Bradford method for measuring protein concentration Principle: This method is based on Coomassie Brilliant Blue G-250, which has two different forms of red and blue. Under a certain concentration of ethanol and acidic conditions, it can be formulated into a light red solution. When combined with protein, a blue compound is produced and the reaction is rapid and stable. The reaction compound has the maximum light absorption value at 465-595nm. The color depth of the compound is proportional to the protein concentration. Therefore, the amount of light absorption at 595nm can be detected to calculate the protein content. Table 4.2 Preparation of standard curve for determination of protein concentration Sample No. Protein amount (ug) Standard solution ZHONGSHAN GRUWILL HARDWARE CO.,LTD , https://www.gwwirebasket.com
Solution:
â‘ Bradford storage solution
100ml95% ethanol
200ml88% phosphoric acid
350mgServaG Blue is stable for a long time at room temperature.
â‘¡ Bradford working fluid
425ml double distilled water
15ml95% ethanol
30ml88% phosphoric acid
30ml Bradford stock solution is filtered with filter paper and stored in a brown bottle at room temperature. It can be stored for several weeks, but it needs to be filtered before use.
â‘¢ Preparation of 1mg / ml bovine serum protein (BSA)
Make a standard curve:
1mg / ml BSA (ul) experimental buffer (ul) Bradford
Reagent (ml) A595
1 0 0 100 3 0
2 2.5 2.5 97.5 3 0.120
3 2.5 2.5 97.5 3 0.130
4 5 5 95 3 0.250
5 5 5 95 3 0.215
6 7.5 7.5 92.5 3 0.331
7 7.5 7.5 92.5 3 0.364
8 10 10 90 3 0.460
9 10 10 90 3 0.442
10 12.5 12.5 87.5 3 0.531
11 12.5 12.5 87.5 3 0.562
12 15 15 85 3 0.633
13 15 15 85 3 0.617
14 17.5 17.5 82.5 3 0.684
15 17.5 17.5 82.5 3 0.650
16 20 20 80 3 0.721
17 20 20 80 3 0.727