4 ℃ pre-chilled PBS to rinse the tissue blood stains, filter paper to absorb residual PBS;
Weigh, add lysate homogenate (on ice);
The obtained homogenate was repeatedly frozen and thawed 3 times with liquid nitrogen;
Let stand at room temperature for 30mins, centrifuge at 15000r / min at 4 ℃ for 1h, take the supernatant, divide and store at -80 ℃.
Introduction: Objective To establish an immunodetection analysis method for P53 protein in animal tissues, and to provide a detection method for the metabolic distribution study of P53 biological drugs. Methods Horseradish peroxidase (HRP) labeled anti-P53 protein monoclonal antibody 3P40, using the labeled antibody and recombinant P53 protein detection to establish a competitive ELISA detection method; using this method to establish a standard curve of P53 protein in serum samples, The concentration of P53 in the serum of mice at different times after intraperitoneal injection of P53 was determined. Results The 3P40 purified by affinity chromatography was used for HRP labeling, and the titer of 3P40HRP was detected by ELISA up to 1: 200 000; the established competitive ELISA method can detect P53 protein in PBST with a sensitivity of 30 ng / ml, and the intraday precision High, with a relative standard deviation (RSD) of less than 5%, reflecting good reproducibility; using normal mouse serum and PBST diluted recombinant P53 protein samples for competitive ELISA testing, the results show that mouse serum components have P53 protein detection Certain influence; the standard curve of P53 protein was prepared by serum, and the concentration of P53 protein in the serum samples of mice at different time points after intraperitoneal injection of P53 protein was detected. Conclusion The HRP labeling of anti-P53 monoclonal antibody 3P40 was carried out, and a competitive ELISA method was established, which can be used to detect the plasma concentration of P53 protein drugs.
When the sample is temporarily unpredictable, it will be stored at minus 80, generally it can be stored for about 6 months, and minus 20 can be stored for 3 months. The extract is not the most suitable environment for the substance to be tested) 1 Accurately weigh the tissue weight (g): volume (ml) = 1: N ratio plus N volumes of normal saline (or PBS) (recommended N = 9) , Ice water bath homogenization (mechanical, manual, etc., ultrasound is not recommended), prepare a homogenate with a certain concentration, centrifuge (according to the test requirements of the tested substance, it is recommended to be about 3000 rpm), take the supernatant for testing 2 Due to certain systematic errors that may occur during the homogenization (extraction) process, it needs to be corrected according to the protein concentration in the supernatant.
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