【Purpose】
Master the principle of BCA method to determine protein concentration.
【Principle】
A reagent composed of BCA (bicinchoninincacid) and other reagents such as copper sulfate of divalent copper ions, when mixed together, it becomes apple green, that is, BCA working reagent. Under alkaline conditions, when BCA binds to protein, the protein reduces Cu2+ to Cu+, one Cu+ chelates two BCA molecules, the working reagent forms a purple complex from the original apple green, and the maximum light absorption intensity is proportional to the protein concentration.
【Operate】
Drawing of standard curve: take seven test tubes numbered and operate according to the following table:
【Calculate】
(a) Plot the standard curve.
(2) Use the absorbance value of the measuring tube to find the standard curve to obtain the protein concentration (g/L) in the serum to be tested.
(3) Select a tube from the standard tube that is close to the optical density of the measurement tube, and obtain the protein concentration (g/L) in the serum to be tested.
【Advantages and disadvantages】
(1) The operation is simple and fast, and the measurement is completed within 45 minutes, which is 4 times faster and more convenient than the classic Lowary method;
(2) Accurate and sensitive, the reagent has good stability, the protein measurement range of BCA reagent is 20-200μg/ml, and the micro BCA measurement range is 0.5-10μg/ml.
(3) Economical and practical, in addition to test tubes, the measurement can be carried out in the microplate wells, which greatly saves the amount of samples and reagents;
(4) Strong anti-reagent interference ability, such as detergents, urea, etc. have no effect
【Equipment】
(1) 7220 Spectrophotometer
(2) Constant temperature water bath
(3) 7 medium test tubes
(4) Pistol pipette
【Reagent】
1. Reagent A: 1% BCA disodium salt
2% anhydrous sodium carbonate
0.16% sodium tartrate
0.4% sodium hydroxide
0.95% sodium bicarbonate
Mix and adjust the pH to 11.25.
2. Reagent B: 4% copper sulfate.
3. BCA working solution: mix reagent A100ml + reagent B2ml
4. Protein standard solution: prepare 1.5mg/ml protein standard solution with crystalline bovine serum albumin according to its purity and with physiological saline. (Purity can be determined by Kjeldahl determination of protein content)
5. Sample to be tested: diluted with the sample of the biuret assay method.
This method for the determination of protein concentration has been widely used by scientific researchers in recent years. Currently, kits for the BCA method are commercially available.
In conclusion, although there are many methods for the determination of protein content, there is no perfect method. When selecting the assay method, it can be determined according to the experimental requirements and laboratory conditions.
