Anti-Mad2L1 antibody (ab24588)

Sorry for the delay in replying, but I have now had a few opportunities to test the replacement Mad2 antibody, with success. The kinetochore signal is certainly quite weak, compared to other similarly localised proteins, but I was still able to get the imformation I required. I will now submit an Abreview for this antibody.

Regards,

ANSWER:

I am very happy to hear the replacement vial worked for you and look forward to reading the Abreview. Please do not hesitate to contact me if I can be of further help.

I wish you all the best with your research,

We want to stain chromosomes with MAD2 anti body.Is immunoflouroscent more sensitive than ABC.

ANSWER:

Thank you for your enquiry.

In general we recommend that users apply the ABC technique as this amplifies the signal significantly. However, on this occasion the antibody has been optimised using fluorescence detection and therefore I would like to recommend this approach and the following protocol:

http://www.abcam.com/index.html?pageconfig=protocols&pid=460&intAbID=24588&strTab=protocols&mode=prot

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

BATCH NUMBER 164635 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM Non-specific staining I get a diffuse cytoplasmic staining, and fail to see localisation of the portein to kinetochores during mitosis.

SAMPLE breast cancer cell line

PRIMARY ANTIBODY 1/100 Mad2 antibody in appropriate blocking buffer for 1h (or, when 3% BSA was uswed for blocking, antibodies were diluted with 0.6% BSA/PBS) wash: 3x 3 min PBS

DETECTION METHOD fluorescence microscopy

POSITIVE AND NEGATIVE CONTROLS USED none

ANTIBODY STORAGE CONDITIONS stored in aliquots at -20deg

FIXATION OF SAMPLE have tried numerous fixation/staining procedures, including: 4% paraformaldehyde 10 min Ice cold ethanol 10 min

ANTIGEN RETRIEVAL none

PERMEABILIZATION STEP have tried numerous steps associated with paraformaldehyde fixation, including: 0.2% tween 20 min 0.5% triton X-100/PBS 5 min 0.1% SDS/PBS 5 min permeabilisation prior to fixation: 0.1% triton X-100 for 30 sec (done in PEM microtubule stabilising buffer)

BLOCKING CONDITIONS various tried, all in PBS: 3% BSA 30 min 0.1% BSA 5 min 5% milk/0.2% tween 1h

SECONDARY ANTIBODY 1/50 polyclonal anti-rabbit immunoglobulins 30 min (dako) - I successfully use this antibody to detect many other primary antibodies

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 7 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? described in detail above

ANSWER:

I have just received feedback from the source of ab24588 and can confirm that the laboratory would expect the antibody to work in one of the many conditions you have tested. I suspect the antibody was damaged during storage or shipping and can confirm that we have not received any other complaint about this popular antibody. I was able to source the protocol which was used to test ab24588 in ICC and it is now available on the datasheet online.

I would like to offer you a replacement vial or refund, whichever you prefer, if the antibody was purchased in the last 90 days. Can you please let me know which you would prefer and the order details and I can arrange this immediately?

I look forward to hearing from you,