For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
If you continue without changing your cookie settings, we'll assume you’re happy with this.
I ordered Anti-Epac1 antibody (ab21236). I have since tried to use the antibody but am unable to get a signal. I have used the entire range of concentrations listed on the abcam websiteand in the primary literature.I have tried to use the antibody for both immunofluorescence and western blotting on endothelial cells. In the immunofluorescence, I also stained for another molecule and got a signal there, but no signal for the Anti-Epac1. In the western blot, I blotted for 3 other proteins and they all showed up on the blot, but the Epac1 band was not present. Based on my troubleshooting, I think the problem may lie with the antibody itself. Could you provide me with assistance on this matter?
Asked on Mar 26 2012
Thank you for bringing this to our attention. I am sorry to read that the antibody is giving you unexpected results in these two applications.
There may be a modification I can suggest to improve your result but it may be the case that, as you suspect, the antibody is defective. The other possibility, assuming your protocol is correct, is that the samples do not contain Epac1, or in amounts too low for the the antibody to detect. Do you have any other evidence, your own or from the litereature, that your cells contain Epac1?
For our records, could you please answer a few questions about your protocol?
Can you please tell me the species of the endothelial cells, and their origin? That is, is this a primary culture, or a continuous cell line?
Can you please tell me what you used for a blocking solution for the western blotting, and the antibody diluent?
Are you confident the transfer from gel to blot was successful? Finally, are you confident that the secondary antibody is effective?
If I cannot offer any suggestions to improve the protocol, and you are confident the cells express Epac1 protein, I think it will be best to replace the antibody, either with a vial from a different lot, if available, or a different Epac1 antibody. This replacement will be free-of -charge, assuming your samples are of one of the species listed on the datasheet (Human, Mouse, Rat and Rabbit).
Answered on Mar 26 2012