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I am using abcam antibody for Western blotting. Anti-GPCR GPR30 antibody (ab39742) Lot GR43581-1 However, as shown in attached file, we cannot see the bands for GPR30m while we can see may of non-spedific bands. Western sample from brain was prepared standard procedure and resolved in 1-% SDS-PAG gels. The cell was used for centrifuged supernatant of whole cell. To detect GPR30, membran fraction must be needed? In my opinion, we did not need such a preparation, because brain tissue lysate can be used for positive control as described in your datasheet.
Asked on Nov 28 2011
Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab39742 in western blot. Since you mentioned that you are getting expected results in immunohistochemistry but not in western blot (WB), the problem may be with the WB application. If you would instantly like a replacement with a different lot or product, I will be more than happy to arrange that for you. Please provide the name of your sub-dealer and sales person so that I can trace your original order number. If you would like some troubleshooting tips, here are some suggestions. 1) I can see from the image that the whole blot is rather dark and have streak of bands. Here at Abcam , we normally use 5% BSA to block membranes and do get cleaner images. If you have BSA in your lab, you may want to try it out. Also, performing a no-primary antibody experiment will be helpful in determining if your secondary antibody is causing the non-specific bands or not. 2) Although we normally recommend loading about 20-40ug of protein, this sometimes is more than enough and cases multiple bands and /or high background. If possible, could you try 10ug sample? 3) Lastly, boiling of sample is done at 98°C for 10 min to ensure that the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody. I hope the tips may be helpful. If not, then please do not hesitate to contact me. Also, please advise me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement to you. I look forward to hearing from you again in order to resolve the matter.
Answered on Nov 28 2011