Anti-NFkB p100 / p52 antibody - ChIP Grade (ab7972)

I am interested in purchasing anti-NFkB p52 antibody (ab7972) in a carrier-free formulation (no gelatin, no BSA, no sodium azide, no Tris), ideally in PBS alone. I need a minimum of 300 ug. Would you be able to provide us with this antibody in the formulation that we need ? I assume, based on the antibody description found on your web site that the antibody has been purified. If, so can you please send us a quote ? Could you please also indicate what will be the delay for delivery. Thank-you very much

ANSWER:

Thank you for your enquiry. We do not supply this antibody in a carrier free formulation I'm afraid. You should be able to dialyze the antibody to remove the azide and the antibody already comes without BSA or Tris (it is in PBS buffer)). The antibody has indeed been affinity purified.

Please do not hesitate to contact us again if you have further questions on our products,

BATCH NUMBER 88970 ORDER NUMBER 050425

DESCRIPTION OF THE PROBLEM No signal, Insoluble material was obsereved.

SAMPLE cell extract from murine B cell

PRIMARY ANTIBODY tried various concettration, for 2 hours at room temp wash 4 times with PBS+0.1% tween

DETECTION METHOD ECL plus

POSITIVE AND NEGATIVE CONTROLS USED Positive control used in this experient was B cell lysate.

ANTIBODY STORAGE CONDITIONS at 4 degrees, but -20 degrees on delivery

SAMPLE PREPARATION Lysis buffer (1% TritonX-100, 0.1% SDS, 50mM Tris Hcl, 150mM NaCl) with heating

AMOUNT OF PROTEIN LOADED equal to 10e4 cels

ELECTROPHORESIS/GEL CONDITIONS Reducing, 10% gel

TRANSFER AND BLOCKING CONDITIONS 5% skim milk

SECONDARY ANTIBODY Anti rabbit IgG-HRP conjugated from Amersham, for 2 hours at room temp Wash 4 times with PBS+0.1% Tween

ADDITIONAL NOTES This is an inquiry from Cosmo in Japan. Our customer would like to try another lot of this antibody. Please let me know how we advise him.

ANSWER:

I'm sorry to hear that your customer is having a problem with ab7972.

I would like to suggest the following modifications to your protocol: 1) the datasheet specifically states "do not freeze", therefore freezing the antibody may have been a problem. Gently finger-vortex it before using it and 2) use the antibody at 1:200 overnight at 4C, and 3) load more cells onto the gel, for example 10e6 cells.

Please let me know if this helps and do not hesitate to contact us for further advice,

Sorry I did not have time to e-mail you last week. Thanks for your reply. I have followed your suggestions for changing the western blot conditions. Unfortunately, they only helped in detecting the incorrect 30kD band. In fact, at this point I'm really not having any trouble with background at all - I'm just very specifically recognizing a band of the wrong size. I would love to have a positive control, however all the ones that you have mentioned are lysates of human cells. I am working with mouse cells, and would therefore like to have a mouse cell lysate positive control. As far as I can tell, your technical literature says that this antibody should recognize mouse p52.

ANSWER:

We have asked the source of ab7972 for some suggestions and here is what they say:

" NFkB p52 is a tricky devil! I wish I had a readily available mouse cell line positive control, but I do not. The best mouse lysates are thymocytes that have been stimulated with PMA and PHA for 6 hours. It really should not matter if the positive control you are using is a human cell line, because the immunogen is taken from the mouse p52. But if you have to have a mouse positive that is the best I can suggest, besides recombinant protein. I hope this helps."

We have in our catalogue some cell lysates of Jurkat, Daudi or Raji cells, I would like to help you and send you a vial free to test the antibody on those, which one would you prefer?

I look forward to hearing from you,

Follow-up to previous correspondance:

Thank you for your suggestions. I will try them, however I have reservations about whether they will work. The suggestions seem more inclined to give me a signal where I see none, rather than getting rid of an intense wrong signal. In fact, as I stated in the query, I see no relevant signal at all at the right size - neither at the 100 nor at the 52 kD marks. I also noticed that when I sent the query, three previously asked questions about the antibody popped up. One of the answers to those questions stated that this antibody recognizes the phosphorylated version of the protein and that this is why you would counsel against using milk as a blotting agent. Nowhere in your datasheet does it say that this antibody recognizes a phosphorylated version of p52, otherwise I would not have bought it. Furthermore, p52, as far as I know, is not phosphorylated; its preprocessed form, p100, is phosphorylated on its c-terminus prior to being cleaved to produce p52 and a c-terminal product that is then degraded. Please let me know if this information about phosphorylation was written in error. Sincerely,

ANSWER:

Thank you for your enquiry.

We are not sure how the statement about the phosphorylation of NFkBp52 became linked to this antibody, but everything that you say is true, only the p100 form possesses residues that can be phosphorylated (however the datasheet states that the antibody recognises both p52 and p100). We have confirmed this with the source of ab7972 and they cannot find anything that states that this antibody is phospho-specific.

They were also able to suggest a few more modifications to your protocol:

"the main difference between their blotting and ours is the Pierce super signal. We use standard ECL. To be honest I have found that the super signal can be very touchy, and if conditions are not just right you can get very dirty blots. Altering the blocking conditions will help eliminate some of the non specific bands, but other adjustments may be needed to help detect p52. I have done some searching and I have not found anyone publishing the detection of p52 in RAW264.7. Could it be that p52 is being expressed at low or un-detectable levels in the lysate? A positive control of Raji, Daudi or Jurkat lysates may be useful. Another detail that might help is that we used PVDF membranes when testing the antibody".

We hope this information will be useful, please do not hesitate to contact us if you need further advice,

BATCH NUMBER 48989 ORDER NUMBER 58248

DESCRIPTION OF THE PROBLEM Non-specific band. I'm seeing multiple bands, none of which correspond to p52. The strongest band, by far, runs at about 30kD. The other very weak bands are only detected after 6 minutes of exposure.

SAMPLE mouse RAW264.7 whole cell extract

PRIMARY ANTIBODY ab7972 diluted in blocking buffer 1:500. Incubated 1 hour. Washed 3 times in TBST.

SECONDARY ANTIBODY goat anti-rabbit (Jackson Laboratories) 1:10,000 in blocking buffer. Incubated 1 hour. Washed 3 times in TBST.

DETECTION METHOD Pierce supersignal west pico

POSITIVE AND NEGATIVE CONTROLS USED p65, cRel, and RelB were all detected in this same extract using the same secondary antibody in the same experiment.

ANTIBODY STORAGE CONDITIONS 4 degrees

SAMPLE PREPARATION diluted in 4X sample buffer to 1X, also added DTT to 100mM, and boiled 5 minutes.

AMOUNT OF PROTEIN LOADED 30 micrograms

ELECTROPHORESIS/GEL CONDITIONS 10% SDS-PAGE

TRANSFER AND BLOCKING CONDITIONS Transferred in 1X transfer buffer at 100V for 1 hour, with ice block. Blocked in TBST with 7.5% milk for two hours.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? none

ANSWER:

I'm sorry to hear you are having a problem with ab7972.

I would like to suggest the following modifications to your protocol: -block the membrane in 5% BSA (filtered) in TBST for 1hr -incubate overnight the primary antibody at 4C in TBST -wash extensively the membrane in TBST -add the secondary antibody diluted in TBST (no milk).

Please let me know if this helps and do not hesitate to contact us for further advice,