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Want to detect oligomers


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#1 vojera

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Posted 13 July 2009 - 06:19 AM

Hi all.

I've only ever done straightforward SDS PAGE before but I'm interested now in finding out if my protein (which is being expressed in the tobacco plastid) is forming oligomers.

I know I can't boil my samples or use any reducing agent in preparing my samples, but would I be right in thinking there's more to it than that? No one else in my lab has any protein experience so I'm a bit stuck! I'd appreciate any advice you could offer. :)

Also, I have another area I'm interested in looking at but my protein is bound to a membrane so I've always used an extraction buffer with SDS in it to get it off. Am I going to be in trouble if I want to do native PAGE with this? B)

#2 mdfenko

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Posted 13 July 2009 - 07:27 AM

Also, I have another area I'm interested in looking at but my protein is bound to a membrane so I've always used an extraction buffer with SDS in it to get it off. Am I going to be in trouble if I want to do native PAGE with this? B)


yes. sds will denature your protein. you can solubilize your membrane with non-ionic detergent (eg triton x-100). this should allow the protein to remain in its native form (you can even add some to the native gel, if necessary).

as for native gels, you can try a method similar to laemmli, just leave out the sds from the gel and buffers and use bromphenol blue and glycerol as sample "buffer" (not necessary to buffer but you can make sample buffer without sds and reducing agent, don't heat sample).

or you can try the formulation that i posted here:

biotechniques forum

the formulation is in the final post.
talent does what it can
genius does what it must
i do what i get paid to do

#3 vojera

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Posted 14 July 2009 - 07:38 AM

Okay, thank you so much for your help. I'll definitely try the triton with my membrane bound samples and I'll definitely give those gels a go.

Thanks!




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