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.
Confocal immunofluorescent analysis of MCF-7 cells using ESET (C1C12) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).
Confocal imunofluorescent analysis of HeLa cells using ASH2L (D93F6) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).
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.
Confocal immunofluorescent analysis of HeLa cells using SET7/SET9 Antibody (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).
Western blot analysis of extracts from various cell lines using SMYD2 (D14H7) Rabbit mAb.
Western blot analysis of extracts from various cell lines using SUV39H1 (D11B6) Rabbit mAb.
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).
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.
Confocal immunofluorescent analysis of HeLa cells using G9a/EHMT2 (C6H3) Rabbit mAb (green). Actin filaments have been labeled with DY-555 phalloidin (red).
Western blot analysis of cell lysates from MCF7 and 293 cells using ESET (C1C12) Rabbit mAb.
Western blot analysis of extracts from various cell lines using ASH2L (D93F6) XP® Rabbit mAb.
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.
Western blot analysis of cell lysates from HeLa, NIH/3T3, C6 and COS cells using SET7/SET9 Antibody.
Western blot analysis of extracts from HCT116 and RAW cells using SET8 (C18B7) Rabbit mAb.
Western blot analysis of extracts from HeLa and 293 cells using G9a/EHMT2 (C6H3) Rabbit mAb.
Western blot analysis of extracts from various cell lines using RBBP5 (D3I6P) Rabbit mAb.
|G9a/EHMT2 (C6H3) Rabbit mAb 3306||20 µl||
||H M R Mk||160,180||Rabbit IgG|
|ESET (C1C12) Rabbit mAb 2196||20 µl||
||H Mk||180||Rabbit IgG|
|ASH2L (D93F6) XP® Rabbit mAb 5019||20 µl||
||H M R Mk||80, 65||Rabbit IgG|
|RBBP5 (D3I6P) Rabbit mAb 13171||20 µl||
||H M R Mk||70||Rabbit IgG|
|SET7/SET9 Antibody 2813||20 µl||
||H M R Mk||48||Rabbit|
|SMYD2 (D14H7) Rabbit mAb 9734||20 µl||
||H M R Mk||49||Rabbit IgG|
|SUV39H1 (D11B6) Rabbit mAb 8729||20 µl||
||H M R Mk||48||Rabbit IgG|
|SET8 (C18B7) Rabbit mAb 2996||20 µl||
||H M R Mk||43||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
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.
Each antibody in this kit recognizes only the specific target protein and does not cross-react with other family members.
Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence of human ASH2L protein, the carboxy terminus of human ESET protein, the carboxy terminus of human G9a/EHMT2 protein, human SET8 protein, residues surrounding Val414 of human SMYD2 protein, residues surrounding Val408 of human RBBP5 protein, or residues surrounding Asp380 of human SUV39H1 protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues the human SET7/SET9 protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.
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).
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