Methodology for Amino Acids and Proteins

Protein Estimation – Bradford Method


 
The protein in solution can be measured quantitatively by different methods. The method described by Bardford uses a different concept – the protein’s capacity to bind a dye, quantitatively. The method is simple, rapid and inexpensive.




Principle
The assay is based on the ability of proteins to bind coomassie brilliant blue G 250 and form a complex whose extinction coefficient in much greater than that of the free dye.


Materials
» Dye concentrate
Dissolve 100mg of coomassie brilliant blue G 250 in 50mL of 95% ethanol. Add 100mL of conc. (ortho) phosphoric acid. Add distilled water to a final volume of 200mL. store refrigerated in amber bottles; the solution is stable at least 6 months.
» Mix 1 volume of concentrated dye solution with 4 volumes of d

istilled water for use. Filter with Whatman No. 2 if any precipitate occurs.
» Phosphate-buffered saline (PBS).


Procedure
1.
Prepare a series of protein samples in test tubes in the concentration. This is preferably prepared in PBS.
2.
Prepare the experimental samples (a few dilutions) in 100mL of PBS.
3.
Add 5mL of dilute dye binding solution to each tube.
4.
Mix well and allow the color to develop for at least 5min but no longer than 30min. the red dye turns blue when it binds protein.
5.
Read the absorbance at 595nm.
6.
Plot a standard curve using the standard protein absorbance Vs concentration. Calculate the protein in the experimental sample using the standard curve.



Notes
1.
It is important to use a protein as similar in its properties to your sample as possible! If your sample is unknown, use antibody protein as reference. BSA usually gives a 2-fold higher value in this assay and therefore cannot be used as a general standard.
2.
As in the case of Lowry’s protein assay procedure detergents such as SDS, Nonidet P40, Triton x 10 etc. interfere with this protocol, too.
3.
Serva blue G dye is another dye used in place of coomassie blue G 250 with similar properties.
4.
The dyes exist as two forms (blue and orange) in acid solution. The proteins bind the blue form preferentially.
5.
Check the absorption of working dye solution at 550nm is 1.18, if necessary, adjust either with the powder or water as required.
 


References
Bradford, M M (1976) Anal Biochem 72.