Nitric Oxide
The Nitric Oxide Quantitation Kit is faster and easier-to-use than existing methods for measuring the production of NO
in biological samples. The kit employs an innovative cofactor technology that reduces the time and number of steps
required, while increasing the dynamic range of NO measurement. The NO Quantitation Kit can be used with a wide
variety of samples, including cell or tissue extract, urine, plasma, serum and saliva.
dynamic range, which increases therange of sample concentrations that canbe measured accurately (Figure 1). This
saves time and money as fewer samples
will need to be diluted and re-assayed toobtain accurate results. To make it easierto process large numbers of samples,the NO Quantitation Kit is provided in aconvenient 96-well format that facilitates high-throughput automation. For abetter method to measure NO, try the
Nitric Oxide Quantitation Kit today.

Product

FormatCatalog No.

Nitric Oxide Quantitation Kit

2 x 96-rxns

40020

1.2
1.0
OD540 nmActive Motif’s Nitric Oxide Quantitation
Kit offers advantages in time, ease-ofuse and accuracy. It employs an innovative cofactor technology that accelerates
the conversion of nitrate to nitrite while
simultaneously degrading NADPH. Thus,
the time required for the reductase
reaction is reduced to only 30 minutes,
and sensitive colorimetric quantiﬁcation
can be performed without the need for
LDH treatment. Moreover, the Active
Motif method is linear over a broader

Active Motif's NO Kit
LDH method

0.8
0.6
0.4
0.2
0.0

0 5 10 15 20 25 30 35 40 45 50 55 60

Concentration of Nitrate (µM)
Figure 1: Dynamic range of nitrate standard curves.
Nitrate standard curves produced using the Nitric Oxide
Quantitation Kit and a conventional Three-Step
LDH-method kit. The NO Kit provides linear, accurate
measurement over a broader range of concentrations.

Efﬁcient, Extremely Affordable Competent E. coli in a More Convenient Format
RapidTrans™ are high-efﬁciency competent E. coli supplied in a convenient
96-tube tray. Ideal for cloning, plasmid preparation and library construction,
RapidTrans cells are affordably priced and packaged in a format that provides
maximum ﬂexibility while eliminating waste. Each tube contains 50 µl of cells
for one transformation reaction. This enables the use of as few or as many
reactions as needed, without thawing the other cells. This eliminates wasted
cells and the reduced efﬁciencies caused by repeated freeze/thaw cycles.
Use only what you need
Unlike other 96-well formats, which
force you to thaw all 96 wells, RapidTransis a tray of 12 x 8 tubes that can be usedsingly or with multi-channel pipettors.
So, you can thaw only what you’ll use.
Don’t pay for unused capacity
Compared to other suppliers, RapidTransare very affordable, primarily because
the transformation efﬁciency of TAM1
E. coli is slightly less, at > 1 x 108 cfu/µg

10supercoiled pUC19 DNA. However, this is
more than adequate for most uses. So,
don’t pay more for super competency
cells that impact your lab’s budget, but
not your results. You wouldn’t buy a
Ferrari to go to the supermarket, wouldyou? Spend wisely; try RapidTrans today.

TAM1 genotype and contents

mcrA ∆(mrr-hsdRMS-mcrBC) Φ80lacZ∆M15∆lacX74 recA1 araD139 ∆(ara-leu)7697 galU
galK rpsL endA1 nupG

For your convenience, cells include SOC
media, pUC19 DNA and sterile reservoirs
for use with multi-channel pipettors.

Product

FormatCatalog No.

RapidTrans™ TAM1 Competent E. coli

1 x 96 rxns
5 x 96 rxns

11096
115096Toll Free — 1 877 222 9543

June 2009 • volume 10 • number 2

GTPase Activation & SUMOylation Assays

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 speciﬁcally to 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, glutathione-coated assay plate.
GST-Raf-RBD is ﬁrst incubated on theplate for one hour to immobilize thiscapture protein. Addition of sample
to the plate results in the binding ofactivated Ras to the Raf-RBD. A primary
antibody speciﬁc for Ras is then added,
followed by an HRP-conjugated secondary antibody and developing reagent.
The plate is then read on a luminometer,
which provides a sensitive, quantitative
chemiluminescent readout of activatedRas GTPase (Figure 1).

Ras GTPase ELISA advantages• More sensitive – assay uses only
25 µg of extract, or 20-fold less thanpull-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
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 1: Quantiﬁcation of activated Ras GTPase.
Increasing amounts of whole-cell extract from HeLa
cells either unstimulated or stimulated 2 minutes with
5 ng/ml EGF are assayed using the Ras GTPase ELISA Kit.

Product
Ras GTPase Chemi ELISA Kit

FormatCatalog No.

1 x 96-well plate

52097

Investigate the Effects of SUMOylation with SUMOlink™
SUMOlink™ Kits are a simple, effective method for generating SUMOylated
proteins in vitro. These fast, efﬁcient and robust assays contain all necessary
reagents for SUMOylation of target proteins, and include positive andnegative controls that help to ensure your success.

MW

1

2

3

1

2

3

98
64
50
36

The SUMOlink method
SUMOlink™ Kits enable you to easily
perform and detect post-translational
modiﬁcations by SUMO (small ubiquitinlike modiﬁer). With SUMOlink, 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 (Figure 1).
With the kit’s p53 antibody and either
SUMO-1 or SUMO-2/3 antibodies, you
can easily see the extent to which yourtarget protein has been SUMOylated.

www.activemotif.com

Everything you need to study SUMO
The kits contain E1 activating and E2
conjugating enzymes along with wildtype and mutant SUMO-1 (SUMO-1 Kit)
or SUMO-2 and -3 proteins (SUMO-2/3
Kit). Antibodies for SUMO-1 or SUMO2/3 modiﬁcations, as well as control p53
protein and antibody, are included. For
complete information, please give us acall or visit us at www.activemotif.com.

30
16
p53 antibody

SUMO-1 antibody

Figure 1: Enzymatic digestion of assembled chromatin.
Western blot analysis of in vitro SUMOylation of p53
protein by wild-type and mutated isoforms of SUMO-1:
The two Western blots were incubated with p53 antibody(1:5000 dilution) and SUMO-1 antibody (1 :4000 dilution).
p53 is SUMOylated only by wild-type SUMO-1 protein.
Lane 1: Wild-type SUMO protein conjugation reaction.
Lane 2: Mutated SUMO protein conjugation reaction.
Lane 3: No p53 control protein used in conjugation.

Product

FormatCatalog No.

SUMOlink™ SUMO-1 Kit

20 rxns

40120

SUMOlink™ SUMO-2/3 Kit

20 rxns40220

11

Epigenetics, Chromatin
Biology & Cell Signaling

THE NEWSLETTER OF ACTIVE MOTIF — June 2009 • volume 10 • number 2• Aberrant DNA Methylation and Pediatric Syndromes• MethylCollector™ Ultra for Enhanced Methylation Enrichment

PRSRT STD
US POSTAGEPAID
PERMIT #94
SAN DIEGO, CA• UnMethylCollector™ Positively Identiﬁes Unmethylated CpG Regions• Identify Protein Co-localization In Vivo Using Sequential Chromatin IP• Histone Puriﬁcation Made Simple in Your Choice of Assay Format• TransAM™ Transcription Factor DNA-binding ELISAs• Improve Luciferase Assay Response with RapidReporter®
• Recombinant and Puriﬁed Proteins for Your Biochemical Assay Needs• ChIP-validated Antibodies for Your Chromatin Experiments• Faster, More Accurate Measurement of Nitric Oxide• Efﬁcient, Affordable Competent E. coli in a More Convenient Format• ELISA for Quantiﬁcation of Activated Ras GTPase• Investigate the Effects of SUMOylation with SUMOlink™

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

Products for Epigenetics,
Chromatin Biology &Cell Signaling