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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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RE:Trouble with large protein transfer
Posted by: lpissarra (IP Hidden, New member, 0)
Date: February 20, 2004 10:06AM
I've been having similar problems trying to detect proteins of the 150-180 kDa range. I transfer in a semidry system to nitrocellulose membranes. I've optimized the transfer protocol and now seem to get good transfers which I assess by coomassie staining the gels after transfer (which give very faint bands). I also use a coloured protein marker ladder that
transfers with no problem (the 220kDa marker is fainter on the membrane and gets more retained on the gel, but that's quite acceptable since the "cut-off" limit for semidry transfers is around 200kDa). The problem is no matter how good the transfer is I don't seem to get any signal after trying different primary antibodies which seem to work fine in a neighbour lab. We share secondary antibodies and detecting reagents, so they should work fine and I also use about twice the amount of protein extract to start with. Have you any suggestions?
Re: RE:Trouble with large protein transfer
Posted by: abdel (IP Hidden, Unregistered user, )
Date: May 25, 2005 06:04PM
make sure your protein samples are completly reduced
good luck
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