how to make neutrophil cell lysates for Western blot? - (May/11/2006 )
I need to make cell lysates of neutrophils for detection of a membrane bound receptor (CXCR2) on Western blots. Until now I was unsucceful.
I need to know optimal lysis buffers and amount of cells necessary and what volumes to use.
CXCR2 is easily detected by flow cytometry with FITC or PE conjugated abs. If you don't have access to a flow cytometer, lyse the cells in in 2X gel sample buffer alone by counting your cells and resuspending them in 50µl PBS/1,000,000 cells and add a equal volume of 2XGSB (100mM Tris-Cl pH6.8, 20% Glycerol, 0.2M DTT, 4% SDS, 0.02% bromophenol blue). The DTT can be replaced with 0.4M MESNA or 10mls Beta-2 mercaptoethanol. I haven't done blots with neutrophils but this works for B-lymphocytes.
Or you could lyse in RIPA buffer (50mM Tris-Cl pH7.5, 150mM NaCl, 0.1% SDS, 0.5% NP40, 0.5% Na deoxycholate, 5mM EDTA), I guess 50-100µl per 1,000,000 cells should be OK and quantify the protein by bradford assay and then adjust the protein concentrations with RIPA so you load equal amounts of protein (10-50µg upwards).
Hope that helps,
thanks for the advice, but the problem with neutrophils is that their granules are full of proteins, which makes it difficult to lyse them - the lysis bufer becomes very slimy, and the pellet of cells can't be well ruptured. (i do add protease inhibitors to teh lysis buffer as well)
another thing is that i want to do immunoprecipitations with the lysates. anyone experience here with neutrophils???
If you're having problems making the lysates, flow cytometry seems a good option since your studying a surface receptor. Unless you don't have access to a flow cytometer. A few people in the lab work with neutrophils but aren't doing westerns. I'll ask them how they lyse the cells for their biochemical studies.