[Protein-analysis] Blue native PAGE problems

Hubert Mayerhofer via proteins%40net.bio.net (by m_hubert from gmx.at)
Thu Jun 21 02:33:14 EST 2007


Hi!

I'm trying to carry out a blue native PAGE as described in the paper by
Ilka Wittig, Hans-Peter Braun and Hermann Schägger in Nature Protocols
1, - 418 - 428 (2006).

Yet if I don't load any protein just to test everything the running
front looks nice. When I load my protein I don't get a sharp band
normally I get smearing over the whole separation gel. I could reduce
this by diluting the protein and now I'm getting something like a zigzag
band.

I prepared the buffers accordingly to the protocol in the paper yet I
didn't cast a gradient gel but a homogenous one as this was also
described to work in a different paper (Reif, S., Voos, W. & Rassow, J.
Intramitochondrial dimerization of citrate synthase characterized by
blue native electrophoresis. Anal. Biochem. 288, 97–99 (2001).) (and to
simplify the casting as I'm lacking the equipment).

Yet I got the impression that this type of gel is normally used for
membrane proteins while I try to use it with soluble proteins. Could
this be the reason that the coomassie doesn't find the required amount
of hydropobic patches on the protein preventing the formation of a sharp
band?

Are there any changes required for soluble proteins or does anyone have
some other suggestions?

thanks in advance
Hubert



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