Protein purification is a popular general direction in biological research area at present, in which protein content determination is a very important content in the purification process.
The most commonly used methods for protein content determination are: Bradford method, Lowry method, BCA method and so on.
Lowry method : Protein in alkaline solution peptide bond and Cu2+ chelation, forming protein-copper complex, reducing phenolic phosphomolybdic acid, producing blue compound, blue depth and protein concentration is linear relationship. The sensitivity is 1-5μg/mL, which is the most sensitive method at present, but it takes a long time, the operation needs strict timing, and the color depth varies with different proteins. Because the potassium tartrate-copper sulfate reagent is difficult to be prepared and preserved, it is gradually replaced by Coomassie bright blue method.
Bradford method: Coomassie bright blue g-250 dye is blue when combined with protein, and there is an absorption peak at 595nm, which shows a linear relationship with protein content within a certain range. Sensitivity in 25-250μg/mL, widely used, color stability, poor specificity, interference material.
BCA method: BCA method is based on the principle of biuret. Under alkaline conditions, the protein reduces Cu2+ to Cu+, and BCA chelates Cu+ as a color developing agent, producing blue-purple and having an absorption peak at 562nm. Unit price of Cu+ is dose-dependent with the protein. Sensitivity between 0.5-20μg/mL, used for rapid detection, strong anti-interference ability, but affected by metal chelating agent.
Each determination method is not perfect and has its advantages and disadvantages. In the selection, the following should be considered:
① Sensitivity and accuracy required by the test;
② Protein properties;
③ Interfering substances in the solution;
④ Factors such as the time spent in the determination.
Select the most appropriate test method according to the specific situation.
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