Revision 1

#8694Store at -20C

1 Kit

(8 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
G9a/EHMT2 (C6H3) Rabbit mAb 3306 20 µl 160,180 kDa Rabbit IgG
ESET (C1C12) Rabbit mAb 2196 20 µl 180 kDa Rabbit IgG
ASH2L (D93F6) XP® Rabbit mAb 5019 20 µl 80, 65 kDa Rabbit IgG
RBBP5 (D3I6P) Rabbit mAb 13171 20 µl 70 kDa Rabbit IgG
SET7/SET9 Antibody 2813 20 µl 48 kDa Rabbit 
SMYD2 (D14H7) Rabbit mAb 9734 20 µl 49 kDa Rabbit IgG
SUV39H1 (D11B6) Rabbit mAb 8729 20 µl 48 kDa Rabbit IgG
SET8 (C18B7) Rabbit mAb 2996 20 µl 43 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

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Description

The Lysine Methyltransferase Antibody Sampler Kit provides a fast and economical means to evaluate endogenous levels of lysine methyltransferases. The kit contains enough primary antibody to perform two western blot experiments per primary antibody.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

SET domain-containing proteins are potential histone methyltransferases (HMTases), which are classified into subgroups by their putative substrate specificities. Histone H3 Lys9 (H3-K9) methyltransferase group genes include Suv39h1, Suv39h2, G9a, G9a related protein (GLP) and SETDB1/ESET (1). The H3-K9 methylation mark plays an important role as a binding site for the chromo-containing protein, resulting in chromatin compaction and heterochromatin generation (2). Histone H3-K4 methylation is exclusively associated with actively transcribed genes (2). The first H3-K4 methylase complex, COMPASS, was identified in the yeast S. cerevisiae and consists of Set1/KMT2 and seven other polypeptides, Cps60-Cps15 (2). Set1/KMT2 functions within COMPASS and is capable of mono-, di-, and trimethylating H3-K4 (2). There are several Set1 related proteins in mammals including WDR5, RBBP5, ASH2L, CXXC1, and DPY30 (2,3). SET7/SET9 is a member of the SET domain-containing family that can specifically methylate H3-K4, Lys189 of the TAF10, a member of the TFIID transcription factor complex, and Lys372 of the p53 tumor suppressor protein (4-6). SET domain-containing lysine methyltransferase 8 (SET8), also known as PR/SET domain-containing protein 7 (PR/SET7), is a single-subunit enzyme that mono-methylates histone H4-K20, preferably on nucleosomal substrates (7-9). SET and MYND domain-containing protein 2 (SMYD2), also known as lysine methyltransferase protein 3C (KMT3C), functions to repress transcription by interacting with the Sin3A repressor complex and methylating H3-K36 (10). SMYD2 also methylates H3-K4 through interaction with HSP90α, and methylates p53 at Lys370 to repress p53-mediated transcriptional activation and apoptosis (11,12).

  1. Tachibana, M. et al. (2005) Genes Dev 19, 815-26.
  2. Shilatifard, A. (2008) Curr Opin Cell Biol 20, 341-8.
  3. Lee, J.H. et al. (2007) J Biol Chem 282, 13419-28.
  4. Nishioka, K. et al. (2002) Genes Dev 16, 479-89.
  5. Kouskouti, A. et al. (2004) Mol Cell 14, 175-82.
  6. Chuikov, S. et al. (2004) Nature 432, 353-60.
  7. Fang, J. et al. (2002) Curr Biol 12, 1086-99.
  8. Xiao, B. et al. (2005) Genes Dev 19, 1444-54.
  9. Couture, J.F. et al. (2005) Genes Dev 19, 1455-65.
  10. Brown, M.A. et al. (2006) Mol Cancer 5, 26.
  11. Abu-Farha, M. et al. (2008) Mol Cell Proteomics 7, 560-72.
  12. Huang, J. et al. (2006) Nature 444, 629-32.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #8694

    Lysine Methyltransferase Antibody Sampler Kit

    Lysine Methyltransferase Antibody Sampler Kit: Image 1 Expand Image
    Western blot analysis of extracts from various cell lines using RBBP5 (D3I6P) Rabbit mAb.
    Lysine Methyltransferase Antibody Sampler Kit: Image 2 Expand Image
    CUT&RUN was performed with HeLa cells and RBBP5 (D3I6P) Rabbit mAb #13171, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding across CDKN2C gene, a known target gene of RBBP5 (see additional figure containing CUT&RUN-qPCR data).
    Lysine Methyltransferase Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of cell lysates from MCF7 and 293 cells using ESET (C1C12) Rabbit mAb.
    Lysine Methyltransferase Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of cell lysates from HeLa, NIH/3T3, C6 and COS cells using SET7/SET9 Antibody.
    Lysine Methyltransferase Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from HCT116 and RAW cells using SET8 (C18B7) Rabbit mAb.
    Lysine Methyltransferase Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from HeLa and 293 cells using G9a/EHMT2 (C6H3) Rabbit mAb.
    Lysine Methyltransferase Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from various cell lines using ASH2L (D93F6) XP® Rabbit mAb.
    Lysine Methyltransferase Antibody Sampler Kit: Image 8 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Lysine Methyltransferase Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from various cell lines using SUV39H1 (D11B6) Rabbit mAb.
    Lysine Methyltransferase Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from various cell lines using SMYD2 (D14H7) Rabbit mAb.
    Lysine Methyltransferase Antibody Sampler Kit: Image 11 Expand Image
    Immunoprecipitation of RBBP5 from C2C12 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or RBBP5 (D3I6P) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using RBBP5 (D3I6P) Rabbit mAb.
    Lysine Methyltransferase Antibody Sampler Kit: Image 12 Expand Image
    CUT&RUN was performed with HeLa cells and RBBP5 (D3I6P) Rabbit mAb #13171, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome #1 (upper), including CDKN2C (lower), a known target gene of RBBP5 (see additional figure containing CUT&RUN-qPCR data).
    Lysine Methyltransferase Antibody Sampler Kit: Image 13 Expand Image
    Confocal immunofluorescent analysis of MCF-7 cells using ESET (C1C12) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).
    Lysine Methyltransferase Antibody Sampler Kit: Image 14 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with 0.5% Triton X-100 for 10 minutes prior to fixation according to Tardat et al. (2007) J. Cell Biol. 179, 1413-26 (right), using SET8 (C18B7) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).
    Lysine Methyltransferase Antibody Sampler Kit: Image 15 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using G9a/EHMT2 (C6H3) Rabbit mAb (green). Actin filaments have been labeled with DY-555 phalloidin (red).
    Lysine Methyltransferase Antibody Sampler Kit: Image 16 Expand Image
    Confocal imunofluorescent analysis of HeLa cells using ASH2L (D93F6) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).
    Lysine Methyltransferase Antibody Sampler Kit: Image 17 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either RBBP5 (D3I6P) or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human CDKN1B Promoter Primers #11951, human CDKN2C promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
    Lysine Methyltransferase Antibody Sampler Kit: Image 18 Expand Image
    CUT&RUN was performed with HeLa cells and RBBP5 (D3I6P) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human CDKN2C promoter primers and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
    Lysine Methyltransferase Antibody Sampler Kit: Image 19 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from K562 cells and either G9a/EHMT2 (C6H3) or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human ZNF19 Intron 3 Primers #98428, human VRK3 intron 11 primers, and SimpleChIP® Human GAPDH Exon 1 Primers #5516. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.