Oct 25, 2022

Public workspaceWestern Blotting (Fly Heads) V.2

DOI
dx.doi.org/10.17504/protocols.io.8epv5j96jl1b/v2
  • 1Brigham and Women's Hospital;
  • 2Harvard Medical School
Daniel El Kodsi
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DOI: dx.doi.org/10.17504/protocols.io.8epv5j96jl1b/v2
Protocol Citation: Mel Feany 2022. Western Blotting (Fly Heads). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5j96jl1b/v2Version created by Daniel El Kodsi
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 25, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 71786
Keywords: ASAPCRN
Abstract
This protocol describes how to perform a Western Blotting technique using fly heads.
Homogenize desired number of fly heads in 1 X Laemmli sample buffer.
Heat samples to Temperature100 °C for Duration00:10:00 , spin briefly before loading.

10m
Load premade gel into western blotting apparatus. Fill reservoir with Running Buffer:
Running Buffer:
Amount6 g Tris-HCL
Amount28.9 g glycine
Fill to Amount1 L with distilled water
Add Amount5 mL 20% SDS

Load samples on gel and attach electrodes.
Run gel at 120 V until dye front reaches the bottom of the gel, Duration01:00:00 . Run longer for greater separation.

1h
Remove gel and transfer using Trans-Blot Turbo.
Perform antigen retrieval by microwaving Duration00:09:00 in PBS.

9m
Block membrane in 1X PBS with 0.05% Tween-20 and 3% dry milk for Duration01:00:00 .

1h
Add primary antibody at correct dilution in PBSTween + milk and incubate with shaking DurationOvernight at Temperature4 °C .

Wash blot 3x in PBSTween, Duration00:05:00 each, with shaking.

5m
Add secondary antibody at the correct dilution in PBSTween + milk, incubate with shaking at TemperatureRoom temperature for Duration03:00:00 .

3h
Wash blot in PBSTween Duration00:30:00 with frequent wash changes.

30m
Develop with ECL substrate or image fluorescence, as appropriate.