Trypsin digestion

Trypsin digestion for IP3R1

- resuspend cells in prewarmed complete medium with or without (for NT=not treated sample)50 mM DTT

- incubate 15’ 37°C

- wash 2 x ice-cold PBS, at 4°C

- resuspend cells in DMEM (25°C)

à keep 0’ 1’ 2’ 4’ 8’ at 25°C for reoxidation

- stop in ice by adding 20 mM NEM

- wash 2 x ice-cold PBS + 10 mM NEM, at 4°C

- lyses in RiPA buffer + 10 mM NEM + Protease inh cocktail

- ConA precipitation O/N 4°C

- wash beads 1 x PBS

- trypsin digestion on beads:

0.1 mg/ml 10’ 25°C

Add 100 ml buffer/ 10 ml beads

Buffer x digestion: 120 mM NaCl

20 mM Tris-HCl pH 7.4

1 mM EDTA

(Mikoshiba’s digestion buffer: 120 mM KCL

20 mM Tris-HCL pH 8.0

1 mM EDTA

1 mM DTT)

- stop digestion by adding soybean trypsin inhibitor (0,4 mg/ml) and proteases inhibitors cocktail (from 50x stock)

- wash beads 1 x PBS, divide samples into 2 during wash

- elute with 10 ml PBS + 7 ml 4x sample buffer (R or NR), at 60°C 10’ / 95°C 5’

- before loading, add 7 ml 1 M IAA both to reduced and non-reduced samples

- load sample in 6 % gel (1x10⁶ cell/ lane)