native gels

From: anita p
Date: 19 January 2012 13:03

Hi All,
Has anyone run a native gel for proteins at pI>8 .
I want to pour my own native gel. Do I run a discontinuous page or a continuous one?? Please help with regards to the buffer system to be used, and the dye to be used.
With regards
Rashmi

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From: Miguel Ortiz Lombardía


Le 19/01/12 14:03, anita p a écrit :

Hi Rashmi,

Try the blue-native method:

Peltier, J. B., Ytterberg, J., Liberles, D. A., Roepstorff, P., and van
Wijk, K. J. (2001) Identification of a 350-kDa ClpP protease complex
with 10 different Clp isoforms in chloroplasts of Arabidopsis thaliana.
J. Biol. Chem., 276: 16318–16327.

Wittig, I., Braun, H.-P., and Schagger, H. (2006) Blue native PAGE. Nat
Protoc, 1: 418–428.

Best regards,


--
Miguel

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From: Katherine Sippel


Hi Rashmi,

In my experience native (even blue native) on proteins around that pI is sketchy at best. The electrophoretic mobility once it gets past the stacking gel goes to crap meaning long electrophoresis times and it needs to be done on a chillable system or in a cold room. If this is a multimer issue I'd suggest trying analytical ultracentrifugation, analytical size exclusion (with the caveat that buffer, temperature and protein shape will affect the output/interpretation), or SAXS first. If native is the only alternative you'll probably get better results changing up the buffer system from traditional tris-glycine or those listed in the blue native protocol keeping in mind that you'll still need to stack the bands.

Good luck,

Katherine

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From: Jacob Keller


I have actually done this by running a normal PAGE gel without
stacking gel and switching the electrodes, which seemed to work
swimmingly.

JPK

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*******************************************
Jacob Pearson Keller

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From: Federica Basilico


Hi Anita,

I have recently performed it following this protocol I found in the Internet:

http://wolfson.huji.ac.il/purification/Protocols/PAGE_Acidic.html

I hope this helps! Good luck,

Federica

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Federica Basilico

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From: Shaun Lott


I've used the beta-alanine buffer system to use native PAGE for a protein with a pI>10. I used an old Phast system (sadly since deceased in a puff of smoke) for screening for heavy-atom derivatives in a buffer-free system. Hopefully this might provide some useful hints. Sorry about the big attachment but the old Pharmacia/Amersham technical notes are not easy to come across.

On an related note... is anyone aware of a bufferless system for doing NATIVE PAGE? There are a few cassette systems (eg E-PAGE from invitrogen) that do SDS PAGE but none to my knowledge that do native PAGE. Not often needed, but useful to have!

cheers

Shaun