How to prepare high quality brain tissue lysate? - Will high salt lysis buffer be helpful? (May/04/2011 )
My final purpose is to find some new interacting protein of my protein of interest.
I want to prepare some high quality mouse brain lysate,then apply them to the strep-tactin resin with my double(strep/flag) tag fusion of interest(from E.coli. expression) on it.
Then I will elute the potential interacting protein complex with D-biotin and apply the eluate on the anti-flag M2 resin,then elute with flag peptide(two step purification to reduce non-specific purification).
Does anyone know some good protocol or lysis recipe to prepare mouse brain lysate?
Which will release great majority of protein,on this regard,will high salt buffer be helpful?which will later be diluted to normal salt concentration.
and will homogenate or sonication be helpful?
and does anyone have any suggestion on my overall interacting protein discovering approach?
You may want to try to use the spin columns (filter cartridges) for fast and clean protein extraction. This format can handle very small sample amount. Depending on the lysis buffer you want to use, you can have denatured (for SDS-Page), or native proteins prepared in a few minutes. I believe the kits are available. Just google "spin column protein extraction" and you should be able to find the source.