mPEG protocols

Hela cells (500.000 cells/500 l in DMEM 5%FCS)

• 1. 50 mM DTT

  2. 10 mM DIA

  3. –

15 min 37°C

Inverse mPEG RIPA Lysis

Wash PBS NEM 20 mM

Incubate 10 min ice cold PBS-NEM 50 mM

Lysis RIPA 500 l 40 mM NEM + cocktail protease inhibitor (spin 6000 rpm Eppendorf 5 min)

In vitro reduction: 50 mM DTT (final) 20 min RT

TCA precipitation: add 500 lTCA 25% 4°C 30 min

Spin 13000 rpm Eppendorf 10 min 4°C

2 washes 100% aceton (do not dry too much pellet)

Resuspend dried pellet in 50 l water and then add -/+ mPEG 2x buffer (160mM Tris pH=8; 4% SDS; 25 mM mPEG 5000)

Incubate 37°C 1h then O/N RT rotating

SDS-PAGE 7,5% not reducing

Inverse mPEG (TCA on plate)

Wash PBS NEM 20 mM

Incubate 10 min ice cold PBS-NEM 50 mM

TCA precipitation: add 500 l TCA 10% 4°C 30 min + 500 l TCA 25% washing the well

In vitro reduction: 50 mM DTT (final), Tris 50mM pH 7,4, 2% SDS 50 l 20 min RT

Add Tris 50mM pH7,4 (no SDS), 50 mM DTT (final) 450l

Add 10% TCA 500 l

Incubate 4°C 20 min

Spin 13000 rpm Eppendorf 10min 4°C

2 washes 100% aceton (do not dry too much pellet)

Resuspend dried pellet in 50 l water and then add -/+ mPEG 2x buffer (160mM Tris pH=8; 4% SDS; 80 mM mPEG 5000)

Incubate 37°C 15 min then 20 min RT rotating

SDS-PAGE 7,5% not reducing

Direct mPEG (TCA on plate)

Wash PBS

TCA precipitation: add 500 l TCA 10% 4°C 30 min + 500 l TCA 25% washing the well

Add 10% TCA 500 l

Incubate 4°C 20 min

Spin 13000 rpm Eppendorf 10min 4°C

2 washes 100% aceton (do not dry too much pellet)

Resuspend dried pellet in 50 l water and then add -/+ mPEG 2x buffer (160mM Tris pH=8; 4% SDS; 80 mM mPEG 5000)

Incubate 37°C 15 min then 20 min RT rotating

SDS-PAGE 7,5% not reducing