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 (prepared using methylated EtOH) then rinse it with water.

3. Soak the membrane for 5 min in 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 in 100 ml each time. Shake at 70 RPM.

6. Block with 1xPBS containing 0.5% 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 PBST.

10. Add the secondary antibody. Incubate 30 min to 1 hour at RT with 70 RPM shake.

11. Wash 4 times with PBST.

12. Wash 6 times more with 100 ml of PBS.

13. Add the ECL substrate.

14. Scan the blot on C-Digit at High resolution.