Protease Protection Assay
30*106 NSO cells
wash twice with PBS
resuspend in 500 ml of homogenisation buffer1, 2 mM (final) Iodoacetamide
20 shots with a 2 ml syringe and 22G needle (in a 15 ml Falcon tube)
spin 3 times 10 min 4°C 1000 xg
divide supernatant in 3 aliquots:
1. add Proteinase K2 0.1 mg/ml (final) + PMSF 2 mM (final)
2. add Proteinase K 0.1 mg/ml
3. add Proteinase K 0.1 mg/ml + NP40 1% (final)
incubate 1h 4°C
Add PMSF 2 mM (final) to sample 2, 3.
Dilute with PBS and immunoprecipitate according to your goal (if labelled cells) or load on a SDS-PAGE the equivalent of 50.000 cells (in the case of NSO cell line).
Stock Solutions: 1: Homogenisation Buffer: Tris pH=7.4 10 mM, Sucrose 250 mM,
2: Proteinase K: 1 mg/ml in 50mM Tris pH=8.0, 20 mM CaCl2