During prolonged washing steps or overnight incubations of Western blots, are proteins fixed to the PDVF membrane sufficiently ? Are there some optimal methods of fixation like air-drying after protein transfer of gels ? Is the PVDF membrane absorbant enough to hold the proteins from the gels ?
Proteins bind to the PVDF via hydrophobic interactions. There are also ways to cross link proteins onto the membrane using, for example, glutaraldehyde. We have used this with mixed success. With some proteins it increases the sensitivity of the IB, in other cases there is no improvement. Also, in one case the target protein could not be detected - perhaps the epitope had been modified during cross linking. For method, see Connern, C.P., and Halestrap, A.P. 1996. Chaotropic agents and increased matrix volume enhance binding of mitochondrial cyclophilin to the inner mitochondrial membrane and sensitize the mitochondrial permeability transition to [Ca2+]. Biochemistry 35:8172-8180
Hi, I remember my beginning of WB when I thought the same as You, but the binding is really strong. But it's a bad idea to let the membrane going dry, don't do that.
Good luck!
Steffi
Thanks Stefanie. Ordinarily I would never let the membrane dry out, but I have been reading that this was a way to fix the proteins in some protocols. However, I think I am just going to increase membrane transfer times to ensure maximum protein availability.
After transfer, the proteins bind very strongly to PVDF (through hydrophobic interactions, as mentioned above). So there is no need to fix them to the membrane for immunoblotting purpose.
Your question implies that you are concerned about losing proteins from the membrane due to lifting out of the proteins during washing and incubation. There will probably be some loss, which is why blot interpretation is usually confined only to the results from lanes of the same blot. You might expand the comparison from one blot to another by loading aliquots of the exact same sample on the different blots, then doing the electrophoresis, transfer and blot development at the same time, using the same volumes of wash, the same configuration of wash container, on the same shaker. Besides losing proteins in the wash and incubations, you can also lose proteins going through to the other side of the membrane and back out to the transfer buffer. This happens most the lower the mol wt, the higher the voltage and the longer the transfer time. You can estimate proper transfer conditions for proteins of the same size as your protein of interest by loading pre-stained molecular weight protein markers and trying out different conditions, then scan and quantifying the bands.
I'm also interested in this because I'm trying to detect a 6kD peptide by western blot. Agitation seems to cause the peptides to float off membranes (Article A new approach to detect small peptides clearly and sensitiv...
) so I would like to try fixing the peptides to the membranes before doing the blot. I do acknowledge that there is a chance that the fixation process may damage the epitope that the antibody recognizes, but if it doesn't, then it would be much better for detection of peptides!
Diana, you could also try a smaller pore-size membrane. It may, or may not, help. I'm not sure about PVDF, but smaller pore-size forms of nitrocellulose are available.
Actually, i started to work recently with PVDF membrane, an important step to do is to incubate the PVDF membrane with methanol for 5 min prior to the transfer. Also the blocking shoudl be for 45 min with 5%milk TBST, and its better to incubate the membrane with the antibodies that are diluted with TBST without milk.
Best of luck
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