Daily cells (500ml) pelleted and resuspended in a- 10ml BBM, b- 10ml MBBM. After overnight incubation cells were pelleted and supernatants were collected (secretome). 1ml aliquots were TCA precipitated. Pellets were suspended in 30 microliters of cracking buffer/w 2mM IAm. Samples were heated at 100C for 4 min, 3 microliter and 12 microliter aliquots were applied to gel.
Cells were washed with 6ml of neutral MOPS buffer, reuspended (2ml) in MOPS. LiCl was added to 1M. Samples were incubated 10 min at RT. Cells were pelleted. One ml aliquots of supernatant were TCA precipitated and prepared for PAGE as above.