Anti-Rad51 antibody (ab63801)

No signal. After irradiation (8Gy) he couldn’t confirm Rad51 foci formation induced by DNA damage in the nucleus. Whereas he confirmed gamma H2AX in the same condition and used same secondary antibody.
1) Abcam product code
ab63801
2) Abcam order reference number or product batch number

2-1) storage temperature of antibody
4℃
3) Description of the problem
After irradiation, Rad 51 was not stained at Double strand breaks of chromosome DNA,
but cytoplasm.
4) Sample preparation:
Species Human
Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed
paraffin embedded sections, cells in culture, other:
Sample preparation
Positive control HeLa Cell+Irradiation (8Gy)
Negative control HeLa Cell + No irradiation
5) Fixation step
Yes
If yes: Fixative agent Methanol and concentration 100%
Fixation time 5 minutes
Fixation temperature -20℃
6) Antigen retrieval method No
7) Permeabilization method:
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution
buffers? We do not need permeabilization step because of methanol fixation, but we use
antibody dilution buffer contained 0.2% Triton-X 100 and washing buffer supplemented
0.2% Triton-X 100.
Permeabilizing agent and concentration: Triton-X 100 and 0.2%
8) Blocking agent (eg BSA, serum…): BSA
Concentration 1%
Blocking time 30 minutes
Blocking temperature Room temperature
9) Endogenous peroxidases blocked? No
Endogenous biotins blocked? No
10) Primary antibody (If more than one was used, describe in “additional notes”) : Anti-Rad 51
ANTIBODY (Rabbit)
Concentration or dilution: 1:100
Diluent buffer 1%BSA/0.2% Triton X-100
Incubation time Overnight at 4℃
11) Secondary antibody: Alexa Fluor 488 (A11008)
Species: Goat
Isotype: IgG
Reacts against: Rabbit
Concentration or dilution 1:500
Diluent buffer 1%BSA/0.2% Triton X-100
Incubation time: 1 hour
Fluorochrome or enzyme conjugate: Alexa Fluor 488
12) Washing after primary and secondary antibodies:
Buffer 0.2% Triton X-100
Number of washes 3 TIMES
13) Detection method Microscope (Fluorescence)
14) How many times have you run this staining? 3 times
Do you obtain the same results every time? Yes
Have you run a "No Primary" control?(yes/no) No
What steps have you altered to try and optimize the use of this antibody?

ANSWER:

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab63801 Anti-Rad51 antibody. I would also appreciate if you can confirm some further details:

1. Please be aware that non-radiated HeLa cells are not a fitting negative control, as they are given as a positive control on our datasheet. However, in this light it is even more odd that the antibody doesn't give the expected results and rather stains the actin filaments.

2.Triton can be quite harsh on some epitopes, therefore, youincrease the clarity of the staining by reducing the Triton concentration down to 0.025%, especially when you incubate over night.

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

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.

Dear Sirs,

I would like to order anti-RAD51 antibodies from you company (through

Sellex here in Brazil). A coleague used the ab46981 and it work against

Leishmania.

However, looking your catalog I found the ab63801, that was used to

labels focci induced by DNA damage.

My question is?

1- Are these two just the same antibody with different purification

steps (the first is whole serum and the second protein A purified) as

the same immunogen was used?

2- They both work binding to focci? (in the catalog figure you have the

46981 labels extensively the cell cytosol).

ANSWER:

Thank you for your enquiry. Regarding question 1: No, these are two different rabbit anti-Rad51 antibodies. Regarding question 2: Both antibodies bind Rad51 and should label foci of cells induced with DNA damage. The ICC image on the datasheet of ab46981 may be showing heavy staining on the edges of the nuclei, rather than cytoplasmic staining. Although I can't confirm that. I hope this is helpful. Please contact me again if you have any further questions.

I was very interesting in using your rabbit poly-clonal RAD51 antibody (ab63801)for immunoflouresence, however i have experienced a lot of difficulty with other RAD51 antibodies from other companies and was wondering if it would be possible if I could get a sample before i buy?

ANSWER:

Thank you for contacting us.

We do not have small samples of any of our antibodies but ab63801 is covered by our Abpromise guarantee for ICC on human, mouse, or chicken samples. If you do not obtain the results you expect (e.g., staining of nuclear foci) and you contact us within 6 months of purchase, and your protocol is similar to the recommended protocol linked on the datasheet, we will issue a replacement or credit or refund.

We have had two reports in the past two years of cytoplasmic staining with this antibody but that is a small percentage (<2%) of the total number of orders.

We have an ICC protocol linked on the online datasheet of the antibody:

http://www.abcam.com/index.html?pageconfig=protocols&pid=1248&intAbID=63801&strTab=protocols&mode=prot

The most recent publication for this antibody is:

Vaz F et al. Mutation of the RAD51C gene in a Fanconi anemia-like disorder. Nat Genet 42:406-9 (2010). PubMed: 20400963.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.