Overview

  • Product name
  • Description
    Rabbit polyclonal to EHMT2/G9A
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IP, ChIP, IHC-P, WBmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Fusion protein corresponding to Human EHMT2/ G9A aa 831-1001.
    Database link: Q96KQ7

Properties

Applications

Our Abpromise guarantee covers the use of ab40542 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IP Use at an assay dependent concentration.
ChIP Use at an assay dependent concentration.
IHC-P 1/2000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB 1/2000 - 1/2500. Predicted molecular weight: 132 kDa. For endogeneous detection (without G9a over expression): use nuclear extracts and block overnight or perform IP prior to WB.

Target

  • Function
    Histone methyltransferase that specifically mono- and dimethylates 'Lys-9' of histone H3 (H3K9me1 and H3K9me2, respectively) in euchromatin. H3K9me represents a specific tag for epigenetic transcriptional repression by recruiting HP1 proteins to methylated histones. Also mediates monomethylation of 'Lys-56' of histone H3 (H3K56me1) in G1 phase, leading to promote interaction between histone H3 and PCNA and regulating DNA replication. Also weakly methylates 'Lys-27' of histone H3 (H3K27me). Also required for DNA methylation, the histone methyltransferase activity is not required for DNA methylation, suggesting that these 2 activities function independently. Probably targeted to histone H3 by different DNA-binding proteins like E2F6, MGA, MAX and/or DP1. May also methylate histone H1. In addition to the histone methyltransferase activity, also methylates non-histone proteins: mediates dimethylation of 'Lys-373' of p53/TP53. Also methylates CDYL, WIZ, ACIN1, DNMT1, HDAC1, ERCC6, KLF12 and itself.
  • Tissue specificity
    Expressed in all tissues examined, with high levels in fetal liver, thymus, lymph node, spleen and peripheral blood leukocytes and lower level in bone marrow.
  • Sequence similarities
    Belongs to the class V-like SAM-binding methyltransferase superfamily. Histone-lysine methyltransferase family. Suvar3-9 subfamily.
    Contains 7 ANK repeats.
    Contains 1 post-SET domain.
    Contains 1 pre-SET domain.
    Contains 1 SET domain.
  • Domain
    The SET domain mediates interaction with WIZ.
    The ANK repeats bind H3K9me1 and H3K9me2.
  • Post-translational
    modifications
    Methylated at Lys-185; automethylated.
  • Cellular localization
    Nucleus. Chromosome. Associates with euchromatic regions. Does not associate with heterochromatin.
  • Information by UniProt
  • Database links
  • Alternative names
    • Ankyrin repeat containing protein antibody
    • Bat 8 antibody
    • Bat8 antibody
    • C6orf30 antibody
    • DKFZp686H08213 antibody
    • EHMT 2 antibody
    • Ehmt2 antibody
    • EHMT2_HUMAN antibody
    • Euchromatic histone lysine methyltransferase 2 antibody
    • Euchromatic histone lysine N methyltransferase 2 antibody
    • Euchromatic histone-lysine N-methyltransferase 2 antibody
    • FLJ35547 antibody
    • G 9a antibody
    • G9 a antibody
    • G9a protein antibody
    • G9A antibody
    • G9A histone methyltransferase antibody
    • GAT8 antibody
    • H3 K9 HMTase 3 antibody
    • H3-K9-HMTase 3 antibody
    • Histone H3 K9 methyltransferase 3 antibody
    • Histone H3 K9 methyltransferase3 antibody
    • Histone H3-K9 methyltransferase 3 antibody
    • Histone lysine N methyltransferase antibody
    • Histone lysine N methyltransferase EHMT2 antibody
    • Histone lysine N methyltransferase, H3 lysine 9 specific 3 antibody
    • Histone lysine N methyltransferase, H3 lysine 9 specific3 antibody
    • Histone-lysine N-methyltransferase EHMT2 antibody
    • HLA B associated transcript 8 antibody
    • HLA-B-associated transcript 8 antibody
    • KMT 1C antibody
    • KMT1 C antibody
    • Lysine N methyltransferase 1C antibody
    • Lysine N-methyltransferase 1C antibody
    • NG 36 antibody
    • NG36 antibody
    • Protein G9a antibody
    see all

Images

  • All lanes : Anti-EHMT2/G9A antibody (ab40542) at 1/2500 dilution

    Lane 1 : Untransfected HeLa cell extract
    Lane 2 : G9a transfected HeLa cell extract

    Predicted band size: 132 kDa
    Observed band size: 132 kDa
    Additional bands at: 60 kDa. We are unsure as to the identity of these extra bands.

  • ab40542 (1:2000) staining EHMT2/ G9A in human prostate using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

References

This product has been referenced in:
  • Anderson EM  et al. Overexpression of the Histone Dimethyltransferase G9a in Nucleus Accumbens Shell Increases Cocaine Self-Administration, Stress-Induced Reinstatement, and Anxiety. J Neurosci 38:803-813 (2018). Read more (PubMed: 29217682) »
  • Zhang L  et al. Overexpression of PRDM13 inhibits glioma cells via Rho and GTP enzyme activation protein. Int J Mol Med 42:966-974 (2018). Read more (PubMed: 29767251) »
See all 23 Publications for this product

Customer reviews and Q&As

1-10 of 13 Abreviews or Q&A

Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (brain)
Permeabilization
Yes - Triton 0.5%
Specification
brain
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Sep 08 2015

Answer

Thank you for your reply. I am sorry that this product has not met your needs.Your credit note ID is xxxxx. I have asked our accounting department to issue a credit note for you, which can be redeemed against the invoice of a future order by passing it on to your purchasing department. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. If you have questions on how to use the credit note, please contact our accounting department. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department. The credit note ID is for your reference only and does not automatically guarantee the credit. I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

Read More

Answer

Thank you for contacting Abcam.

I am sorry about that you are experiencing some problems with ab40542 in ChiP. The antibody is covered under our Abpromise for six months and is guaranteed to work in ChIP on human samples . If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.

When you say that the antibody did not work as well as previous lots, were you still getting a signal using the current lot of ab40542, but just had to use a higher concentration of the antibody, or did the antibody not produce the expected results at all. Also, did both of the antibody vials you received have the same issue?

Would you also be able to provide the order or PO number used to purchase these vials of ab40542, so that I can locate your order details.

I look forward to your reply.

Read More

Answer

Thank you for contacting us.
For ab40542: As immunogen, the recombinant fusion protein, corresponding to amino acids 831-1001 of Human G9a was used to raise the antibody. The epitope has not been mapped.
As for the DNMT1 (DNA methyl transferase 1) antibody, coud you please let me know which antibody (catalog number) you are refering to? Thank you.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Answer

Thank you for your reply. I am sorry that this antibody is producing poor results in WB. Based on the images provided, I suspect the antibody has lost specificity with age. It is possible that the results may be improved with troubleshooting, but it would bechallenging given the intense background andweak specific band. The following may help:

1)Given that KMT1C is a nuclear protein, I would recommend preparing the lysates with RIPA buffer. The specific band may be strengthened and the background reduced by enriching the samples in a nuclear extract. Sample degradation could account for some of the background bands, so fresh lysates prepared on ice with protease inhibitors would be recommended.

2) What blocking agent was used? This antibody was validated using an overnight block with milk, which is recommended for keeping the background low.

3) How much primary antibody was used and how long was it incubated for? We recommend using a 1:2500 dilutionfor 2 hours at room temperature. If you continue to seebackground at thisdilution, you could try reducing the secondary antibody concentration.

I hope this helps, please let me know if you need any additional information or assistance.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (human pancreatic ductal adenocarcinoma)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate pH=6
Permeabilization
Yes - PBS+0.025% TritonX100
Specification
human pancreatic ductal adenocarcinoma
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jan 28 2011

Application
Western blot
Sample
Human Cell lysate - other (A549)
Gel Running Conditions
Non-reduced Denaturing (8-16)
Loading amount
75000 cells
Treatment
multiple (DFO & CoCl2)
Specification
A549
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 50000µg/mL · Temperature: 22°C

Mr. Ted Natoli

Verified customer

Submitted Nov 16 2010

Abreviews
Abcam has not validated the combination of species/application used in this Abreview.
Application
ChIP
Sample
Rat Tissue lysate - whole (muscle)
Negative control
Genes in an active state of transcription, negative for H3K9me2 and H3K27me2.
Specification
muscle
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde
Positive control
Genes that are positive for H3K9me2 and H3K27me2, i.e. in a repressed state of transcription.

Abcam user community

Verified customer

Submitted Jul 19 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - nuclear (Hela)
Gel Running Conditions
Reduced Denaturing
Loading amount
60 µg
Specification
Hela
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jan 15 2010

Abreviews
Application
Western blot
Sample
Human Cell lysate - nuclear (HeLa)
Gel Running Conditions
Reduced Denaturing (4-20% gel)
Loading amount
25 µg
Treatment
G9a specific siRNA
Specification
HeLa
Blocking step
Milk as blocking agent for 15 minute(s) · Concentration: 2% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 08 2009

1-10 of 13 Abreviews or Q&A

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