Measurement of superoxide dismutase (SOD) concentration
Measurement of superoxide dismutase (SOD) concentration
Mouse SOD was determined by enzyme-linked immunosorbent assay (ELISA). By double-antibody one-step sandwich method, in a 96-well plate coated with SOD antibody, the specimen, standard and HRP-labeled detection antibody were added, incubated by a constant temperature incubator and washed thoroughly.
The substrate TMB is added to develop the color. TMB is converted to blue by catalysis of peroxidase, and converted to final yellow by the action of acid. The color is positively correlated with the SOD in the sample, and the concentration of the sample is calculated by measuring the absorbance (OD) at 450 nm using an enzyme counter.
Operation method
Mouse Superoxide Dismutase (SOD) Assay - ELISA
Materials and Instruments
Test samples, pipette guns, enzyme markers, constant temperature incubators, mouse superoxide dismutase (SOD) ELISA test kits, etc. Move 1. Sample collection and processing Mouse serum: Blood was collected in tubes free of heat and endotoxin and centrifuged at 3,000 rpm/min for 10 minutes to rapidly and carefully separate serum from erythrocytes. Plasma: Clean EP tubes are moistened with sodium heparin, blood is collected, centrifuged at 3,000 rpm/min for 10 minutes, and the supernatant is carefully collected. Tissue homogenate: take mouse tissue, add appropriate amount of saline and mash, centrifuge at 3,000 rpm for 10 minutes and collect the supernatant. Cell culture supernatant: 1) To detect secreted factors, collect the cell supernatant and centrifuge at 2,000~3,000 rpm/min for 20 minutes; 2) To detect intracellular factors, freeze-thaw the cells repeatedly with liquid nitrogen to induce the cells to destroy and release the intracellular factors. 2,000~3,000 rpm/min centrifugation for 20 minutes, then collect the supernatant. If the test is not timely, freeze at -20 ℃, thaw at room temperature and ensure that the sample is thawed uniformly and fully when the test is performed. 2. Kit Storage Store the mouse superoxide dismutase (SOD) ELISA kit at 2~8 ℃ and equilibrate at room temperature for 20 minutes before use. 3. Reagent Preparation Prepare washing buffer according to the instruction of the kit and dilute 1:20, i.e., 1 part of 20 x washing buffer plus 19 parts of distilled water. 4. Detection steps (1) Remove the plate after equilibrating for 20 min at room temperature, and seal the remaining self-sealing bag and put it back to 4 ℃. (2) Set up the standard wells, sample wells and blank wells, and add 50 μL of different concentrations of standards to each standard well. (3) If the content of the detection factor in the sample is too high, it is necessary to dilute the sample, i.e., add 10 μL of the sample to be tested, and then add 40 μL of the sample diluent to the sample well; if not, add 50 μL of the sample to be tested to the sample well, and do not add it to the blank well. (4) In addition to the blank wells, add 100 μL of HRP-labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with a sealing film, and incubate at 37 ℃ for 1 h in a sealed thermostat. 5) Discard the liquid, pat dry on absorbent paper and add washing solution, leave for 1 min, shake off the washing solution and pat dry, repeat the plate washing 5 times. (6) Add 50 μL of substrate A and B to each well and incubate at 37 ℃ for 15 min under low light. (7) Add 50 μL of termination solution to each well, and measure the OD value of each well at 450 nm within 15 min. 5、Results judgment Draw the standard curve, take the concentration of the standard as the horizontal coordinate and the corresponding OD value as the vertical coordinate, draw the linear regression curve of the standard, and calculate the concentration value of each sample according to the curve equation. Caveat 1. High centrifugation speed during sample preparation, resulting in hemolysis. 2. the test kit was not completely dissolved at room temperature before use. 3. the incubation operation was not carried out according to the time, amount and sequence of liquid addition indicated in the instruction manual. 4. liquid reagents were not shaken well before use. For more product details, please visit Aladdin Scientific website.