Principles of Biochemistry (2+1)

Procedure

• Remove the animal tissue and quickly transfer it into a beaker containing a premeasured volume of chilled homogenization buffer 5mM Tris-HCl buffer (pH7.4).
• Weigh and slice into fine pieces with the help of a pair of dissecting scissors at 0-4ºC. Drain the excess buffer.
• Add a calculated volume (5ml/g tissue) of homogenization buffer into the tissues and pour the homogeniser tube.
• Operate the homogeniser at about 500 rpm. Push the glass tube up and down 8-10 times to ensure adequate mechanical breakage of cells.
• Filter the homogenised tissue through 4 layers of fresh cheese cloth, moistened with the homogenization medium.
• Pour the filtrate into centrifugation tubes and centrifuge at 1000xg for 10min. Carefully separate the supernatant from the loose pellet, P₁ that settles down.
• Recentrifuge the supernatant at 3000xg for 10min. again carefully collect the loose and fluffy pellet and label it P_2.
• Centrifuge this supernatant once again at 10,000 xg for 30 min. and recover the pellet gently and label it P₃
• Similarly, centrifuge the supernatant at 50,000xg for 30min. and lastly at1,00,000xg for 40min. Recover pellets P₄ and P ₅ respectively. Store all the pellets in an ice jacket.