native PAGE - (Jul/11/2006 )

Hi,

Does anybody have a protocol/suggestions/tips for native protein PAGE?I found a short mention in a book, but not very helpful...

Thanks a lot in advance

Miguelon

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do you want high pH (standard), neutral pH or low pH formulation. what do you need to separate?

i can give you several formulations and protocols when i am at work tomorrow, just let me know what you need.

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Thanks mdfenko,

I would like to try to resolve two small complexes of my protein, and I just wanted to know a little more about this approach. I suppose that standard (pH8.8) would be ok, at 4% (those are the conditions I´ve seen for similar approaches (EMBO J 2004 Apr 21;23(8):1782-91), but I don´t know how to do it, the compositions of buffers, conditions of running) ?

Just a few more questions: I´ve also seen mentions to native agarose gel electrophoresis of proteins. How´s that made? Can you separate whole protein complexes for further characterization after staining? and, Is it possible to visualize fluorescent fussion proteins from an extract in polyacrylamide gels?

Thanks in advance and sorry for the mess of questions

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I would like to try to resolve two small complexes of my protein
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I would like to separate them after affinity competition in an antibody-coupled column. Is that feasible?

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the Ornstein and Davis formulation:

8X resolving gel buffer:
48 ml 1N hcl
36.3 gm tris base
0.23 ml temed (you can leave this out if you wish to add later)
dw to 100 ml
(pH should be 8.8-9.0)
use 37.5:1 acrylamide:bisacrylamide ratio (30% acrylamide solution- 30:0.8 acryl:bis)

8X stacking gel buffer:
48 ml 1N hcl
5.98 gm tris base
0.46 ml temed (see above)
dw to 100 ml
(pH should be 6.6-6.8)
stacking gel should be photopolymerized with riboflavin and fluorescent light
acrylamide for stacking gel should be 10% acryl, 2.5% bisacryl (will polymerize cloudy or nearly milky white), high crosslinker concentration is for mechanical stability.

10X electrode buffer (might be 1x but my protocol calls it "10x (as used)" and is probably correct):
3.0 gm tris base
14.4 gm glycine
dw to 1L

the standard acrylamide concentration is 7% but you can use what you need (or a gradient might be nice).

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