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Thank you for the prompt reply. Here are the answers to your questions:
1) Yes, I have evidence from the literature that show that Epac1 is present in detectable levels (via Western blot and immunofluorescence) in humanendothelial progenitor cells. There havebeen some papers that specifically use the abcam Epac1, which is why I chose it in the first place.
2) I am using human endothelial progenitor cells. They are primary culture cells and not a cell line.
3) I block in 5% milk/TBST for the western and the antibody is diluted in 1% milk/TBST.
4) The transfer from the gel to the blot is successful and Iam confident in the secondary as it has worked in the past and it worked for other bands in the gel.
I am open to any suggestions or a replacement from a different lot. Thanks for your help.
Asked on Mar 27 2012
Thank you for your thorough reply. One other question I meant to ask was how much sample you load per lane of the gel. We recommend at least 20ug per lane, especially if no signal is evident.
Assuming you are loading enough sample, I suggest trying a different antibody as a free-of-charge replacement for ab21236. We do have another lot of ab21236 but there have been other reports of batch inconsistency and I think a rabbit monoclonal such as ab109415 will be a better choice for you. Please let me know if you would like to try this.
Click here (or use the following: https://www.abcam.com/index.html?datasheet=109415).
Answered on Mar 27 2012