Nov 08, 2022
Western Blot
- 1Icahn School of Medicine at Mount Sinai
- Ahfeldt Lab
Protocol Citation: Elena Coccia, gustavo.parfitt 2022. Western Blot. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7y428gwz/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: October 27, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 71931
Keywords: ASAPCRN, western blot, protocol
Abstract
Western Blot Protocol
Cell lysis preparation
50m
Prepare the appropriate amount of RIPA buffer with Protease and Phosphatases Inhibitor Cocktail (Roche) and maintain On ice
Collect cells/organoids in PBS On ice in an eppendorf and spin down 500 rpm, 4°C, 00:05:00
5m
Add appopriate amount of lysis buffers (1:50 v/v proportion between cell pellet and lysis buffer) to the pellet and pipette up and down
For organoid samples use an homogeniser
Incubate samples for 00:30:00 On ice . Spinning every 00:10:00
40m
Centrifuge at 15000 x g for 00:10:00 at4 °C and collect the supernatant in a new tube.
10m
If SDS fraction is desired, add 2 Mass / % volume SDS to RIPA buffer and resuspend pellet from step 5.
Sonicate the sample a 3 times for00:00:10 to ensure fragmentation of genomic DNA released during lysis and consequently reduce viscosity
10s
Quantify protein concentration.
SDS-PAGE
Mix 20-30 µg of protein with Laemmli's loading buffer
Denaturate the proteins by incubation at 95 °C for 00:05:00 , spin briefly before loading
5m
Load pre-cast gel into Western Bloat apparatus and fill with Running Buffer (BioRad).
Load samples and protein ladder into gels, Run the gel at 120V for 00:45:00 to ensure protein separation.
45m
Protein Immunodetection
Transfer proteins into a PVDF membrane (previously activated with methanol and hydrated in ddH2O)
Remove membrane from transfer and place into a box with blocking buffer: 5% BSA In TBS-T (20mM Tris-HCl, 150mM NaCL pH8, 0.1% Tween20). Block for 01:00:00
1h
If alpha-synuclein is to be blotted, fix the membrane with 4% PFA in PBS for 00:30:00 prior to blocking
30m
Once blocked, sequentially probe the membrane for antibody staining and detection. Antibody dilution might need optimization.
Prepare primary antibody in 5% BSA in TBS-T and left incubating Overnight at 4 °C
30m
Wash membranes 3x 00:10:00 in TBS-T and then incubated for 01:00:00 at Room temperature with the appropriate HPR-tagged secondary antibody.
1h 10m
Membrane was washed 3x 00:10:00 in TBS-T
10m
Mix EZ-ECL solution 1:1 (v/v) and incubate on the membrane for 00:01:00
1m
Image membranes using a Licor Odyssey Chemioluminescence Imager.