Anti-BRCA1 antibody [MS110] (ab16780)

No staining of formalin-fixed paraffin-embedded breast carcinoma tissue sections

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number ******. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.  

LOT NUMBER GR36461 ORDER NUMBER 897575 DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE whole cell lysates human RPE cells PRIMARY ANTIBODY Concentration or dilution 1:100 ~ 1:1000 Diluent buffer 5%skim milk + 10%sodium azide or 5% BSA Incubation time overnight Incubation temperature: 4c Washing Buffer: 1x TBST Number of washes 3 times (10min each time) DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED - ANTIBODY STORAGE CONDITIONS -20c SAMPLE PREPARATION Lysis buffer : HEPES lysis buffer Protease inhibitors: Aprotimin, Leupepin, pepstatin A, NaF, PMSF Phosphatase inhibitors: Phosphatase inhibitor cocktail 3 (sigma). Reducing agent: 6X sample buffer Boiling for ≥5 min? yes AMOUNT OF PROTEIN LOADED 20ug/lane ELECTROPHORESIS/GEL CONDITIONS Percentage of gel : 10% SDS-PAGE gel TRANSFER AND BLOCKING CONDITIONS Type of membrane Nitrocellulose Protein transfer verified Blocking agent and concentration 5% Skim milk Blocking time 30min Blocking temperature room temp SECONDARY ANTIBODY WashingBuffer: 1x TBST Number of washes 3 times (10min each time) Washing after primary and secondary antibodies: Washing Buffer: 1x TBST Number of washes 3 times (10min each time) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? antibody dilution factor and incubation time ADDITIONAL NOTES

ANSWER:

Thank you for your enquiry regarding ab16780and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody. Additionally, thank you for supplying an image, as this has been very beneficial in understanding your concerns.

The protocol looks fine to me however I would like to make few comments/suggestions that might help to improve our results and that you could consider trying:

- I would recommend using positive control lysates from breast cancer, prostate cancer or ovarian cancer cells. These cell lines show very high expression of BRCA1 protein. - Try switching lysis buffer from HEPES to RIPA. - I would also recommend using 2X loading buffer instead of 6X. - Use 0.1% sodium Azide if there is requirement else you can avoid using it.

I also would like to ask few questions which would help me identify the source of the problem;

- What was the secondary antibody used? - At what dilution secondary was used and how long did you incubate?

I hope this will be useful for you. Should you still have any problem with this antibody after following my suggestions, and then please do not hesitate to contact our Technical Department again.

I am studying the chicken meiosis and I would need to know the specific secuence of the synthethic peptide of the Brca1 antibodies (ab16781 and ab16780) to decide the buying of these antibodies. thank you in advance

ANSWER:

Thank you for your enquiry.

Unfortunately, the only information we have is that the target epitopes are within the N-terminal 304 amino acids of BRCA1. No further characterization was done in-house, and I have not been able to identify citations of more extensive characterization.

I am sorry I can't be of more assistance in this occasion. Should you need any further information, please do not hesitate to contact me.