Western Blot on an IEF separated recombinant protein - (Sep/22/2006 )
I tried to blot proteins from IEF gel, which I cast my own to PVDF membrane. But, I failed to detect any bands on the film. I transfer the gel with tris, glycine and methanol buffer...Am i using the wrong buffer?? :huh: :(
what do you mean by "IEF gel"? is it a native PAGE slab gel or tube gel? analytic or even preparative IEF is commonly performed with IEF acrylamid gel fixed on plastic strips; because of the plastic on one side they cannot be blotted; your transfer buffer system should contain SDS; anyway, your "IEF gel" should be soaked in your SDS-containing transfer buffer before blotting
Thanks for your reply Kosmodrom..
U are rite, there is always a plastic strips attached to one side of IEF acrylamide gel..but I prepare myself using Biorad electrophoresis unit (mini protean III) which used to run SDS-PAGE. Therefore, blotting was performed w/o plastic. But, I never add SDS in transfer buffer,so I agree with you that should add SDS in the buffer to re-charge the proteins. Do you have any idea what's the concentration of SDS in buffer??
My transfer buffer is made of 100 mL methanol, 100 mL premade buffer TGS from biorad (composed of 25mM tris, 192 mM glycine, 0,1% w/v SDS, pH 8,3) and bring up to 1 L with distilled water. It has always worked fine.
Hope this helps
Hope this helps
I will give a try !
for SDS-gels we use a self-mixed transfer buffer modified and adapted from Towbin et al., PNAS (1979), containing 48 mM Tris, 30 mM Glycin, 0.037 % (w/v) SDS, 20 % methanol;
however, samples were boiled in 5 % SDS, and in running buffer is about 1.25 % SDS; soaking your native gel in original Towbin-buffer will not contain enough SDS; therefore, add 5 % SDS in an aliquot of transfer buffer for only some minutes to incubate the gel immediately after PAGE ( years ago I´ve done a similar experiment and incubated too long: smaller polypeptides were washed out of the gel!); wash than quickly in a transfer buffer aliquot of 1.25 - 2% SDS; this last mentioned buffer should also be used for transfer; use cooled tank blotting for several hours