Oct 25, 2022

Western Blotting (Fly Heads) V.2

DOI
https://dx.doi.org/10.17504/protocols.io.8epv5j96jl1b/v2
  • 1Brigham and Women's Hospital;
  • 2Harvard Medical School
  • Daniel's workspace
Daniel El Kodsi
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DOI: https://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, western blotting technique, using fly head, western blotting, blotting technique, fly head, head, protocol
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 100 °C for 00:10:00 , spin briefly before loading.

10m
Load premade gel into western blotting apparatus. Fill reservoir with Running Buffer:
Running Buffer:
6 g Tris-HCL
28.9 g glycine
Fill to 1 L with distilled water
Add 5 mL 20% SDS

Load samples on gel and attach electrodes.
Run gel at 120 V until dye front reaches the bottom of the gel, 01:00:00 . Run longer for greater separation.

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

9m
Block membrane in 1X PBS with 0.05% Tween-20 and 3% dry milk for 01:00:00 .

1h
Add primary antibody at correct dilution in PBSTween + milk and incubate with shaking Overnight at 4 °C .

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

5m
Add secondary antibody at the correct dilution in PBSTween + milk, incubate with shaking at Room temperature for 03:00:00 .

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

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