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|Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.|
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I use Tris-HEPES gels from Pierce, run in Tris-HEPES-SDS running buffer from Pierce, in a BioRad TransBlot mini-TETRA system. I often do 2-3 gels, based on what I'm running that day. I blot with BioRad Immun-blot 0.2 um PVDF in a regular Tris-Glycine transfer buffer, with 20% MEtOH and 0.5% SDS in the same system, with 1 or 2 systems depending on the # of gels.
Recently, I have had 2 separate instances in which 1 of my membranes has shown absolutely no transfer. The first time, it was a membrane that was in its own tank when I was running 3 gels (needing 2 tanks for the transfer). I know it was in the correct orientation for proper transfer, and the cassette was in position 1 of the cassette tank, closest to the black, negative part of the tank. The transfer cassette was tight, and there were no bubbles between the membrane and gel (I always roll bubbles out with a 25 mL aspirating pipette). After transfer was finished (45 mins @ 0.2 A, followed immediately by 45 mins @ 0.25 A, our standard protocol), I removed the membrane from the cassette and there wasn't even evidence of our pre-stained molecular marker. I still blocked the membrane and probed it with a duplex of b-actin (for housekeeping) and another total protein of interest. The b-actin showed up faintly, but the other protein did not show up at all when viewed on a fluorescent machine. Note that the gel, when stained, looked perfectly normal (some higher weight proteins still in the gel, no lower weight proteins left; this is normal for us).
The second time, I was only running 2 membranes, same conditions and in the same tank. The membrane that showed no transfer was the membrane further from the negative area (position 2 in the cassette tank), and my other membrane showed perfect transfer. Again, after staining the gels, both looked exactly the same. I still blocked and probed this membrane, the same as before (beta-actin and a total protein of interest) and saw b-actin faintly, and the other protein not at all. The other membrane looked perfect and as expected.
Has anyone else experienced this? Can anybody help me? This is extremely frustrating to troubleshoot, and BioRad's tech support has not had a good answer for me.
|problems , pvdf membrane , transfer , transfer problem , western transfer|
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