I am looking for protein complexes and was wondering whether I can run a native gel followed by western from previously frozen protein lysates which are in -20 for 2 weeks.
I would say a qualified "yes".
I expect there will be some aggregation from the freeze-thaw, this will depend on the the original material, the method of homogenization, the concentration of proteins in the lysate and the buffer it is in (pH, salt, osmolytes, non-ionic detergents). A brief clear-spin should sediment these aggregates however you may deplete your complex (this would be dependent on the size of the complex). Any aggregates loaded on the gel would appear as high MW smears at the top of your blot. You might want to run +/- spin lanes to evaluate freezing as part of your workflow.
If you are going to do this routinely in the future I would recommend freezing pelleted material at -80 and homogenizing just before you are ready to run your gel.
Hi Soham,
I think it would depend on many different factors, but it's impossible to say without you trying it. It would likely depend on the method of freezing the sample, the buffer composition, the strength of the interaction between the components of your complexes. Unfortunately, I think you could run the gel and see no sign of any complex, but I don't think it would be hard proof of anything.
You could also argue if you did see anything on a native gel that they are aggregates formed as a result of the freezing process - you'd probably have to compare it with something that had not been frozen.
Best wishes,
James
Hi James and Micah,
Thank you for your response. I would also prefer to run native from a fresh lysate and had speculated the caveats of running frozen ones. Since I do not have experience with native gels, just wanted to be sure if can do a quick and dirty one.
Thanks again.
Regards
Soham
Sure you can !
I did a comparison of fresh vs. frozen. It works equally well from frozen. I was looking at mitochondrial complexes and supercomplexes. Check out the quality in these two papers. They are done on frozen tissue samples .
https://www.ncbi.nlm.nih.gov/pubmed/27284200
https://www.ncbi.nlm.nih.gov/pubmed/26928661
Cheers
Pooja
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