Anti-p53 antibody (ab1101)

Does this antibody recognize both mutant as well as wild type form?

ANSWER:

Thank you for contacting us.

This antibody was made against recombinant full-length human p53 corresponding to the sequence of native human p53. Positive controls can be whole cell lysates of 293T cells or A431 cells. The specific epitope recognized by this antibody is at the N-terminal end aa 20-25, so depending on where in the protein a mutation occurs, this antibody may or may not recognize a mutant form of the protein.

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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A researcher wants to know whether p53 antibody (ab1101) reacts with wild or mutant protein. Could you please inform me about this?

ANSWER:

ab1101 has been developed against the recombinant protein and has alsobeen tested with the endogenous protein (see http://www.abcam.com/p53-antibody-ab1101.html#p53-Primary-antibodies-ab1101-2.jpg). Therefore this antibody recognises both recombinant and endogenous p53.

p53 mutants have not been tested.

It has also been determined thatab1101 recognises an epitope within aa 20-25 at the N-terminus of human p53.

sincerelly
Last month I asked ten anitbody with Kimera ltd in istanbulthere was a serious problem about a antibody ( ab1101-anti p 53 antibody) because mistakenly I asked this product (ab1101-anti p53) but I needed mutant p 53 antibody for my working project
I need a mutant p 53 antibody, does included this product namely ab1101mutantp 53 protein

ANSWER:

Thank you for contacting us.
Could you please contact and discuss this case with our distributor Kimera Medical Laboratory Supplies.
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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http://www.abcam.com/abreviews

BATCH NUMBER 95016 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM I am obtaining no signal whatsoever. I have used this antibody at x 200 and x500 dilution to probe nitrocellulose membrane with 20ug/lane upto 160ug/lane of HL60 cell lysate. Protein inhibitors were added on preparation of whole cell extract.

SAMPLE Whole cell extract prepared from HL60 cell line. (Untreated HL60 vs. HL60 treated with Sodium Valproate.)

PRIMARY ANTIBODY Incubation with primary ab (anti p53 - ab1101) for 1 hour with agitation at x200 and x500 and x1000 and x2000 dilutions. Diluted in 10% marvel TBS/Tween solution.

Three washes in 1xTBS/Tween solution (0.1% Tween) for 15minutes each.

SECONDARY ANTIBODY Incubation with secondary antibody (goat anti-mouse horseradish peroxidase) for 1hour at x1000 dilution in 10%marvel 1x TBS/Tween solution with agitation.

Three 15minute washes in 1xTBS/Tween.

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED None used.Just ran with Rainbow Molecular Marker

ANTIBODY STORAGE CONDITIONS -20 degrees C. Thawed only once so as to use.

SAMPLE PREPARATION Preparation of whole cell extract using lysis buffer (containing protease inhibitor tablets) - made up fresh.On loading of 10% sds gel samples were heated to 100 degrees for 10min and then kept on ice for 5min.

AMOUNT OF PROTEIN LOADED 20 micrograms per well upto 160micrograms per well.

ELECTROPHORESIS/GEL CONDITIONS 10% SDS gel.Transfer checked by Ponceau and found to be sufficient.

TRANSFER AND BLOCKING CONDITIONS Blocked in 10% marvel 1xtbs/tween (0.1% tween) for 1 hour at room temperature with agitation.

ECL reagents used to visualise bands.

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? Have altered the concentration of the antibody from x1000 dilution to x200 dilution. Have also loaded more cell lysate per lane.

ADDITIONAL NOTES At incubation stages with primary/secondary antibodies agitation is quite vigorous due to the speed of the machine being quite fast.Material is transferred sufficiently so it is present on the membrane. Is there a sufficient amount of p53 transcribed in HL60 cell line that should be detectable or would it be necessary to transfect with p53 constuct containing plasmid to ensure sufficient p53 levels are generated.

Also used the ab 4267 (rabbit polyclonal to p53 Acetyl K373 + K382 - this antibody picked up numerous bands when used at x 200 dilution/20ug per well of cell lysate yet only picked up a very faint band when used at x 500 and x1000 dilution/20 ug per well of cell lysate.The same procedure as described above was used. at x 2000 dilution no image was visualised.

ANSWER:

I'm sorry to hear you are having a problem with ab1101 and ab4267.

I would like to suggest the following modifications to your protocol:

-p53 is in general nuclear so I suggest a nuclear extraction to maximise the amount of protein in your samples.

-run a positive control of nuclear lysate of HBL100 cells along with your samples

-block the membrane in 5% milk in TBST (1hr, RT) (also try 5%BSA for the problem of high background with ab4267)

-wash for a few seconds in TBST

-incubate the antibody overnight at 4C in TBST (try 1:100-1:200)

-Use ECl+ rather than ECL as it has a higher sensitivity.

It is possible that indeed levels of p53 in your samples are below detection level by Western blotting, hence a positive control will help you determine if you have sufficient p53 protein in your samples.

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

Could I use this antibody in Western blotting to localize/visuakize p53 levels in cell culture media /cell samples after radiation treatments? Sincerely yours Raimo J Pollanen PhD

ANSWER:

This antibody is suitable for Western blotting using nuclear or whole cell lysates.