Lysis buffer selection - (Oct/19/2010 )
There are a lot of different recipes for a lot of different lysis buffers. Does anyone have a flow chart or general rules of thumb about which lysis buffer to choose? Does it depend on the type of cells that are being prepared (tissue or cell culture), the protein of interest, protein determination assay, antibody, etc? Any help will be GREATLY appreciated! Thanks.
All of the above really. For tissue you will need a fairly strong lysis buffer to get the proteins out compared to what you would need to lyse cultured cells. Some proteins are not readily soluble so you need to add more detergents to get them to solubilise. Some reagents, usually detergents interfere with protein quantitation with particular assays. Some antibodies detect only native protein, some only denatured protein...the lysis buffer can affect the conformation of the protein.
Generally the more detergent and the less salt (i.e. more hypotonic) a lysis buffer has in it, the stronger it is.
Here is some idea about different buffers. With higher salt and detergent you increase stringency.