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It's been a little while since I emailed you, so to refresh your memory I'm Andrew Knapp from Dr. Pilar Alcaide's lab. We're moving back toward VAP-1 western blotting experiments, and we're again having issues with finding VAP-1 in lysate tissues. I wanted to write you with the problems we've been having, and see if there were any solutions or ideas you might have.
1) When running our samples against the lysate you sent us as a control, our samples come out around 75 kDa and the lysate has a band around 37 kDa. Neither of these are in the 85-100 kDa range that your website suggests for VAP-1.
2) When running immunoprecipitate samples, we see a band around 150 kDa. Have you ever done this or seen this result?
3) Do you typically recommend running in reduced or non-reduced sample buffer? We have been running them in reduced sample buffer, and were not sure if that makes a difference.
4) Would it be possible for us to have another lysate from you to use as a control? You originally sent us a lung cell lysate (ab29297) to try. Could we have another of these, or is there a different lysate you have been able to detect VAP-1 in that might work for us?
Asked on Aug 15 2012
Thanks for your email.
The data you are seeing do appear unexpected.
Reducing conditions are used during western blotting.
Before I believe a loading control western blot was producing expected banding pattern so I don't think this is an issue with the lysates. More likely it appears to be an issue with the antibody.
Have you used any other anti-VAP-1/AOC3 antibodies to test for reactivity via western blotting?
Answered on Aug 15 2012