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| Section: Plant Lab Protocols |
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| Methodology for Amino Acids and Proteins |
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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.
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Prepare a series of protein samples in test tubes in the concentration. This is preferably prepared in PBS.
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2.
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Prepare the experimental samples (a few dilutions) in 100mL of PBS.
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3.
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Add 5mL of dilute dye binding solution to each tube.
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4.
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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.
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5.
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Read the absorbance at 595nm.
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6.
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Plot a standard curve using the standard protein absorbance Vs concentration. Calculate the protein in the experimental sample using the standard curve.
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Notes
1.
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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.
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2.
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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.
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3.
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Serva blue G dye is another dye used in place of coomassie blue G 250 with similar properties.
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4.
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The dyes exist as two forms (blue and orange) in acid solution. The proteins bind the blue form preferentially.
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5.
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Check the absorption of working dye solution at 550nm is 1.18, if necessary, adjust either with the powder or water as required.
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References
Bradford, M M (1976) Anal Biochem 72.
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