Overview

  • Product name
    Anti-acetyl Lysine antibody - ChIP Grade
    See all acetyl Lysine primary antibodies
  • Description
    Rabbit polyclonal to acetyl Lysine - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    Recognises proteins acetylated on lysine residues. Tested: acetylated histone, acetylated BSA, and acetylated MBP, no reaction to the non acetylated proteins.
  • Tested applications
    Suitable for: IP, IHC-P, ChIP, ICC/IF, WB, ELISAmore details
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    Acetylated KLH conjugates.

  • Positive control
    • Subconfluent MMRU cells with or without HDAC inhibitor treatment
  • General notes


    Use to detect acetylation of lysine.

Properties

Applications

Our Abpromise guarantee covers the use of ab21623 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.
IHC-P Use at an assay dependent concentration. PubMed: 24160175
ChIP Use 2-4 µg for 25 µg of chromatin.
ICC/IF 1/100 - 1/250. Subconfluent MMRU cells seeded in a 6-well plate with or without HDAC inhibitor treatment were fixed and permeabilized with 1% formaldehyde and 0.5% Triton X-100 for 10 minutes and blocked with 10 mg mL-1 BSA for 1 hour at room temperature. Cells were stained with FITC-conjugated anti-acetylated lysine antibody at 1:100 dilution with PBSt 1 % BSA for 1 hour at room temperature.
WB 1/1000 - 1/2500.
ELISA 1/5000.

Target

  • Relevance
    In the nucleus, DNA is tightly packed into nucleosomes generating an environment which is highly repressive towards DNA processes such as transcription. Acetylation of lysine residues within proteins has emerged as an important mechanism used by cells to overcome this repression. The acetylation of non-histone proteins such as transcription factors, as well as histones appears to be involved in this process. Acetylation may result in structural transitions as well as specific signaling within discrete chromatin domains. The role of acetylation in intracellular signaling has been inferred from the binding of acetylated peptides by the conserved bromodomain. Furthermore, recent findings suggest that bromodomain/acetylated-lysine recognition can serve as a regulatory mechanism in protein-protein interactions in numerous cellular processes such as chromatin remodeling and transcriptional activation. The reversible lysine acetylation of histones and non-histone proteins plays a vital role in the regulation of many cellular processes including chromatin dynamics and transcription, gene silencing, cell cycle progression, apoptosis, differentiation, DNA replication, DNA repair, nuclear import, and neuronal repression. More than 20 acetyltransferases and 18 deacetylases have been identified so far, but the mechanistic details of substrate selection and site specificity of these enzymes remain unclear. Over 40 transcription factors and 30 other nuclear, cytoplasmic, bacterial, and viral proteins have been shown to be acetylated in vivo.
  • Alternative names
    • pan acetyl Lysine antibody

Images

  • Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 2µg of  ab21623 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.    

  • Primary: All Lanes: Anti acetyl Lysine antibody (ab21623) at 1:1000. Lane 1: Marker. Lane 2: HeLa cells vehicle-treated (ab139414). Lane 3: HeLa cells, trichostatin A-treated (ab139414). Lysates at 20 ug/lane . Secondary: All Lanes: Goat anti-Rabbit IgG 1:10000. Performed under reducing conditions. Blocking buffer: 5% milk in PBS. Observed band sizes: 11 kDa 15kDa 45kDa 50 kDa.
  • Lane 1 = Extract of Mcf7 cells incubated with vehicle ? 20 ug. Lane 2 = Extract of Mcf7 cells incubated with trichostatin A ? 20 ug. Lane 3 = Extract of Mcf7 cells incubated with EX527 ? 20 ug. Lane 4 = Extract of Mcf7 cells incubated with nicotinamide ? 20 ug. Lane 5 = Extract of Mcf7 cells incubated with camptothecin ? 20 ug. Lane 6 = Extract of Mcf7 cells incubated with camptothecin and trichostatin A ? 20 ug. Lane 7 = Extract of Mcf7 cells incubated with camptothecin and EX527 ? 20 ug. Lane 8 = Extract of Mcf7 cells incubated with camptothecin and nicotinamide ? 20 ug. Lane 9 = Extract of 293T cells incubated with vehicle ? 20 ug. Lane 10 = Extract of 293T cells incubated with camptothecin ? 20 ug. Lane 11 = Extract of 293T cells incubated with camptothecin and trichostatin A ? 20 ug. Lane 12 = Extract of 293T cells incubated with camptothecin and EX527 ? 20 ug. Lane 13 = Extract of 293T cells incubated with camptothecin and nicotinamide ? 20 ug
    SDS PAGE performed under reducing conditions (100mM DTT ? Sample heated at 50?C). Primary : Lanes 1-13: Rabbit anti acetyl Lysine antibody (ab21623) at 1/500 dilution. Secondary : Lanes 1-13: Goat anti rabbit IgG(H&L)-IR680 at 1:10,000 (in green). Developed: Oddysey. Blocking: in 5% Milk + PBS for 3 hours at RT. Primary antibody: in 5% BSA + 50mM Tris pH 7.5 + 150 mM NaCl + 0.05% Tween-20. Secondary antibody: in 5% Milk + PBS + 0.1% Tween-20 + 0.01%SDS for 2 hour at RT. Predicted band size : multiple. Observed band size : multiple
  • All lanes : Anti-acetyl Lysine antibody - ChIP Grade (ab21623) at 0.5 µg/ml

    Lane 1 : Untreated Human melanoma cell lysate
    Lane 2 : TSA-treated Human melanoma cell lysate

    Lysates/proteins at 75 µg per lane.

    Secondary
    All lanes : Goat anti-rabbit IgG HRP at 0.25 µg/ml

    Developed using the ECL technique.

    Observed band size: 16-18 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 12-14 kDa. We are unsure as to the identity of these extra bands.

  • Immunofluorescent staining of Human melanoma cells, using Rabbit polyclonal to acetyl Lysine (ab21623) at 1:100 dilution.

    1: Untreated cells

    2: TSA treated cells

  • p53 acetylation upon Doxorubicin treatment in human melanoma cells (MMRU cells). MMRU cells were treated with 0.5 ug/ml Dox for various times and lyzed for whole protein. Immunoprecipitation was performed with ab21623. Western blot was performed to detect the immunoprecipitated p53 with anti-p53 antibody.

References

This product has been referenced in:
  • Xu D  et al. SIRT2 plays a novel role on progesterone, estradiol and testosterone synthesis via PPARs/LXRa pathways in bovine ovarian granular cells. J Steroid Biochem Mol Biol 185:27-38 (2019). Read more (PubMed: 30009951) »
  • Joung H  et al. Sumoylation of histone deacetylase 1 regulates MyoD signaling during myogenesis. Exp Mol Med 50:e427 (2018). Read more (PubMed: 29328071) »
See all 35 Publications for this product

Customer reviews and Q&As

1-10 of 11 Q&A

Answer

Thank you for contacting us.

Technically yes!, the antibody should be able to pull down the peptides with acetylated lysine.

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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Answer

Thank you for contacting us.
The immunogen for ab221623 is chemically acetylated kehole limpet hemocyanin (KLH) and the antibody was affinity purified with Ne-Acetyl-L-lysine on Agarose.
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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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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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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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.

Read More

Answer

Thank you for your reply.

Since the antibody you bought is not from our catalogue, I cannot comment of the suitability for your planned experiments.

If this antibody is tested and guaranteed (as we do with our antibodies) for IP and CHIP, then this antibody should most probablybe fine. But of course I will not be able to guarantee this and therefore suggest to get in contact with the company that produces this antibody and requestmore information on it.

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

Read More

Answer

Thank you for your inquiry.

I am assuming that before subjecting the samples to LC-MS/MS, the enrichments of the acetylated lysine containing peptide is planned to be done with immunoprecipitation?

I am happy to confirm that we offer two antibodies specific for acetylated lysine that are tested and guaranteed for IP (ab21623 is also CHIP tested and guaranteed).

Please follow these links to review the respective datasheets:

ab21623

https://www.abcam.com/index.html?datasheet=21623 (or use the following: https://www.abcam.com/index.html?datasheet=21623).

ab80178

https://www.abcam.com/index.html?datasheet=80178 (or use the following: https://www.abcam.com/index.html?datasheet=80178).

We also have extensive protocols for IP on our homepage:

https://www.abcam.com/index.html?pageconfig=resource&rid=11385

https://www.abcam.com/index.html?pageconfig=resource&rid=11382

I am not especially familiar with the requirements that samples have to adhere to for LC/MS/MS. But maybe elution of protein with little antibody contamination is preferable? Therefore I suggest the following protocol:

https://www.abcam.com/index.html?pageconfig=resource&rid=11415

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

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Answer

Merci de nous avoir contactés.

L'anticorps ab21623 est en effet un bon choix pour l'immunoprécipitation de peptides acétylés. Nous n'avons pasd'information supplémentaire sur le rendement de cet en anticorps en IP, cecidoitêtre déterminé par l'utilisateur.

ab21623 est un IgG de lapin, nous recommandons donc l'utilisation de billes couplées à laprotéine A. Malheureusement nous n'avons pas encore ce genre de produit à notre catalogue.

Je vous envoie séparément à cet email un devis pour une unité de ab21623.

J'espère que ces informations vous seront utiles. N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions.

Read More

Answer

Thank you for contacting Abcam.

The antibody ab21623 has been tested on acetylated histone, acetylated BSA, and acetylated MB and has no reaction to the non acetylated proteins. There are alsoafew references on our website that have used this antibody in ChIP and so you may be able to find more information there:

https://www.abcam.com/acetyl-Lysine-antibody-ChIP-Grade-ab21623-references.html



Please let me know if there is anything else I can help you with.

Read More

Answer

Thank you for your patience in this matter. The originator of ab21623 (acetyl Lysine antibody - ChIP Grade) has confirmed that this product recognizes mono, di, tri- and multi-acetylated proteins (on lysine residues). The more acetylated residues are in a protein (on lysine residues), the greater detection sensitivity. I hope this information helps. Please don't hesitate to contact us again if you have further questions.

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1-10 of 11 Q&A

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