Osenses Protocols: Western Blot

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1. Run the SDS-PAGE at 180V.

2. Soak the gel for 15 min in transfer buffer (prepared using methylated EtOH) then rinse it with MQ water.

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

4. Semi-dry transfer at 10 V (run for 2 hours for 4 gels/blots; 1.5 hours for two gels/blots)

5. After transfer, rinse once the membrane with 200 mM NaCl and then wash 3x, 5 min each with 200 mM NaCl in 100 ml. 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 200 mM NaCl.

8. Assemble the MPX (from LiCore) and add the primary antibodies (170 µl/channel). Incubate O/N in the fridge (3-5C).

9. Aspirate the antibodies and dissemble the MPX. Wash the membrane 5 times (5 min each) with 200 mM NaCl-T containing 0.1% Tween-20. Wash in 100 ml with shake 70 RPM.

10. Add the 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 70 RPM shake.

11. Wash 4 times (5min each) with shake 70 RPM with 100 ml of 200 mM NaCl-T containing 0.1% Tween-20.

12. Wash 6 times more with 100 ml of 200 mM NaCl with shake 70 RPM.

13. Prepare the ECL substrate from BioRad. Use 800 µl per membrane (400µl+ 400 µl).

14. Pipette 800 µl of the substrate on the glass 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).