western-blotting and urea gel

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Laia
Laia's picture
western-blotting and urea gel

I'm trying to do Western-blot from a 5% polyacrilamide/8M urea gel (with NO SDS) . pI of my protein is about 5.7 so it should enter the gel, run properly and separate according to its charge. I see no bands in the Western and after staining with Comassie brilliant blue both gel and membrane no bands either. What could be the problem? And how to solve that?
Thanks,
Laia

vanishing
vanishing's picture
are your samples in 8M urea

are your samples in 8M urea as well?
(by the way: do NOT boil these samples, you will get unspecific protein aggregation)

try pre-running (the empty gel-no samples loaded yet) for 2-3 min, rinse out the wells just before loading, then load the samples

have you thought about the polarity, when you plug it into the transformator?

vanishing
vanishing's picture
and check out the link for a

and check out the link for a protocol

http://www.raydreams.com/docs/ureapage.html

Laia
Laia's picture
Yes, my samples have also 8 M

Yes, my samples have also 8 M urea (and I didn't boil them, just heated at 37C for 10 min).
Regarding polarity, my running buffer is Tris/glicine (no acetic acid), so I guess my protein should be charged negatively and run to the positive. I saw in the protocol they make acid/urea gel so I wonder if is it better to make acid running buffer and run the contrary?
Thanks a lot for suggestions,
Laia

vanishing wrote:

are your samples in 8M urea as well?
(by the way: do NOT boil these samples, you will get unspecific protein aggregation)

try pre-running (the empty gel-no samples loaded yet) for 2-3 min, rinse out the wells just before loading, then load the samples

have you thought about the polarity, when you plug it into the transformator?