Anti-XIAP antibody [2F1] (ab28151)

Thank you for the quick follow up. The samples were lysed using lysis buffer consisting of a final concentration of 0.2% NP40, 20mM Tris, 2mM sodium orthovanadate, 10% glycerol, 150mM NaCl, and 1 tablet of Roche protease inhibitor/25ml of buffer. This is pretty standard for our lab and I have managed to IP other antibodies, such as caspase-3 as I have shown in the blot I sent previously. I used 30ul of Protein A magnetic beads purchased from Millipore and 4-6ul of XIAP antibody. Wash steps utilized the same lysis buffer used to lyse the cells. My problem with moving forward with the IP is that it appears the antibody doesn’t even detect the protein by Western blot (as compared to the antibody I purchased from a different company against XIAP speaking to the fact that it is not a problem of the lysate but of the antibody). If the negative finding by western blot using your antibody is at all indicative of the specificity of the abcam antibody to XIAP, I’m willing to bet that doing the IP with either Protein G beads or more antibody will not make a difference. Again, any suggestions will be helpful.

ANSWER:

Thank you for your reply. I recommended the Protein G beads because they bind more strongly to mouse IgG1 than Protein A does. The image you sent does seem to show the expected 50 kDa band in most of your samples when the Caspase 3 IP was probed with ab28151. If you are not satisfied with your results, I am more than happy to offer you a free of charge replacement with a new lot or a credit or refund. Please let me know how you would like to proceed and I will be happy to assist you further.

I am having difficulties immunoprecipitating XIAP as well as detecting it by immunoblot from mouse T cells with the Ms monoclonal anti-XIAP antibody clone [2F1] I obtained from your company. The catalog number is ab28151. I have attached the immunoblots for troubleshooting. The top blot shows an IP with anti-caspase-3 (denoted as C3) blotted with your XIAP antibody on the top and caspase-3 antibody on the bottom of the membrane. WCL denotes mouse whole cell lysate from T cells. This showed a band for full length caspase-3 but not XIAP (using abcam anti-XIAP). The bottom blot is an IP using the same lysates as the top membrane with your anti-XIAP antibody and immunoblotted with XIAP (from a different vendor) on the top and caspase-3 on the bottom. As you can see, XIAP was not detectable in the IP (suggesting that the antibody did not pull any XIAP down) but was detectable in the WCL and postdepletions. This detection was only made apparent using a different vendor’s antibody (which is from a rabbit host) to detect by Westerns. Unfortunately, this competitor’s antibody is not able to perform well for IP, which was why I purchased the XIAP antibody from abcam. Any help would be great.

ANSWER:

Thank you for contacting us. I am sorry that this antibody is not working in IP. Could you please provide some details about how your samples were prepared and what buffers were used? What type of beads did you use? I would recommend using Protein G beads with this mouse IgG1 antibody. How much antibody was used and how long was it incubated?I would recommend using ab28151 at a concentration of5 - 10ug/mL. I hope this helps, if not, please let me know and I will be happy to offer you a free of charge replacement, credit, or refund.

What size band is seen in Western blot with this antibody in monkey samples?

ANSWER:

Thank you for your enquiry.

The protein should be ~54 kDa.

Please let me know if I can be of further assistance.