Bovine serum albumin extraction procedure:
1, salting out take a centrifuge tube to add bovine serum 2mL, add an equal amount of PBS (phosphate-buffered saline) to dilute the serum, shaking well, drop by drop to add pH7.2 saturated ammonium sulfate solution 2mL, shaking as it is added, mixing well, and then let stand 10 minutes, and then centrifuged (2000r/min) Then centrifuge (2000r/min) for 10min, and pour the supernatant into the test tube.
2. Take a clean colorimeter plate, add one drop of Nasrid reagent in each well?
3, take a piece of cellophane, folded into a bag, the supernatant after centrifugation poured into the bag, with a line tied tightly to the upper mouth (pay attention to leave a gap), with a glass rod hanging in a 100mL beaker containing half a cup of distilled water, so that the lower half of the dialysis bag invades the water, the dialysis of protein solution, commonly used glass rod stirring the liquid outside the bag (in the beaker) to shorten the time of dialysis.
4, check every 2 minutes, replace the distilled water many times, check the NH4+ of the liquid outside the bag with Nasher's reagent, observe the color change and record it until the salt in the bag is dialyzed.
5, the bag of liquid poured into the test tube, that is, bovine serum albumin solution.
The identification method of bovine serum albumin:
1, spot sample take a film strip, put the film lusterless side down, put the film into the petri dish in the barbiturate buffer to make the film strip fully saturated, take it out, use the clean filter paper to suck away the excess buffer, take the lusterless side of the film to make a spot sampling line from the end of 1.5㎝, and put the sample of bovine serum and bovine serum albumin to be measured in the same film's spot sample line with the spotting device respectively. The bovine serum sample and bovine serum albumin solution to be tested were spotted at different positions on the spotting line of the same film. The standard solution was spotted once and the solution to be tested was spotted three times.
2, electrophoresis will be ordered after the membrane strips to the electrophoresis tank rack, placed in the film strips of the lusterless face down, the end of the sample to the cathode, to be equilibrated for 5min, turn on the power supply, power supply adjustment, electrophoresis instrument voltage of 160 volts, energized for 60min, turn off the power supply, and then use tweezers will be taken out of the mold strips.
3, staining the film strip directly immersed in the staining solution containing aminoblack 10B, stained for 2 minutes and then removed, immediately immersed in the rinsing solution, respectively, in the rinsing solution 1, 2, 3 each rinsed for 5min, until the background rinsed clean until the film was blotted dry with filter paper.
4. Identify and compare the electrophoresis results of albumin in the sample and the solution to be tested on the film to see if the position is consistent.
Expanded information:
Bovine serum albumin
Simple proteins in bovine serum It contains only hashish and hashish amine, and contains only 0.2% of lipid. Albumin consists of 581 amino acid residues, of which 35 cysteines form 17 disulfide bonds, and there is a free sulfhydryl group at the 34th position of the peptide chain. Albumin binds to a wide range of cations, anions, and other small molecules.
Albumin in blood mainly plays a role in maintaining osmotic pressure, PH buffering, carrier and nutrition. In the serum-free culture of animal cells, the addition of albumin can play a physiological and mechanical protective role and carrier role. Bovine serum albumin (BSA), also known as the fifth fraction, is a globulin in bovine serum, containing 583 amino acid residues, with a molecular weight of 66.430 Da and an isoelectric point of 4.7. BSA has a wide range of applications in biochemical experiments, such as as a Blocking agent in western blot.
? Baidu Encyclopedia - Bovine Serum Albumin