I am working with HL-1 cardiomyocytes. I have been troubleshooting a subcelluar fractionation protocol FOREVER. I have tried douncing and sonicating (a combination of the two). And still the cells remain in tact. All the literature says that the cells should by put in a hypotonic buffer to allow swelling before lysis. but I worry that using the hypotonic buffer will in some way alter the proteomic state of the cells before I get a chance to find my protein of interest.
Are my worries uncalled for? Should I just use the hypotonic buffer? Will it alter anything drastically?
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