Anti-Rad51 antibody [14B4] (ab213)

I need your help to address the problem, the customer us Abcam( cat #: ab213) primary antibody for western blotting, and the experiment didn’t work, could you help him find out what is the problem? Please see the attached for the protocol he follows ,



Thanks a lot , your quick response will be greatly appreciated!

ANSWER:

Thank you for contacting Abcam.

I just had a few more protocol questions for you regarding the problems you are having with ab213 in western blot.

1 - Why was 2 different lysis buffers used in the extraction protocol? Either of them should have been suffcient on their own.

2 - Whenyou say that the wetern blotting did not work. Does that means there was no signal, high background, weak signal?

3 - Could you please provide the lot number of the antibody you are using?

I look forward to your reply and helping resolve this situation.

Thank you so much for your help. I will sent you update of the ab63801. And the secondary antibody we used was 1:2000 for ab213.

ANSWER:

I am happy to help. Best of luck in your experiments!

I have used this antibody to stain MCF7 cells successfully via ICC/IF but WB with MCF7 lysate from 50ug protein extract, with primary at 1:250 and 1:500, barely showed a band after 45' exposure

ANSWER:

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 for one vial of ab63801.

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.

ICC: sees big red blob in nucleus (correct location) MCF7 cells, treatment with mitomicine C Ab:1/500 1h secondary: 1/200 Texas red (goat anti-mouse), works well otherwise

pre-extraction with 0.25% TX fixation: 3% PFA perm step: 0.5% TX no separate blocking step, but done with primary incubation in 3% BSA

suggestions: use less antibody (secondary, maybe also primary), include separate blocking step, omit pre-extraction step)

ANSWER:

Thank you for contacting us. I am sorry to hear that the antibody is giving problems.

As we discussed yesterday over the phone, the following suggestions might help to get rid of the red blob you are seeing in the nuclei.:

1) use less antibody (for the secondary: e.g. 1/500 or even 1/1000, maybe also for the primary: e.g. 1/1000) 2) include a separate blocking step (e.g. 3-5% BSA or 10% goat serum) 3) omit the pre-extraction step

Please let me know if these if these suggestions are of help or not. If the tips do not help, please let me know your PO or order number. If the product was purchased within the last 6 months, we can discuss a free of charge replacement, credit or refund according to our Abpromise guarantee.

I wish you good luck with your experiments and look forward to hear back from you.

We would like to use Anti-RAD51 for IF and we would like to know if there is at your solution any carrier protein and if you can produce anti-RAD51 without any additional carrier protein. Also if you are able to manufacture an Anti-RAD51 with a GFP / RFP conjugation? What will be the additional cost for these request's? Thank you very much for your time.

ANSWER:

Thank you for your inquiry. The Rad51 antibody [14B4] ab213 is supplied in Phosphate-buffered saline, pH 7.4, containing no preservatives or stabilizers.

Unfortunately we are only able to provide this product in the unconjugated form indicated on our datasheet.

I'm sorry I cannot help you more on this occasion.