Blue Native-GE & Blotting
Paul S. Brookes.
brookes at uab.edu
Tue Mar 27 09:20:17 EST 2001
Geert Persiau (geper at gengenp.rug.ac.be) wrote:
I'm currently doing some preliminary
experiments with Blue native-GE.But to optimize the
conditions I would like anybody who has some
experience in this technique to share his
knowledge in this field.
So any referrence,protocol,articles...on as
well Blue-native-GE as the blotting of these gels
We tried blotting blue-native gels and didn't have a lot of
success. Certainly, it is possible to blot the gel after it has been run,
but the coomassie blue tends to interfere with Ab binding for the
immunodetection. I'm currently messing with a protocol to denature the
proteins on the membrane, and remove the coomassie, so that the Ab will
bind just like a regular SDS-PAGE blot. We have had more success by
running a 2nd dimension tris-tricine gel (as per Schagger & Von Jagow
method), then blotting that. The 2nd dimension run removes a lot of
coomassie, and the proteins blot across better. Ab binding to the 2D blot
doesn't seem to be any different from a regular SDS gel. As for the
blot of the 2D gel itself, we use a regular Towbin (tris-glycine) buffer
system with 0.03% SDS, 2hrs at 100V in the cold room, after pre-soaking the
gel for 20min in this transfer buffer. If you absolutely have to blot only
the first dimension, then try replacing the cathode buffer with dye-free
buffer about half-way through the run. This cuts the amount of dye,
although you need to check that your proteins don't precipitate, because
they need a certain amount of dye to remain complexed.
BTW, what samples are you running?
Dr. Paul S. Brookes. (brookes at uab.edu)
UAB Department of Pathology, G004 Volker Hall
1670 University Blvd., Birmingham AL 35294 USA
Tel (001) 205 934 1915 Fax (001) 205 934 1775
Freudian slip - when you say one thing but mean a mother
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