Non-denaturing western blot - I need a transfer protocol for non-denaturing gel (Aug/20/2008 )
I am afraid I have not been able to find this in the forum.
I want to perform a South Western blot (hybridizing labelled oligonucleotide to proteins fixed in the membrane).
I already performed a gel-shift assay using different protein extracts and there was a difference, so there is some specific interaction.
I did a first SW-blot using SDS page and the usual methods, but there was no interaction at all. I believe this lack of answer may be caused by the disruption of the protein complexes the oligos I am using bind to.
So now I would like to do a SW-blot using a non-denaturing gel, but I do not know how I could transfer the proteins from the gel to the membrane.
I have a paper describing the SW-blot technique, but as it is from 1989 and there were no electric transfer apparatus then, I wonder If anyone had come with a better (and faster - their transfer takes 48h) solution. They use 0.05 M NaCl, 2 mM Na-EDTA, 0.1 mM DTT and 10 mM Tris pH 7.
Hi, you should be able to transfer the non-denaturing gel in the same way as you would an SDS gel. Here is also a general protocol on non-denaturing, or Native Gels.