Osenses Protocols: Western Blot

 

 .................................................................................................................................................................................

 

1. Run the SDS-PAGE at 150 V.

 

2. Soak the gel for 15 min in transfer buffer then rinse it with MQ water.

 

3. Soak the membrane for 5 min in MQ water. Then dip it in the transfer buffer. We use NC membrane, pore size: 0.2 um.

 

4. Semi-dry transfer at 10 V (run for 90 minutes).

5. After transfer, rinse once the membrane with PBS and then wash 3x, 5 min each with PBS in 100 ml each time. Shake at 70 RPM.

 

6. Block with 1xPBS containing 1% LFDM for 30 min at RT with shake at 70 RPM.

 

7. Rinse with 70 ml of PBS.

 

8. Add the primary antibodies. Incubate O/N in the fridge (3-5C).

 

9. Wash the membrane 5 times, 5 min each with PBS-T containing 0.1% Tween-20. Wash in 100 ml with shake 70 RPM.

 

10. Add the Sigma HRP-Goat anti Rb (diluted 1: 13,000). Add 6 ml per membrane (6x8 cm). Incubate 30 min to 1 hour at RT with 70 RPM shake.

 

11. Wash 4 times, 5 min each with shake 70 RPM with 100 ml of PBS-T containing 0.1% Tween-20.

 

12. Wash 6 times more with 100 ml of PBS with shake 70 RPM.

 

13. Prepare the ECL substrate from BioRad. Use 1000 µl per membrane (500µl+ 500 µl).

 

14. Pipette 1000 µl of the substrate on the glas of the C-Digit. Place the membrane face down on the substrate, avoid air bubbles. Cover the membrane with Gladwrap and incubate 2 minutes. Then roll over the membrane. Scan the blot on C-Digit at High resolution (12 minutes).