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 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 shake for 30 min.
  6. Using Hielscher 200St-T sonicator, sonicate the samples for 1x + 1x +1x set at: time: 40 sec; AMPL: 100%; C (Pulse) at 80% (Power will vary accordingly, the amplitude is constant). 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 cycle of sonication, invert the vial 3 times and then keep the vial on ice horizontally and shake for 60 minutes.
  7. And finally, sonicate one more time after the 60 minutes incubation. Incubate on ice for 5 minutes.
  8. 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.
  9. Centrifuge at 4C for 30 minutes at 20000 xg.
  10. Carefully, collect the supernatant and discard the pellet.
  11. Add the loading buffer. At this stage, we typically aliquot (in 1.6 ml and 200 ul), label and store the lysates at -20C. When needed, take one vial out and heat it with shake for 15 minutes at 70C (start the timer once you place the lysate in the heating block ie starting at room temperature). After this step, add the Osenses' Pink-2 colour.
  12. Sample is ready now for WB. The remaining can be refrozen.