1) Bacteria or cells: collect bacteria or cells into the centrifuge tube, discard supernatant after
centrifugation. According to the proportion of bacteria or cells number (104): Extraction reagent Ⅰ
volume (mL) of 500-1000-1 to extract. It is suggested that 5 million of bacteria or cell amount with 1
mL of Extraction reagent Ⅰ. Split the bacteria or cell with ultrasonication (placed on ice, ultrasonic
power 200W, working time 3s, interval 10s, repeat for 30 times). Centrifuge at 8000 g for 10 minutes
at 4℃ to remove insoluble materials, and take the supernatant on ice before testing.
2) Tissue: According to the proportion of tissue weight (g): Extraction reagent Ⅰ volume (mL) of 1:5-10
to extract. it is suggested that 0.1 g of tissue with 1 mL of Extraction reagent Ⅰ and fully homogenized
on ice bath. Centrifuge at 8000 g for 10 minutes at 4℃ to remove insoluble materials, and take the
supernatant on ice before testing.
3) Serum (plasma) sample: detect sample directly. Centrifuge before detect if there are precipitation.
2. Cu/Zn SOD activity
1) Take the supernatant from the previous step. According to the proportion of supernatant volume (mL):
Extraction reagent II volume (mL) of 2:3 to mix. it is suggested that 0.2 mL of supernatant with 0.3
mL of Extraction reagent II and fully mixed for 1 minute. Centrifuge at 4000 g for 10 minutes at 4℃
to inactivate Mn SOD, and take the uppermost layer of the solution (Treated supernatant) to
determinate Cu/Zn SOD activity.
Note: there are three layers in the solution after centrifuge and tests only need the uppermost
layer.
2) According to the proportion of water volume (mL): Extraction reagent II volume (mL) of 2:3 to mix. it
is suggested that 0.2 mL of water with 0.3 mL of Extraction reagent II and fully mixed for 1 minute.
Centrifuge at 4000 g for 10 minutes at 4℃, and take the uppermost layer of the solution (Treated
water) to be the blank tube of Cu/Zn SOD activity detection.
II. Determination
