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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.


Thanks!!!!

-jovi-

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,
Ceri

-Ceri-

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???

-jovi-

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.

Ceri

-Ceri-