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.
If you have any problem or require further information, please contact info@welchmat