Western Blotting

1. Run and SDS-PAGE gel with the samples to be analyzed. Use a pre-stain marker for the molecular weight standards.

2. Prepare transfer solution using cold H2O. Pour into a deep tray and allow the bubbles to disperse. Separately soak the white pads in the buffer.

3. Cut the PVDF membrane to the appropriate size and wet it in methanol for 15 seconds, followed by 2 minutes in H2O. Leave it submerged in transfer buffer until ready to make the western sandwich.

4. After the gel has finished running, carefully remove the front glass plate and remove the stacking gel. Equilibrate the gel 10 minutes in transfer buffer.

5. Prepare the western sandwich in the deep tray containing the transfer solution. Pt down a white pad, 3 sheets of 3 mm Whatman paper, the PVDF membrane, the gel with the visible molecular weight markers to the right (this will transfer the proteins in left to right orientation onto the membrane – the way they were loaded), 3 more sheets of 3 mm Whatman paper and a second white pad. Remove all bubbles from within the sandwich by rolling a pipette over the set-up. Then put the sandwich within the holder, with the PVDF membrane closest to the transparent side.

6. Mount the sandwiches in the transfer device, putting the black sides of the holders near the black side of the transfer device.

7. Fill the device with the transfer solution; add an ice pack (to cool down the buffer during the transfer) and a stir bar (to mix the buffer during transfer, allowing it to cool better, and preventing it from fractionating). Transfer at 90 V for 90 minutes. The transfer can also be preformed overnight at 25 mAmps at RT.

7. Dry the membrane to insure the proteins stay bound throughout the protocol. Dip the membrane 10 seconds in methanol and leave to dry for 15 minutes. After wards, reactivate the membrane by dipping it in methanol (10 seconds), water (10 seconds) and PBS-T (10 seconds).

8. Block the membrane in 5% milk in PBS-T for 1 hour at RT with shaking.

9. Add the first antibody in 1% milk in PBS-T and incubate at RT for 1 hour with shaking. Alternatively, the first antibody can be added and left overnight in the cold room with shaking.

10. Wash the membrane 3x5mintes with PBS-T at RT.

11. Add the second antibody in 1% milk in PBS-T and incubate 15 minutes at RT with shaking (the secondary antibody is at a 1:2500 dilution – 2 mL in 5 mL of 1% milk in PBS-T).

12. Wash the membrane 3x5mintes with PBS-T at RT.

13. Detect the protein of interest with Amersham ECL kit.

14. Stain the membrane with amino-black stain solution for recording.

Reagents

Transfer buffer

1x SDS electrophoresis buffer 20% methanol (the concentration of methanol can be varied according to the protein: lower percentage for higher molecular weight proteins)

Amido Black Stain

0.1% Naphthol Blue Black (amido black) 10% Acetic Acid 7.5% Methanol

Amido Black Destain

10% Acetic Acid 7.5% Methanol