Bradford method for measuring protein concentration

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.
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:

Table 4.2 Preparation of standard curve for determination of protein concentration Sample No. Protein amount (ug) Standard solution
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

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