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PROTEIN ASSAY: Folin-lowry method of protein Estimation
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| | | |-+  PROTEIN ASSAY: Folin-lowry method of protein Estimation
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Francis Umeoguaju
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« on: April 10, 2010, 04:48:28 PM »



Folin-lowry method of protein Estimation
 
Protein reacts with the Folin- lowry reagent  to gives coloured complex. The colour so formed is due to the reaction of the alkaline copper with the protein as in biuret test and the reduction of phosphomolybdate by tyrosine and tryptophan present in the protein. The intensity of colour depends on the amount of these aromatic amino acid  present and will vary in different protein (plummer 1978).

REAGENT AND PREPARATION

   200µg/1ml Bovine serum albumin standard (prepared by dissolving 10mg in 50ml of distilled water).
   
   4% Solution of Na2Co3 (i.e dissolve 8  gram of Na2Co3 in 200ml of water)
   
   0.2N NaOH (for a 200ml solution, 16g is dissolved in 200ml of water)
   
   1% CuSO45H2O (for 50ml solution, 0.5g was dissolve in 50ml of water)
   
   2% Na, K tartrate (for a 50ml solution, 1g was dissolved in 50ml of water.)
   
The following mixtures were prepared on the day of assay.
    Solution A; equal volume of 4% Na2CO3 and 0.2N NaOH
   
   Solution B; mix equal volume of 1% CuSO45H2O and 2% Na, K tartrate.

   Solution C; Mix solution A and B in the ratio 50:1
   
   Solution D; 3 time dilute solution of folin Ciocateur’s reagent.

PROCEDURE

   A standard curve is prepared by dispensing varying volumes of the standard protein solution into labeled testtubes and the volumes of  each tubes is then made up to 0.5ml with distilled water. 2.5ml of solution C is added with thorough mixing and the solutions were left  to stand for 10mins. this is followed by the addition of 0.25ml of solution D.
   
   The resultant solution is left to stand for 30min after which the absorbance read at 750nm.


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