Anti-Jarid2 antibody (ab48137)

I am using your ab48137 (Jarid2 antibody) for WB. Besides 3 bands you show in your datasheet, I always got more than 10 bands, and the band at the right size is weaker than others. Also I got dirty background. my samples are nuclear extract from lyphoma cell lines, I used PVDF membrane, and I tried block the membrane with either 5%NF-dry milk or 3%BSA. I loaded 20ug of sample per well, and the samples were denatured too. Please help me to improve my results. Thanks a lot.

ANSWER:

I am sorry to hear that you have been experiencing problems with ab48137 in WB.

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 credit note/refund will be offered.

If you would like some technical support, I have listed some common reasons to why multiple bands are observed.

In most cases, the cause of multiple bands is because the primary and/or secondary antibody concentration is too high and causes non-specific binding. The dilution that we have on the datasheet is a recommended starting dilution and customers are encouraged to determine the optimal concentration/dilution. Please try decreasing the primary (1:2000 ~ 1:5000) antibody concentration. You can also run a no-primary control (without the primary antibody). Running a no-primary control will be able to eliminate the possibility that your secondary antibody is binding non-specifically.

What are the band sizes you obtained? The bands located above the expected molecular weight band could be multimers. Please try boiling your protein in SDS-PAGE for 10 minutes rather than 5 minutes to ensure proper disruption of multimers. This will ensure the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody. Meanwhile, bands located below the expected molecular weight may indicate that your target protein has been digested. Please make sure that you incorporated sufficient protease inhibitors and have kept your samples on ice the whole time.

I am afraid that without more information on the protocol you used, I am not able to provide any more suggestions. Should you require further assistance, please spare some time and fill up our technical questionnaire, which will help you gather you protocol and allow us to provide more help. I have attached the WB link below for your convenience.

http://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=48137&mode=questionaire

If there is any thing else that I can help you with, please do not hesitate to contact me.