I want to verify, by western blotting, the formation of dimers of a 350 kDa protein (dimer is therefore ~700 kDa). Any information about transference of high molecular weight proteins (I use an iBlot dry-transfer system) to nitrocellulose membranes and how to prepare the cell lysates would be great. So far, I tried a Triton X-100-based lysis buffer and made samples for SDS-PAGE with and without B-mercaptoethanol, with no success. Any thoughts on whether the samples should or not be boiled for the sake of preserving the dimers?
Dear Luiz,
By preparing the sample for SDS-PAGE you can obtain an artificial, mysterious protein dimer described below, thus keep it in mind:
https://www.researchgate.net/post/Dimer_at_sds-page
With high MW proteins Western blotting the best advices you will find here:
https://www.researchgate.net/post/Transferring_high_molecular_weight_proteins_in_western_blots
Anyway there is a frustrating job to do for you. Could be your huge protein is degraded by to high temperature (for this can't find the ref.). Such a huge dimer will be extremely hard to show by native electrophoresis. On one hand I suggest you to set - up the WB for detection the denatured monomer. On the secound to follow the oligomerization process by native size-exclusion FPLC with 150mM salt at the beginning, than increasing to destroy the complex. The Superdex 200 300/10 should be suituable. All the details about your transfer buffer, source of your protein and its chemical features will help to help you;)
Good luck - Jan
I agree with Jan and Sven. This would be difficult to do with SDS-PAGE and SEC or AUC are alternatives if you can express enough protein to observe the UV-absorbance signal. Realize that SDS will denature your protein destroying any non-covalent dimers formed. Also it will expose burried Cys residues, if they are present, potentially allowing non-specific di-Cys mediated dimers to form.
Are you trying to detect these dimers in the cell? If so, you could try coexpressing 2 differently tagged forms of the preotein and detecting dimers by CoIP or coexpressing the protein with 2 different fluorophores (GFPs) and detecting a FRET signal.
Size exclusion chromatography is your answer..!! see the following paper which describes 700 kDa complex and 1.4MDa complex
http://www.jbc.org/content/275/9/6067.full
It is definitely a big protein that beyond my experience. Maybe you can try 0.45 um nitrocellulose membrane instead of 0.22 um one. Also cook the lysate at 37C not 95C. Give it a try if you have not done so before.
The hindered diffusion of water in its dilute aqueous solution (if it is a solution?) or in gel state can help to get closer to the problem. Of course, first answer Svens question: what do you want? To verify the dimerization, a concentration dpendence of water diffusion rate seems to help.
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