These questions may sound silly, but this is all quite new to me! I have two queries:
1. If I grow my cells in 24/6-well plates, is it safe to trypsinize the cells first then lyse in buffer? Or should I scrape cells directly off the plate, then lyse?
2. Since the kits I purchased didn't come with lysis buffer- can anybody refer me to a formulation of this?
If the protein you are measuring is a surface protein it will likely be digested by trypsin giving false low values. If it is intracellular trypsin should not affect your measurements as the trypsin will be removed with you wash steps. The choice of lysis buffer may vary depending on the stability of the protein in various solutions. Please see: http://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/lysis_buffer_additives/index.html and http://www.piercenet.com/method/cell-lysis-solutions . I have also attached a publication that discusses mammalian cell culture cell lysis in detail.
Im actually looking at TNF-alpha, MCP-1 and IL-6. I just realized that i don't have any cell scrapers with small head width- thats why i'm asking whether it's safe to use trypsin....
If the cells are not so tightly attached (eg:293T cells) you can harvest them simply by adding PBS solution, and spin down your cells. And if your cells are too strongly bound to the surface (like macrophages) you can simply wash the plate with PBS and lyse them by adding the buffer directly on the plate and mix all over the surface. Scrapping is not desired since you may loose your cells and it may also rupture cells.
As suggested by Frank (his attachment), if the purpose is not for Immunoprecipitation, I would use RIPA buffer.
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