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I attached our previous run of the lysate you sent us next to one we had. The one we’ve been using still produces a 75 kDa band, rather than the 85-100 kDa band that your website lists as VAP-1. As you can see, the lysate you sent us produces a much lower band under the same conditions.
It’s possible that this 75 kDa band is VAP-1, and for whatever reason we’re getting a slightly lower band than we should. We would believe this is the case if we could corroborate it with a positive control, but the lysate you sent us didn’t work as planned, either with the original antibody we bought or with the new antibody you gave us. It produced a much lower, 37 kDa or so band.
Abcam has sent us a free antibody and a free lysate, which we really appreciate, but they just have not worked in our hands the way your website guarantees they should. I’m not sure if this is a problem with the antibody itself, the lysate we had, or with our conditions, but it isn’t working the way we need it to. We think the lysate you gave us may be the issue because its results are very different from anything else we've run with either antibody. We’re currently using reducing conditions, so if there are other suggestions you may have that might help, that would be great.
The other antibody we have for VAP-1 is ab81673, which we haven’t used in western blotting because it isn’t guaranteed for western blot. This one does work fine for us for other uses.
We’d really like a different lysate to use as a positive control, because our other loading controls don’t produce a band in your recommended range.
Asked on Aug 17 2012
Thanks for your email.
Since you have ab81673 I would recommend using it in a western blot with the 2 lysates you have been testing. It is quite likely this antibody will work in western blotting as it has already been shown to work in a diverse application range.
Thanks for all your troubleshooting efforts. I look forward to hearing how this next western blot goes.
Answered on Aug 17 2012