I have around 100ul of HeLa cells pellet. I want to make a very concentrated lysate in around 100ul of vol for westren blot. I want all the proteins (membrane + soluble) but not any lipid or mebrane fraction so that gel should run smoothly and band should be clean. Thank you all in anticipation.
One possibility is to disrupt the cells in Tri-reagent, and follow the protocol for protein isolation. With this method you have a total protein extract by the lipids remain in solution. Take a look: https://www.thermofisher.com/de/de/home/references/protocols/nucleic-acid-purification-and-analysis/dna-extraction-protocols/tri-reagent-dna-protein-isolation-protocol.html#prot11
Add 200 ul of acetone at -20 degrees C (from a freezer) - place the extracts in the freezer for 30 minutes. Remove and centrifuge to pellet the proteins. Air dry the pellets to eliminate residual acetone and add SDS-PAGE loading solution, boil etc. ..... for your SDS-PAGE protocol. This procedures precipitates all proteins but not lipids or salts and so is very compatible with SDS-PAGE.
One possibility is to try TCA (Trichloroacetic Acid) protein precipitation
http://cshprotocols.cshlp.org/content/2011/8/pdb.prot5651.short
Thank you all for your answers. Most of the answers contains the precipitation step of proteins (except the Tri reagent). But I am still not very sure how to lyse the cells (which lysis buffer I should use) to get whole protein content (both the soluble and membrane) and then which protein precipitation step I should follow, which will be compatible with my lysis buffer to get the best result. Thanks
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