J chain folding assay

Transfect cells with PEI 2 days before in 10cm petri dish

• wash cells PBS x2

• add 3 ml Trypsin-EDTA

• 5 min RT

• count cells

• resuspend in 2x10⁶ cells/ml in DMEM -FCS 37°C 5 min + 5mM DTT (final, fresh made) (50 µl DTT 0.1M)

• wash cells 2x PBS 4°C

• centrifuge 4°C pelletting cells

• divide cells in different Eppendorf (1x10⁶ cells/ml) in complete 10% FCS DMEM and put at the same time (few samples) in a 37°C or 24°C water bath

• at different times add NEM (10 µl NEM 1M) and put in ice: 1=0, 2=2 min, 3=4 min, 4=8 min

• wash 4°C PBS 1x

• lysis of cells in RIPA 10mM NEM + cocktail protease inhibitors: 15 min ice - 10x10⁶ cells/ml (tot 100 µl)

• load 10µl on NR SDS-PAGE 12% MIDI