Apr 03, 2023
Western Blot Analysis
- 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Protocol Citation: michela.deleidi, Federico Bertoli 2023. Western Blot Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkwpy5l5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 01, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 79873
Keywords: ASAPCRN
Abstract
Western blot protocol
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.