Blue Native-GE & Blotting

[Paul S. Brookes.]

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
                          are welcome.


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?

Regards
PSB


_________________________________________
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
http://peir.path.uab.edu/brookes

Freudian slip - when you say one thing but mean a mother
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