1, salting-out: add 2mL of bovine serum into a centrifuge tube, add the same amount of PBS (phosphate buffered saline) to dilute the serum, shake well, add 2ml of saturated ammonium sulfate solution with pH 7.2 drop by drop, shake well while adding, mix well, then stand for10min, and centrifuge (2000r/min) 65438+.
2. Take a clean shade guide and drop a drop of Nessler's reagent in each hole?
3. Take a piece of cellophane, fold it into a bag, pour the centrifuged supernatant into the bag, tie the top tightly with a thread (pay attention to leave a gap), and hang it in a 100mL beaker filled with half a cup of distilled water with a glass rod, so that the lower part of the dialysis bag invades the water and dialyzes the protein solution. The liquid outside the bag (in the beaker) is usually stirred with a glass rod to shorten the dialysis time.
4, check once every 2 minutes, change distilled water for many times, use Nessler's reagent to check the NH4+ of the liquid outside the bag, observe the color change and record it until the salt dialysis in the bag is finished.
5. Pour the liquid in the bag into the test tube to obtain the bovine serum albumin solution.
Identification method of bovine serum albumin;
65438+ standard liquid point once, liquid point to be measured three times.
2. Electrophoresis: Place the spotted membrane strip on the electrophoresis tank frame, with the frosted surface facing down and the spotted end facing the cathode. After balancing for 5+/-5 minutes, turn on the power supply, adjust the power supply, adjust the voltage of the electrophoresis apparatus to 160V, and power on for 60 minutes. After turning off the power supply, take out the membrane strip with tweezers.
3. Dyeing: directly immerse the film strip in the dyeing solution containing amino black 10B, take it out after dyeing for 2 minutes, immediately immerse it in the rinsing solution, rinse it in the rinsing solution 1, 2 and 3 for 5 minutes respectively until the background is rinsed clean, and blot the film 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 positions are consistent.
Extended data:
bovine serum albumin
Simple protein in bovine serum is the main component of blood (38g/ 1000ml) with a molecular weight of 68kD. Isoelectric point 4.8. The nitrogen content is 16% and the sugar content is 0.08%. It only contains hexose and hexosamine, and the fat content is only 0.2%. Albumin consists of 58 1 amino acid residues, of which 35 cysteines constitute 17 disulfide bonds, and there is a free sulfhydryl group at the 34th position of the peptide chain. Albumin can bind to many cations, anions and other small molecules.
Albumin in blood mainly plays the role of maintaining osmotic pressure, PH buffer, carrier and nutrition. In serum-free culture of animal cells, adding albumin can play a physiological and mechanical protection and carrier role. Bovine serum albumin (BSA), also known as the fifth component, 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. Bovine serum albumin is widely used in biochemical experiments, for example, as a blocking agent in protein blot.
? Baidu encyclopedia-bovine serum albumin