Western Blotting (Fly Heads)

Homogenize desired number of fly heads in 1 X Laemmli sample buffer.

Heat samples to 100°C for 0h 10m 0s , spin briefly before loading.

Load premade gel into western blotting apparatus. Fill reservoir with Running Buffer:

Running Buffer:

6g Tris-HCL

28.9g glycine

Fill to 1L with distilled water

Add 5mL 20% SDS

Load samples on gel and attach electrodes.

Run gel at 120 V until dye front reaches the bottom of the gel, 1h 0m 0s. Run longer for greater separation.

Remove gel and transfer using Trans-Blot Turbo.

Perform antigen retrieval by microwaving 0h 9m 0s in PBS.

Block membrane in 1X PBS with 0.05% Tween-20 and 3% dry milk for 1h 0m 0s.

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

Wash blot 3x in PBSTween, 0h 5m 0s each, with shaking.

Add secondary antibody at the correct dilution in PBSTween + milk, incubate with shaking at Room temperature for 3h 0m 0s.

Wash blot in PBSTween 0h 30m 0s with frequent wash changes.

Develop with ECL substrate or image fluorescence, as appropriate.