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PROTEIN ASSAY: Bradford method of protein estimation.
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Author Topic: PROTEIN ASSAY: Bradford method of protein estimation.  (Read 5267 times)
Francis Umeoguaju
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« on: April 10, 2010, 04:47:05 PM »



Bradford method of protein estimation

This method relies on the binding of commassie blue G250 to protein molecules. The anionic form of the dye with absorption maximum of 595nm, binds the protein.

The dye appears to bind arginine residue in protein (Okonkwo et al 2006).


REAGENTS AND PREPARATION
   100µg/ml Egg albumin standard solution (prepared by dissolving 5mg of egg albumin in 50ml of distilled water).
   
   Bradford Reagent

PROCEDURE

A standard curve was prepared by dispensing varying amounts (20,40,60,70 and 100µg) of standard protein solution into 5 labeled test tube. The  volume of each tube were made up to 100µl. 1ml of Bradford reagent was then added  to each tube followed by gently shaking. The absorbance of the solution were taken at  595nm. A curve of the absorbance against the concentration of the  standard protein solution were plotter.  A reagent blank comprising of 100µl of distilled water and 1ml of Bradford reagent was used to zero the spectrophotometer.
   
100µl of the sample was mixed with 1ml of Bradford reagent and the absorbance was read at 595nm.
   
The concentration of the protein was extrapolated from the curve.


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