Anti-DDB2 antibody [2246C4a] (ab51017)

BATCH NUMBER 33510 ORDER NUMBER 250825

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE Hela cells, 293T cells

PRIMARY ANTIBODY Abcam Ab 51017. Dilution was 1:1000 incubation for 1hr at RT. Also tried Dilution was 1:1000 incubation for ON at 4 degrees

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Hela and 293 extract where other proteins can be detected bu Western

ANTIBODY STORAGE CONDITIONS minus 20 degrees

SAMPLE PREPARATION treatment of cell pellet in SDS Loading buffer

AMOUNT OF PROTEIN LOADED around 50 microgram

ELECTROPHORESIS/GEL CONDITIONS Reducing, 12 % gel

TRANSFER AND BLOCKING CONDITIONS 5% milk , 1 hr at RT, TBST

SECONDARY ANTIBODY Anti Mouse HRP from Dako catalog # P0161.

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? No

WHAT STEPS HAVE YOU ALTERED? ON and 4 degrees incubation

ADDITIONAL NOTES PO # NII/IMP/161/07-08 Invoice # 1093681 Order Ref # 250825 We have tried this antibody atleast 5 times. it was received by us on July 27, 2007. Please replace this antibody with alternate working DDB2 antibody urgently.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you are experiencing difficulties with this product ab51017 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered.

After looking at the protocols you used, I am surprised that even 50ug of protein, you still did not manage to get any bands. However, it is very hard for me to determine the cause without more details of your WB protocol. I would therefore appreciate if you can please clarify the following items.

According to the datasheet, this product has a recommended dilution of 1:50. Please try this dilution as previous tests have shown that a higher than normal range is needed for this product to work.

Was the lysis buffer you used as strong as RIPA buffer? Please try RIPA buffer as it is the strongest and will ensure that you get all the protein of interest out.

Did you use reduce and denaturing conditions? Please reduce and denature the sample at 95oC for 10 minutes in buffer containing SDS and mercaptoethanol. This will ensure that the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody.

Most customers who use milk as a blocking agent tend to have problems detecting bands. At Abcam, almost all of our products have been tested with 5% BSA. Sometimes, a different blocking buffer might cause cross reaction between the blocking agent and antibodies, therefore causing weak bands or no bands at all. I would recommend trying 5% BSA, if you have not already done so. Also, cross reaction between the antibodies with the diluent could also cause the lack of signals. We would normally use 1% BSA to dilute the primary and secondary antibodies. You can also use PBST only.

Can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? In some cases, the problem may be with the secondary antibody not working.

Could the no signal you are experiencing be due to poor transfer of protein to membrane? Did you check the transfer efficiency? This can be simply done with a reversible stain such as Ponceau S.

I am sorry for the string of questions but I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me with details of your order shipping address/purchasing agent information. Also, please advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund to you.