MW marker for native PAGE - (Mar/26/2008 )

HI ALL!!
just a quick question i want to see if my protein forms a dimer, so i am planning on performing NATIVE gels. any idea what to use as a MW marker (the cheaper the better!!) the only marker i have in the lab is precision plus all blue from biorad. i cannot find if this contains reducing agents so i dont think it is suitable/ or is it? if not what have ye used.
many thanks!

Hello,

I am looking also for a marker, as i am doing WB for proteasome. Anyway, have you find the marker? And, for the native gels, what protocol are you using.. We have two different protocols and we are havinf a lot of trouble.

R

this is probably going to start some new arguments so i will start off with an admonition against using native gels for size determination. in general, don't use native gels for size separations (except for blue-native page).

that being said, you can use it for seeing differences within the same protein (although they may not run true to molecular weight, they should still show differences). you can enhance size separations by using a gradient gel.

you can not use the prestained markers in native gels, they contain sds and reducing agent.

we used marker proteins from gehealthcare (pharmacia) to get an idea of size in gradient native page. the lmw markers do not contain sds or reducing agent.

see these web pages:

lwm-sds and hmw-native markers

and:
protein mw standards

the best standard to use for your purpose would be purified multimers (and monomer) of your protein of interest.

to kikinho:

proteasomes are very high mw and will be difficult to separate on native page. you will need to run a low concentration gel (probably 5-6%). you may be able to use a gradient like 4-10% (oops, just saw your other post, i still advocate a gradient to sharpen your bands but maybe a 3-5%, instead).

which formulation do you use?

you will find the formulations for the ornstein and davis buffer system posted in these fora (i know because i posted them). just search. there are formulae for basic (the normal native page), neutral and acidic buffer systems.

you should probably use the hmw native markers but the proteosomes may come out higher than the highest standard.