Hi. One way to address protein oligomerization is by means of blue native page. I am attaching a link to a relevant article that may give you all the technical info you need. Good luck
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2898288/
percent of gel will depend on the size of the oligomer, but 4% gel should be sufficient, i have used it to resolve proteasome (2.4MDa) , tris borate buffer is any time better.
Thanks George and Amit. My protein homo multimers are 150 kDa and 250 kDa. generally in a normal western blot I pick up all the three bands which is a gel of 8%. I was just curious if I run a gradient gel ,will I have a better profile. My idea is to look at the trimer fraction.
Dear Kalpita,
we may try gradient gel. you may also try few things, if the protein of interest is acidic 4% native gel is good option. We may also try fractionation followed by electrobloting or sucrose /other density gradient to get the most abundant or native oligomeric status of ur protein
Hi,
Amit I didnot get the idea of fractionating followed by electrobloting, how is it done ..may I get an opinion on that?
Hi Kalpita,
gradient gels will sometimes give you worse results than you are hoping for especially at those sizes. Gradients are good for resolving very low and very high MW bands on the same gel - the reason is that the gradient is composed of very small regions of each percentage. For the weights you need, you will be better with a single low percentage gel e.g 4, 8, 10 should do.
If the gel running temperature due to time of run etc is an issue, switch to gel types such as Bis-Tris which are not heat labile and therefore run better for these types of gel .
One way other than native gel is to cross-link the proteins in solution using chemical crosslinkers e.g. BS3 thermo Scientific. these will keep your dimers etc together on a standard gel. there are some caveats of course (other proteins could be bound and therefore cross-linked) but we have used it with a certain amount of success and its fairly easy. a few other things can help i.e. you could pull the protein down on beads from solution.
Hope this helps,
S
Thanks Scott,
I have been using 8% in tons of my westerns and what I have not done till now is below 6% or a crosslinking. My proteins are a result of overexpression post tranfection and so I am not sure how much non-specificity is pulled down with my polyclonal Ab. Thats a thing to do. And even Bis -Tris for which I got to see the composition as I use only tris.
I am already labeling my proteins with biotin which is a sulpho-SS tag so I dont think a second pulled down can be done. I already use Avidin resin to pull down.
I may change the gel may be.
Hi Kalpita,
I only suggest the pull down only to clean up the sample prior to cross link , i.e. remove contaminants. If you are already purifying in one way then that will help. its also not 100% necessary.
We use the Invitrogen Novex Bis-Tris gels (they can also be adapted to use Biorad Gel tanks) Its more expensive to make them than buy them so either way they are definitely not the cheapest of gels.
Another difference can be buffer used for separation, MOPS gives better separation than MES in BIS-Tris gels.
the advantage of crosslinking is that if your protein is mostly in dimer/ trimer, they will stay in that state as you run the gel, increasing the amount available for detection. Its an easy step in your sample processing's, add BS3 then quench prior to running on gel
Good luck,
S
Depending upon the pI of the protein you can hand-pour different pH native gels, or a gradient gel and see for multimers as described in McLellan T, Anal Biochem, 126, 94–99 (1982) OR A concise version in Bio-RAd Mini-Protean Tetra cell manual pg 18.
Another option is to pour a Laemmli gel without SDS. The stacking gel can be 5 % and the resolving gel can be 8 or 10 % whichever works best.
I have had success with pH 4.5, pH 8.1, 8.9 from the paper described above and the Laemmli gel. Make sure gels are run/precoooled at 4 C.
I never had success with any commerical native gels/blue gels.
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