Osenses Protocols: Western Blot

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1. Run the SDS-PAGE at 250V.

2. Soak the gel for 10 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 in 100 ml. 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 with antibody 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 50 RPM.

10. Add Osenses' rabbit anti Gp adaptobody at 1:3000 dilution and incubate 30 min, wash and incubate with Sigma HRP-Goat anti Rb diluted 1: 15,000). 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 PBS-T containing 0.1% Tween-20.

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

13. Prepare the ECL substrate from BioRad.

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