transfer through the PVDF membrane - (Mar/08/2006 )
Hi all, I am having some problems with my transfer. we use semi- dry system and try to develop a protocol for transfering a protein of 42kDa. i tried a number of voltages/amperages and times (the manufacturer recommends 10-15V for 15-30mins) and I still cannot get my protein on the membrane. The gels are clear (checked by coomasie) and the problem is that my proteins actually cross the membrane- most of them is on the second membrane within 10mins on 10V (transfer buffer 48 mM Tris / 39 mM glycin /20% v/v methanol + sodium orthovanadate to inhibit phosphatases). Any idea? (5V wasn´t much better...) Thanks!
what are the properties of your protein? perhaps it needs a different transfer buffer
(I have learned this the hard way once, with a protein that had a high pI; took a few tries to realize that standard transfer buffer wasn't going to work)
Have you tried double layering the PVDF?? meaning....place one more membrane below the one that is in contact with the gel....it might catch it. However, b- actin which is a common control is around 42 kDa. It does not slip out even if you transfer for 1 h at 100 V. So why does ur protein which is also 42 kDa. (This is all with wet transfer)
Therefore me thinks aimikins might be onto something...it might be the nature of your protein and it might require different transfer or preparation. I dont think the V or timings has got to do anything....
try a membrane with smaller pores (0.2 um or smaller).
thanks for all the advice, the protein is ERK1/2. I finally succeeded with the immunodetection with one antibody, although I had to use about 4x higher primary abs concentration than recommended (so I still think, there is some problem with the transfer, but now that I have some signal I can optimize it more easily...). I tried double layering the PVDF, anyway ovalbumin, which I use as a MW marker crosses them both quite easily. Cheers, Busa.
PS any general advice for improving the transfer buffer?
well, we didn't 'improve' the tx buffer, just found a recipe more appropriate to our specific protein, based on the protein's size and pI...it required a bunch of time in google, this site, and some literature mining about the protein itself
but, for ERK you should be able to use anything others are using...that one is published quite a bit and we have blotted it with standard buffers and techniques (using 0.2um PVDF though, better safe than sorry)...we used semi-dry transfer and it worked pretty well