Anti-Cyclin A antibody [E23.1] (ab38)

BATCH NUMBER 30233 ORDER NUMBER 68447

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE C2C12 myoblast and myotube

PRIMARY ANTIBODY abcam, 2ug/ml to 4ug/ml, 4 degree overnight, 3 washesX10mins

SECONDARY ANTIBODY 1:1000 to 1:2000 anti-mouse IgG HRP

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Hela cell lysate as positive control

ANTIBODY STORAGE CONDITIONS 4 degree

SAMPLE PREPARATION Standard cell lysis buffer

AMOUNT OF PROTEIN LOADED 100 microgram

ELECTROPHORESIS/GEL CONDITIONS 12% SDS-PAGE

TRANSFER AND BLOCKING CONDITIONS Tris-Glycine-Methanol

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? Primary and secondary concentration

ADDITIONAL NOTES When we probe a parallel blot with you ab2948 (polyclonal anti cyclinA) we got excellent result. We had problem with your ab39 too.

ANSWER:

I'm very sorry to hear you are having problems with ab38.

We do not have a recommended starting dilution so I would like to recommend trying a higher concentration of antibody (try 10ug/ml). We have only received one complaint regarding this popular antibody. I can certainly offer you a replacement vial of ab38 in case the antibody was damaged during transport, or a credit note or refund. I can also offer you a replacement vial of ab39 if you purchased this antibody in the last 90 days. If this is the case let me know the order details.

Please let me know which you would prefer and I will arrange accordingly.

I look forward to hearing from you,

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 12895470

DESCRIPTION OF THE PROBLEM No signal at all.

SAMPLE We used HeLa cell nuclear extract, A431 cell lysate (positive controls) and other tissue lysates, which all showed cyclin A2 expression on Western Blots with other primary antibodies targeting cyclin A2.

PRIMARY ANTIBODY We have tried 1:250 to 1:100 dilution and also tried incubation times varying from 1 hour at room temp. to overnight at 4'C.

SECONDARY ANTIBODY We used anti-mouse secondary antibody at 1:2000 to 1:5000, which worked well for other primary antibodies.

DETECTION METHOD ECL (Amersham)

POSITIVE AND NEGATIVE CONTROLS USED Antibody did not work on our positive controls (HeLa nuclear extract and A431 cell lysate) or samples.

ANTIBODY STORAGE CONDITIONS 4'C

SAMPLE PREPARATION as per standard protocol. We are experienced with performing western blots and have consistent results with other primary antibodies.

TRANSFER AND BLOCKING CONDITIONS We tried different blocking conditions.

WHAT STEPS HAVE YOU ALTERED? see above

ANSWER:

I'm sorry you are having problems with ab38 and ab39. It is possible that those antibodies have gone off during shipping or storage. I can send you replacements for those antibodies: ab38 a vial of the same lot ab39 a vial of a new batch

The label on ab38 will have a different batch number to the one you have, but it comes from the same batch of the originator.

Please let me know if you are happy with those replacements or would rather have a refund on one or both products. I look forward to hearing from you,

Your datasheet of the antibody AB38 to cyclin A shows two bands. Do you know what the second (smaller) band shows?

ANSWER:

Here is a detailed answer to your query regarding the antibody AB38 (E23.1) to cyclin A showing two bands.

Our scientist source of the antibody has replied as below: "Many years ago when I first looked in to this I thought that the larger protein (running slower than the intense one) might be full length cyclin A and the more intense band is actually a cleaved fragment. Immunoprecipitations of the cyclin A cdk2 complex with either anti cyclin A or anti cdk2 antibodies reveal that both bands are in the complex. A range of monoclonal antibodies (even those that do not react with cyclin A1) also blot these proteins which leads me to think that both of these bands are cyclin A2. Until there is evidence of another cyclin A in somatic cells I probably still have to conclude that both of these fragments are cyclin A2.

You may not see both of these fragments in some blots because it depends a lot on how good the antibody is at detecting cyclin A. Other antibodies which are not as good as ab38 may not blot the less intense band and this may lead one to think that only some antibodies are detecting the less intense band."

I hope this is useful, please do not hesitate to contact us if you need further information,