Anti-Methylated Lysine (di methyl , mono methyl ) antibody (ab23366)

Reviews (8)Q&A (16)References (24)

Overview

  • Product name
    Anti-Methylated Lysine (di methyl , mono methyl ) antibody
    See all Methylated Lysine primary antibodies
  • Description
    Rabbit polyclonal to Methylated Lysine (di methyl , mono methyl )
  • Host species
    Rabbit
  • Specificity
    Recognize proteins methylated on lysine residues (mono- & di-methyl lysine). This antibody was affinity purified with mono-methyl lysine and di-methyl lysine, and may also react with tri-methyl lysine. Does not cross-react with acetylated protein.
  • Tested applications
    Suitable for: IHC-P, WB, ELISAmore details
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    Methylated Lysine conjugated to KLH.

  • Positive control
    • HCL (0.1N) extracted and acetone precipited histone fraction

Properties

Applications

Our Abpromise guarantee covers the use of ab23366 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
IHC-P Use at an assay dependent concentration.
WB Use a concentration of 0.5 - 1 µg/ml. Do not use milk for any of the buffers as complex milk proteins may contain competitive methylated proteins. Use BSA instead. ab23366 incubation time 2h RT. If no or weak signal is obtained overnight at 4deg might help.
ELISA Use at an assay dependent concentration.

Target

  • Relevance
    Many proteins are post translationally modified. Modifications such as phosphorylation, glycosylation, ubiquitination and methylation have been shown to play an important role in the development, physiology and disease of animals and plants. Histone lysine methylation is regarded as a very stable modification with important functions in epigenetic gene control and for organizing chromatin domains.
  • Alternative names
    • Dimethylysine antibody
    • Methyl lysine antibody
    • N epsilon dimethyl lysine antibody
    • N epsilon mono methyllysine antibody
    • N epsilon trimethyl lysine antibody
    • N(epsilon) trimethyl lysine antibody
    • Trimethyl lysine antibody
    see all

Images

  • Western blot - Anti-Methylated Lysine (di methyl , mono methyl ) antibody (ab23366)
    Western blot - Anti-Methylated Lysine (di methyl , mono methyl ) antibody (ab23366)
    Lane 1 : Anti-Methylated Lysine (tri methyl) antibody (does not cross react with mono, or di methyl lysine residues)
    Lane 2 : Anti-Methylated Lysine (di methyl , mono methyl ) antibody (ab23366)

    All lanes : Yeast extract
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Methylated Lysine (di methyl , mono methyl ) antibody (ab23366)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Methylated Lysine (di methyl , mono methyl ) antibody (ab23366)This image is courtesy of an anonymous Abreview
    ab23366 at 1/200 staining human kidney tissue sections by IHC-P. The tissue was formaldehyde fixed a heat mediated antigen retrieval step was performed in citrate buffer. An HRP conjugated goat anti-rabbit antibody was used as the secondary.

    See Abreview

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Methylated Lysine (di methyl , mono methyl ) antibody (ab23366)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Methylated Lysine (di methyl , mono methyl ) antibody (ab23366)This image is courtesy of an anonymous Abreview

    ab23366 staining formalin fixed paraffin-embedded rat kidney tissue sections.  The section was subjected to heat mediated antigen retrieval in citrate buffer (pH 6.0) and blocked with 5% serum for 30 minutes at 20°C, prior to incubation with the primary antibody (diluted 1/200 in PBS + 5% serum) for 45 mintes at 20°C.  An HRP-conjugated goat anti-rabbit antibody was used as the secondary.

    See Abreview

References

This product has been referenced in:
  • Lee SW  et al. MicroRNAs Overcome Cell Fate Barrier by Reducing EZH2-Controlled REST Stability during Neuronal Conversion of Human Adult Fibroblasts. Dev Cell 46:73-84.e7 (2018). Read more (PubMed: 29974865) »
  • Feldman M & Levy D Peptide inhibition of the SETD6 methyltransferase catalytic activity. Oncotarget 9:4875-4885 (2018). Read more (PubMed: 29435148) »
See all 24 Publications for this product

Publishing research using ab23366? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 24 Abreviews or Q&A

Immunoprecipitation abreview for Anti-Methylated Lysine (di methyl , mono methyl ) antibody

 Below Average
Abreviews
Abcam has not validated the combination of species/application used in this Abreview.
abreview image
Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (Hek293 - kidney cell line)
Total protein in input
1000 µg
Immuno-precipitation step
Other - Dynabeads or Protein G-coated magnetic beads
Specification
Hek293 - kidney cell line
Read More

Byron Baron

Verified customer

Submitted Sep 06 2018

Western blot abreview for Anti-Methylated Lysine (di methyl) antibody

 Good
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Hep3B)
Gel Running Conditions
Non-reduced Denaturing (15%)
Loading amount
20 µg
Specification
Hep3B
Blocking step
I-Block(Applied biosystems) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2µg/mL · Temperature: 23°C
Read More

Abcam user community

Verified customer

Submitted Jan 03 2017

Immunoprecipitation abreview for Anti-Methylated Lysine (di methyl , mono methyl ) antibody

 Good
Abreviews
abreview image
Application
Immunoprecipitation
Immuno-precipitation step
Protein G
Sample
Human Cell lysate - whole cell (Human Embryonic Kidney 293 cells)
Specification
Human Embryonic Kidney 293 cells
Total protein in input
1000 µg
Read More

Dr. Yi-Wen Chang

Verified customer

Submitted Jan 26 2015

Question

I ordered the Anti Methylated Lysine antibody (ab23366), but the product size is not listed. Do you mind telling me the molecular weight.

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Answer


The short answer is that there is no predicted MW for this antibody to detect in WB. This antibody will detect any protein of any size that has a methylated lysine. Therefore, depending on your sample you may see no bands, many bands, a smear, etc.

Take a look at this reference. I don't have access to the journal so I can't see if there is a WB image, but we know this ab was used in WB here: http://www.ncbi.nlm.nih.gov/pubmed/21204252?dopt=Abstract

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Question

I need Discount code for the three below antibodies . Please arrange to send the same at your earliest .

ab21623, ab23366 and ab412

for detection of methylation and acetylation of bacterial proteins.

Regards

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Answer

Thank you for contacting us.

These antibodies are raised against targets that are not species specific. The antibodies would detect the proteins irrespective of species so the Abtrial discount will not be valid.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question

I noticed that Abcam has in its inventory anti mono- and di-methyl arginine antibody (ab412), and anti-methylated lysine (mono-methyl, dimethyl) antibody (ab23366).
Could you please send us a small sample of the antibodies mentioned above for a test in our in vitro system? Of course whichever of those antibodies that gives us a positive result will be ordered from you in the future.
Thank you so much for your time and consideration.

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Answer

Thank you for contacting us. Because we carry over 90,000 products, it isn't feasible for us to keep small sample sizes of our products.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase.

Otherwise, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Plus, each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates.

To find out more about our Abreview system, please see the following link:

https://www.abcam.com/abreviews

I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

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Question

Thanks for this information. May I ask two more questions?
How much is the discrimination factor against BSA alone and is this antibody specific to methyllysine, or may it crossreact with methylarginine as well?
Thanks you very much for your help again,
Best,

Read More
Answer

Thank you for your reply.

I can confirm that ab23366 does not recognize free BSA or BSA-linked with methylarginine.

I hope this information is helpful and wish you good luck with your experiments.

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Question

Product code: 23366
Lot number: GR32966-7

Inquiry: Dear abcam-customer service! I actually do not really have a problem with the above-mentioned product, rather a question regarding its specificity. Actually, in a recent publication (Levi et al, Epigenetics & Chromatin 2011, 4:19 doi:10.1186/1756-8935-4-19), two different lots of the above-mentioned antibody were tested using a human epigenome peptide microarray platform with quite different outcome: one (ab23366/904302 Abcam, Cambridge, Cambridgeshire, UK) exhibited methylation-specific reactivity, but recognized only trimethylated peptides (Figure 2C). Relative to the first two antibodies tested, the third antibody (23366/915620 Abcam) was highly specific to monomethylated and trimethylated peptides, and did not crossreact with unmethylated peptides on the array (Figure 2D). I wondered whether lots are systematically tested on a peptide array and whether you can provide me with information according to the lot number I bought (GR-32966-7)? Do you have information if the sequence surrounding the methylated Lysine has an impact on the detection of the Ab and if yes, could you provide me with further information? I would really much appreciate any support according to my questions. Thank you very much in advance, Best regards,

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Answer

Thank you for your inquiry.

I apologize for the delay in my answer. It took some time to pull the data for lot GR32966-7.

I am happy to confirm that we tested lot GR32966-7 not in a peptide array buton the single amino acid linked to BSA such as mono-mehtyllysine-BSA, di-methyllysine-BSA, tru-methyllysine-BSA.

The ELISA titer (OD50) is 80 ng/mL, 200 ng/mL, 50 ng/mL for mono-methyllysine, di-methyllysine and trimethyllysine-BSA, respectively for lot GR32966-7.

We do not know what amino acid affect the binding, presumably the amino acid with a bulky docking side chain such as tyrosine, phenylalanine, isoleucine may contribute to the steric hindrance of the binding.

I hope this information is helpful and wish you good luck with your research.

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Question

Thank you very much for your kind replying and help. I am going to place a order for our substrate specificity studies. However, There are four anti-lysine abs available in Abcam as shown thehere. any difference among these Abs?Which one is the best for substrate specificity studies and the mutations around the lysinewill not affect the antibodies ability to recognize the Lysine sites. I am looking forward to heraing from you soon. Thank you.

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Answer

Thank you for your reply.

Based on the available information, any of those antibodies would work for you, but I would recommend ab21623.

If there is anything else I can help you with, please let me know.

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Question

We would like to order two antibodies with Anti-Methylated Lysine (mono methyl, di methyl) antibody (ab23366) and Anti-acetyl Lysine antibody - ChIP Grade (ab21623). Before the order, I have a few questions would like to ask.



1. Will these Abs recoganize different peptides or proteins with methylated or acetylated Lysine?



2. How the mutationsof singleadjacent amino acid residues oflysineaffecting the recognization of these two Abs?

Ie: ARTKQTARK9STGG(Wild type)

ARTKQTAAK9STGG(mutation)

ARTKQTATK9STGG(mutation)

If these three peptides have been methylated or acetylated in K9 by enzymes, will these two Abs recognize these three peptides?

3. Is immunogen sequence just one Lysine(Me or Ace) or a few amino acids including lysine?What is the immunogen sequence for these antibody?

Read More
Answer

Thank you for contacting Abcam.

Please find the answers to your question below:

1 - For ab21623, it will recognize proteins acetylated on lysine residues. For ab23366, it will recognize proteins methylated on lysine residues (mono- & di-methyl lysine).

2 - The mutations should not affect the antibodies ability to recognize the Lysine sites.

3 - The immunogen sequence of these antibodies is proprietary information and so I am unable to supply you with those sequences.

If there is anything else I can help you with, please let me know.

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