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Manual biochemistry

Manual biochemistry

   First, the steps of biochemical experiments

  Manual biochemistry is operated according to the kit instructions. The experiment operation of different indexes is different, mainly for antioxidant indexes, including SOD, MDA, GSH-PX, GSH, NO, NOS, CAT.

  1. Sample pretreatment

  Serum and plasma: If there is turbidity, need about 3500 rpm, centrifuge for 10 minutes, and take the supernatant for use. Clarified serum blood

  The pulp was diluted with saline to different concentrations for preliminary experiments.

  Tissue: Weigh the tissue accurately and add 9 times the volume according to the ratio of weight (mg): volume (ul) = 1:9

  water, cut the tissue, prepare the homogenate in an ice-water bath, centrifuge at 2500-3000 rpm for 10 minutes, take the supernatant that is 10% of the homogenized supernatant for later use. Tissue samples need to measure protein concentration.

  2. BCA protein concentration determination

  Standard preparation: Take 1ml of protein standard preparation solution and add it to a 25mgBSA protein standard tube to completely dissolve the protein standard, which is a 25mg/ml protein standard solution. The prepared protein standard solution can be stored for a long time at -20℃. Take an appropriate amount of 25mg/ml protein standard solution and dilute to a final concentration of 0.5mg/ml. It is best to dilute the standard with the same solution as the protein sample, usually using 0.9% NaCl or PBS to dilute the standard.

  Sample addition: Add 0,1,2,4,8,12,16,20ul of the standard to a 96-well plate, and add standard diluent to make up to 20ul if it is less than 20ul. Dilute the protein sample to be tested to an appropriate concentration and add it to a 96-well plate to make the total volume of the sample to be tested also 20ul.

  BCA working solution preparation: According to the number of samples, prepare an appropriate amount of BCA working solution according to 50 volumes of BCA reagent and 1 volume of copper sulfate solution (50:1), and mix well. The BCA working fluid is stable within 24 hours at room temperature.

  Reaction and detection: Add 200ul of BCA working solution to each well, mix well (you can put the plate on a shaker for 30s), place it at 60°C for 30 minutes, use standard curve No. 0 as reference, and compare at 562nm wavelength Color measurement, record the absorbance value of each hole.

  Calculation: Use the standard absorbance value as the abscissa and the standard concentration as the ordinate to draw the standard curve. According to the absorbance value of the measured sample, the corresponding protein concentration can be inversely calculated on the standard curve, and the actual concentration of the sample is multiplied by the sample dilution factor.

  3. According to the instructions of different indicators, set the control tube and the blank tube according to the operation table, add the corresponding reagents and samples, measure the absorbance value at the specified wavelength after the reaction, and calculate the sample index content according to the calculation formula.

  Second, matters needing attention

  1. Sample requirements: The sample should be clear and transparent, and the suspended matter should be removed by centrifugation; avoid using hemolytic samples. If the samples are not tested immediately after collection, they should be frozen and stored in a -80° refrigerator to avoid repeated freezing and thawing. The samples should be mixed well before each test and use. After use, record the corresponding layout and sample number on the laboratory book, and put the samples in the refrigerator in time.

  2, preparation before experiment

  Check whether the number of samples, number, and sample volume are sufficient, whether there is hemolysis, and check the shelf life of the kit. Pay attention to the customer's requirements in the remarks column on the order, whether to do re-holes, etc. If there is any problem, communicate with sales in time.

  Third, operation requirements

  1. The reagent preparation must be accurate, the sample amount must be accurate, and the samples and reagents in the pipette must be completely exhausted;

  2. The incubation temperature and time are accurate; strictly follow the steps in the instructions.

  3. Use a multi-channel pipette to add common reagents or substrate application solutions to shorten the time and reduce the error between each well.

  4. Mix thoroughly after adding the sample to ensure full contact between the sample and the reagent.

  5. Some index reagents need to be stored at -20°C, and some reagents will appear turbid or crystallized under low temperature conditions and need to be dissolved at 37°C. Pay attention to the label on the kit.

  6. The NOS accelerator is best prepared for immediate use. After preparation, try to use it up within one day. If there is any remaining, it should be stored below -20°C for no more than a week; before configuration, such as light yellow or white powder becomes brown or tan particles It is invalid and unavailable.

  7. GSH-PX standard products should be prepared and used immediately.

  8. SOD enzyme is unstable in summer, but better in winter, try to control the experiment time in the same time period.

  9. The CAT experiment is prone to bubbles and precipitation. If precipitation occurs, take the supernatant and then test again to avoid sucking bubbles.

  10. Since the microplate reacts in a water bath, there will be water at the bottom of the plate, so before each test, the bottom of the plate must be wiped dry and then placed in the microplate reader for testing. Be careful of scalding when the water bath is boiling.

  Note: The above are the precautions for common indicators in the laboratory. If there is a kit ordered by the customer, please operate in strict accordance with the instructions.

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