Nov 08, 2022

Public workspaceWestern Blot

DOI
dx.doi.org/10.17504/protocols.io.q26g7y428gwz/v1
  • 1Icahn School of Medicine at Mount Sinai
Elena Coccia
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DOI: dx.doi.org/10.17504/protocols.io.q26g7y428gwz/v1
Protocol Citation: Elena Coccia, gustavo.parfitt 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
Abstract
Western Blot Protocol
Cell lysis preparation
Cell lysis preparation
50m
50m
Prepare the appropriate amount of RIPA buffer with Protease and Phosphatases Inhibitor Cocktail (Roche) and maintain TemperatureOn ice

Collect cells/organoids in PBS TemperatureOn ice in an eppendorf and spin down Centrifigation500 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 Duration00:30:00 TemperatureOn ice . Spinning every Duration00:10:00
40m
Centrifuge at Centrifigation15000 x g for Duration00:10:00 atTemperature4 °C and collect the supernatant in a new tube.

10m
If SDS fraction is desired, add Concentration2 Mass / % volume SDS to RIPA buffer and resuspend pellet from step 5.

Sonicate the sample a 3 times forDuration00:00:10 to ensure fragmentation of genomic DNA released during lysis and consequently reduce viscosity

10s
Quantify protein concentration.
SDS-PAGE
SDS-PAGE
Mix Amount20-30 µg of protein with Laemmli's loading buffer

Denaturate the proteins by incubation at Temperature95 °C for Duration00:05:00 , 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 Duration00:45:00 to ensure protein separation.

45m
Protein Immunodetection
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 Duration01:00:00

1h
If alpha-synuclein is to be blotted, fix the membrane with 4% PFA in PBS for Duration00:30:00 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 DurationOvernight at Temperature4 °C

30m
Wash membranes 3x Duration00:10:00 in TBS-T and then incubated for Duration01:00:00 at TemperatureRoom temperature with the appropriate HPR-tagged secondary antibody.

1h 10m
Membrane was washed 3x Duration00:10:00 in TBS-T
10m
Mix EZ-ECL solution 1:1 (v/v) and incubate on the membrane for Duration00:01:00

1m
Image membranes using a Licor Odyssey Chemioluminescence Imager.