Western Blot Analysis
michela.deleidi, Federico Bertoli
Abstract
Western blot protocol
Steps
Wash cells 1X in PBS.
Detach cells using Accutase for 5 minutes at 37°C and collect them.
Spin cells in a centrifuge at 250g for 5 minutes at room temperature.
Remove the supernatant.
Lyse cells in 1% TBS + 0.5% NP40 + PI/PHI (Pierce #A32959).
Determine protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
Dilute samples in 6x Laemmli buffer containing 12.5% β-mercaptoethanol.
Boil samples 5 minutes at 95°C in a thermoblocker.
Load protein in a self-casted 7.5-15% acrylamide gel or in precast NuPage 4-12% Bis-Tris Protein gel.
Run gel at 100V for 30min-2h.
Transfer proteins to PDVF membrane via wet transfer (20% methanol) overnight at 20V at 4°C.
Block membrane in TBS 0.1% Tween containing 5% milk or 5% bovine serum albumin.
Incubate primary or secondary antibodies in 5% Roche Block solution or 5% BSA in TBS-T supplemented with 0.04% sodium azide.
