Protease protection assay

30*10⁶ NSO cells

wash twice with PBS

resuspend in 500 l 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 g/ml (final) + PMSF 2 mM (final)

  2. add Proteinase K 0.1 g/ml

  3. add Proteinase K 0.1 g/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