That should be enough. 1. experimental purpose: (1) to master the general steps of SOD enzyme extraction, separation and detection. (2) Understand two parameters of enzyme extraction: recovery rate and purification multiple. (3) Master the use of centrifuges. Second, experimental principle: Under alkaline conditions, pyrogallol can rapidly self-oxidize, release O2- and generate colored intermediate products. After 30 ~ 45s, the accumulation of intermediate products is linear with time, and the linear time is generally maintained within 4min. The intermediate products have strong light absorption at the wavelength of 420nm. When SOD exists, it can catalyze the combination of O2- and H+ to produce O2 and H2O2, thus preventing the accumulation of intermediate products. Therefore, the enzymatic activity of SOD can be determined by measuring light absorption. 3. Experimental reagents: garlic, cold acetone, phosphate buffer (PH7.8, 0.05mol/L), chloroform-ethanol mixed solution, pyrogallol, concentrated hydrochloric acid, balance, quartz sand, mortar, freezing centrifuge, 8 50mL centrifugal tubes, UV-Vis spectrophotometer, 8 250mL triangular bottles, 8 glass rods and 3 250mL triangular bottles. Experimental steps: (1)SOD extraction: Weigh 5g garlic cloves, add quartz sand to grind and break the cells, add 15mL phosphate buffer with pH of 7.8 0.05mol/L, grind and stir for 20min to fully dissolve SOD, centrifuge at 6000rpm, discard the precipitate, and take the supernatant. (1mL for later use, accurately measure the volume of the remaining supernatant, and record) (2) deproteinization: add 1/4 volume chloroform-ethanol mixed solution to the extract, stir 10min, centrifuge at 6000rpm 15min for precipitation to obtain crude enzyme solution. (take 1mL crude enzyme solution for standby, and accurately measure the volume of the remaining crude enzyme solution) (3)SOD enzyme precipitation separation: add the same volume of cold acetone to the remaining crude enzyme solution, stir 15min, and centrifuge at 6000rpm 15min to obtain SOD enzyme precipitation. Add 2mL phosphate buffer to the precipitate, dissolve it, then add 3mL, and mix well. Centrifuge at 6000 rpm for 65438 05 minutes, and take supernatant to obtain SOD enzyme solution. Take 1 ml for standby, and measure the volume of the remaining part. (4) Determination of activity of crude enzyme solution: detection of SOD activity in extract, crude enzyme solution and enzyme solution, with specific steps as follows. After adding pyrogallol, mix quickly, accurately time for 4min, add a drop of concentrated hydrochloric acid to stop the reaction, and measure the absorbance at 420nm. Reagent/(mL) blank tube control tube OD 1 extract OD2 crude enzyme solution OD2PH8.3 buffer 33333SOD extract 000. 10. 1 distilled water 21.81.71. 5) Determination of soluble protein content in solution: Take 0.3mL of standby extract, crude enzyme solution and enzyme solution from 1mL, dilute them according to the following multiples, measure the absorbance at 260nm/280nm, and calculate the protein concentration according to the formula. Dilution of extractive solution: 50 × crude enzyme solution dilution: 20 × enzyme solution dilution: 10 × 5. Experimental data: the total volume of crude enzyme solution (ml) is 260 nm, and the absorbance value of crude enzyme solution is 280nm. Formula protein concentration (mg/ml) = (1.45a280-0.74a260). × dilution multiple protein concentration (mg/ml) Control tube (OD 1) extract (OD2) crude enzyme solution (OD2) enzyme solution (OD2)420nm absorbance value VI. Experimental results and data processing: (1) enzyme activity unit: = 2 (od 1-od2) 0. 1= crude enzyme activity unit: = 2 (od1-od2) × 5/0.1 Total activity U= activity unit × total volume = total activity protein concentration of crude enzyme =(4) purification times of crude enzyme solution = specific activity of crude enzyme solution/specific activity of extract solution = purification times of enzyme solution = specific activity of enzyme solution/specific activity of extract solution =(5) recovery rate = total activity of crude enzyme solution/total activity of extract solution = enzyme solution recovery rate = total activity of enzyme solution/total activity of extract solution = VII. Error analysis of experimental results: (1) (2) Error caused by measuring instrument precision. (3) During the operation, the pyrogallol is not evenly mixed, which may lead to insufficient chemical reaction, which may be one of the reasons why the experimental data is inconsistent with expectations; (4) During the experiment, the amount of chemical reagents needed is not very accurate, which may be another reason for the problem of experimental data; (5) In addition, the experiment itself is still rough, the extraction, separation and purification of enzyme are not complete, and there are many miscellaneous proteins that interfere with the experiment.