Mar 04, 2022
Western Blot
- 1University of Ottawa
Protocol Citation: Haley Geertsma 2022. Western Blot. protocols.io https://dx.doi.org/10.17504/protocols.io.b5wkq7cw
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: March 04, 2022
Last Modified: March 04, 2022
Protocol Integer ID: 59052
Abstract
This protocol is used to western blot proteins-of-interest.
Add 4X Laemmli buffer to protein samples and incubate at 95oC for 7 minutes.
7m
Load samples into 12% polyacrylamide gel and run in running buffer at 80-100V for 60-120 minutes.
2h
Transfer gel to 0.2μm nitrocellulose membrane at 350mA for 60 minutes at 4oC.
1h
Wash membrane with 1X TBS-T then block in 10% milk for 30 minutes at room temperature.
1X TBS-T: 10mM Tris-HCl + 150mM NaCl + 0.5% Tween-20 in H2O, pH 8.0
35m
Wash membrane with 1X TBS-T for 4x 5 minutes.
20m
Incubate membrane in primary antibody solution overnight at 4oC.
Antibodies diluted to the appropriate concentration in 2% bovine serum albumin in 1X TBS-T with 0.02% sodium azide
1d
Wash membrane with 1X TBS-T for 4x 5 minutes.
20m
Incubate membrane in secondary antibody solution for 1 hour at room temperature.
Secondary antibody (horseradish peroxidase conjugated) is diluted in 10% milk.
1h
Wash membrane with 1X TBS-T for 4x 5 minutes.
20m
Rinse ECL Clarity Solution over membrane 20x then image.
1h
Once imaging is complete, wash membrane with 1X TBS-T x2 then H2O x2 then let dry at room temperature overnight before storing.
10m