Anti-Rad51 antibody [51RAD01] (ab1837)

BATCH NUMBER 169563 ORDER NUMBER 152012

DESCRIPTION OF THE PROBLEM Immediately after arrival, worked fine for Western. A week later the aliquot at 4 deg tested for IF but gave no signal. A couple of days ago we tested fresh aliquot from - 20 deg in Western but obtained no signal. The blots were also stained with two other antibodies and worked fine. Exactly the same problem occurred twice with this antibody bought by my collaborators in Sofia, Bulgaria, within the last 6 months.

SAMPLE HeLa and PC3 cell whole cell extracts for Western. HeLa and PC3 cells irradiated with X-rays tested for IF.

PRIMARY ANTIBODY 1:1000 dilution in TBS, overnight incubation at 4 deg. Washed in TBST, 5 times for 5 min.

DETECTION METHOD Licor Odyssey infrared scanner.

ANTIBODY STORAGE CONDITIONS 1 aliquot stored at 4 deg ALiquots at -20 deg

SAMPLE PREPARATION Cells resuspended in CSK buffer with leupeptin, pepstatin, DTT and complete protease inhibitor (Roche). Standard SDS gel loading buffer, heated at 100 deg.

AMOUNT OF PROTEIN LOADED At least 20 ug loaded per lane.

ELECTROPHORESIS/GEL CONDITIONS Reducing SDS PAGE, 12.5%.

TRANSFER AND BLOCKING CONDITIONS Semi-dry transfer, 25V, 45 min. Blocking: 5% dry skimmed milk in TBS, 1h room temp.

SECONDARY ANTIBODY AlexaFluor 680 conjugated goat anti-mouse IGG, MOlecular Probes. 1:2000 dilution.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? There is no point to alter steps as the protocol we used at first gave good result. Moreover, we did not get immunofluorescence signal with this antibody. Attempts to use lower dilutions were carried out by my collaborators, who experienced similar problems with this antibody in Bulgaria, were unsuccessul.

ADDITIONAL NOTES We have used this antibody extensively in the past. In fact, you are citing two of our papers in the information provided for this antibody. Problems started after you started shipping a diluted version of the antibody.

ANSWER:

I'm very sorry to hear you have experienced problems with ab1837 and thank you for providing your protocol details. I have forwarded your complaint to the source of this antibody for their feedback and take your comments extremely seriously.

Would you like a replacement vial or would you prefer a refund?

I look forward to hearing from you and apologise for this problem,

What is the concentration of this antibody? What dilution has been tested in IF?

ANSWER:

Thank you for your enquiry. The concentration is 100 ug/ml. The antibody has been used at 1/25 to 1/50 using the ABC method. You may wish to start at 1/25 and further dilute from there if necessary.

I hope this information helps. Please do not hesitate to contact us if you need anything furhter.

As you suggested, we have tried the Rad 51 antibody (ab1837) dilluted to a titer of 1:25 with cells fixed in acetone, we also blocked with 10% of normal goat serum. the antibody shows negative results. please let me know if you have any other sugestions or if we can get a refund of this product thank you

ANSWER:

Thank you for contacting us.

I am sorry that we were not able to improve your results to your satisfaction. I have asked our accounting department to issue you a full refund. I hope that this experience will not prevent you from purchasing a product from us in the future as we have many quality products.

If you have any further questions concerning your refund, please contact the accounting department at accounts@abcam.com.

The technical team is always at your service, should you require further expert advice.

Best of luck with your research.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER CA082382

DESCRIPTION OF THE PROBLEM No staining

SAMPLE we test the antibody on cells from mouse testis

PRIMARY ANTIBODY abcam/mouse/PBST +1%BSA/1:25/OVERNIGHT

DETECTION METHOD flurescence microscopy

POSITIVE AND NEGATIVE CONTROLS USED positive control: cells in pahytene stage Negative control: samples without primary antibody

ANTIBODY STORAGE CONDITIONS +4 short term storage -20 for long term storage

FIXATION OF SAMPLE we tried different moethods:parafoaldehyde and paraformaldehyde+ SDS

ANTIGEN RETRIEVAL heat mediated techique

PERMEABILIZATION STEP triton 0.5% +saponin 0.1%

BLOCKING CONDITIONS PBST+1.5% BSA /3 TIMES 30 min

SECONDARY ANTIBODY JACKSON IMMUNORESEARCHLAORATORIES/GOAT/PBST+1.5% BSA/2h washed with PBS 3 times 5 min each

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

I'm sorry to hear you are having a problem with ab1837.

You are using the positive control so you should see a signal. I would like to suggest the following modifications to your protocol: 1) I am concerned that the instructions for using the antibody explicitly state "This antibody may be diluted to a titer of 1:25-1:50 in an ABC method". I would recommend using a biotin conjugated secondary and the ABC method. 2) Are you using paraffin embedded tissue? If not, then you do not need to perform antigen retrieval. 3) If you are using cells, you can fix in acetone and then do not need a permeablization step. 4) I would also recommend trying to block in 10% normal goat serum for 30 min at RT, incubate in primary in PBS for 30 min at RT, and secondary also for 30 min at RT.

Please let me know if this helps and do not hesitate to contact us for further advice.

What is the concentration? Abcam lot 116934.

ANSWER:

The concentration is 100 ug/ml.

Please contact us again if you have any additional questions.