Osenses Protocols: Western Blot

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1. Run the SDS-PAGE at 250V.

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

3. Soak the membrane for 5 min in Di water.

4. Semi-dry transfer at 10 V

5. After transfer, rinse once the membrane with PBS and then wash 3x, 5 min each with PBS. Shake at 50 RPM.

6. Block with 1xPBS containing 1% LFDM for 30 min at RT with shake at 50 RPM.

7. Rinse with 70 ml of PBS.

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

9. Wash the membrane 5 times (5 min each) with PBST containing 0.1% Tween-20. Wash in 100 ml with shake 50 RPM.

10. Add the Sigma HRP-Goat anti Rb (diluted 1: 30,000). Add 6 ml per membrane (6x8 cm). Incubate 30 min to 1 hour at RT with 50 RPM shake.

11. Wash 4 times (5min each) with shake 50 RPM with 100 ml of PBST.

12. Wash 6 times more with 100 ml of PBS with shake 50 RPM.

13. Add ECL substrate.

14. Scan the blot on C-Digit at High resolution (12 minutes).