Osenses Protocols: Western Blot

Lysates were prepared in GLB-1 lysing buffer (containing 1% SDS, 1% SDC, 1% Triton X-100). Tissues were pulverised in liquid nitrogen followed by sonication.

1. Weigh the tissues. Use 1 ml of lysing buffer for every 100 mg tissue.
2. Keep the tissues in liquid nitrogen.
3. Using pestle & mortar pulverise the tissue in liquid nitrogen.
4. Add the the pulverised tissue to lysing buffer and mix well with pipette without making foam.
5. Transfer the homogenate to the tube. Place on ice horizontally and keep shaking until all the tissues are pulverised.
6. Using Hielscher 200St-T sonicator, sonicate the samples for 1x + 1x +1x set at: time: 40 sec; AMPL: 50% in the start and then increase to 100% to avoid foaming; C (Pulse) at 80% (Power will vary accordingly, the amplitude is constant).
7. Place the tip of the probe in the middle of the liquid. Use 10-ml tubes for a volume of 2 ml to 9 ml. For volume more than 9 ml, divide the homogenate in two or more vials. For 1 ml to 1.5 ml use Axygen 2-ml tube. Keep the vial in the Cooler while sonicating and after each cyle of sonication, invert the vial 3 times and then keep the vial on ice horizontally and shake for 60 minutes.
8. And finally, sonicate one more time after the 60 minutes incubation. Incubate on ice for 5 minutes. The homogenate should not be viscous and some homogenates like liver becomes translucent. For volume of 0.5 ml to 2 ml use probe S26d1; 2 ml to 50 ml use S26d2; for 20 ml to 500 ml use S26d7. Centrifuge at 4C for 30 minutes at 20000 xg.
9. Carefully, collect the supernatant and discard the pellet.
10. Add the loading buffer. At this stage, we typically aliquot (in 1.5 ml and 200 ul), label and store the lysates at -20C. When needed, take one vial out and heat it with shake for 25 minutes at 75C (start the timer once you place the lysate in the heating block ie starting at room temperature). After this step, add the Osenses' Red-5 colour.
11. Sample is ready now for WB.
12. The remaining can be refrozen.
    

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1. Run the SDS-PAGE at 100V.

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 (160 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 Licore HRP-Goat anti Rb (diluted 1: 12,000). 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 Licore (Westernsure). 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).