PBS, or Tris-buffer, pH7.2-7.4

For antioxidants

·         Weighted the organs i.e liver.

·         Added phosphate buffer (pH 7.4) 4 times greater than the weight of organ i.e 1:4.

·         Homogenize it for 15 minutes with the help of pestle and mortar or with the help of homogenizer with short intermissions.

·         Passed the homogenized tissues through muslin cloth to remove the biomass.

·         Filtered the liquid obtained from muslin cloth with the help of whatman filter paper no. 1.

·         Centrifuged the filtrate obtained above step in refrigerator centrifugal machine at 10, 000 rotates per minutes for 15 minutes.

·         Both sediments and supernatants separated for further analysis.

·         All the enzyme isolation steps will be carried out at 4°C.

The supernatants and sediments were stored at 4°C until further analysis.

Preparation of 50 mM phosphate buffer (pH 7.0):

0.371 g NaH2PO4 and 0.270 g Na2HPO4 was taken in a flask and added distilled water to make volume upto 100 mL and pH was adjusted 7.0.

thanks a lot