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 1mL 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