Anti-Histone H4 (acetyl K91) antibody - ChIP Grade (ab4627)

I have purchased ab4627 antibody from you. This antibody is stored in a buffer containing 1% BSA. I would like to have this antibody without BSA for a cross-linking experiment. Could you supply this? I would greatly appreciate if you could formulate the storage buffer without BSA. Please let me know about the availability of this antibody without BSA. Thank you very much.

ANSWER:

Thank you for your enquiry regarding ab4627. Unfortunately, we don't supply a non-BSA storage buffer solution version of this antibody. We are also not able to formulate the storage buffer without BSA. Sorry for any inconvenience that this may cause to you. If you require any further information, please contact us again and we will gladly assist you.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 54252

DESCRIPTION OF THE PROBLEM This antibody is purported to specifically recognize histone H4 that is acetylated on lysine 91. On western blots, the antibody recognizes histone H4 from a wild type strain. When the antibody is used to probe histones from a strain in which the H4 lysine 91 is mutated to alanine the signal decreases but does not disappear completely. When histones from a strain carrying a lysine 91 to arginine mutation are tested, the signal is nearly indistinguishible from wild type. This clearly indicates that this antibody is not recognizing acetylated lysine 91 and therefore, invalidates results that would be obtained by use of this antibody.

SAMPLE Purified histones from S. cerevisiae

PRIMARY ANTIBODY 1/150 dilution of Ab4627 in TBS-T incubated O/N at four degrees. Blot washed 4X.

SECONDARY ANTIBODY AP-linked anti-rabbit secondary diluted 1/1000 in TBS-T for 2 hours at room temp. Washed 4X.

DETECTION METHOD BCIP-NBP from Sigma

POSITIVE AND NEGATIVE CONTROLS USED wild type and mutant yeast histones.

SAMPLE PREPARATION histones purified from a whole cell extract. The samples are heated in SDS-PAGE load dye for 5 minutes prior to loading on gel.

TRANSFER AND BLOCKING CONDITIONS Semi-dry transfer for approximately one hour in Tris/glycine/methanol transfer buffer. Blot is blocked using TBT-T with 5% milk.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? ~3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No

ANSWER:

I have consulted our laboratory manager and this is her reply: "This could be a problem caused by not cross-affinity-purifying against the non-modified peptides (and therefore low-level cross-reactivity with the non-modified peptide). We now do cross-affinity purifying as standard but the batch pre-dates this. I would like to re-purify and then send you a new vial free of charge".

I understand this delay can be frustrating but would very much like to help you and also improve the quality of this antibody. Can you please let me know if you would be interested in waiting for a more purified antibody? If so would you be able to send us some of the mutated histones so we can confirm the specificity of the new antibody?

I look forward to hearing from you,