Basic Concepts in Clinical Biochemistry-A Practical Guide.7z

form a coordination complex with four imino groups present in the peptide bonds
and show an absorption maximum at 540 nm. The method is suitable for estimation
of proteins having concentration greater than 1 mg. The intensity of color produced
is directly proportional to the amount of protein present in the sample.

9.4 Reagents........................................

1.20% TCA–Dissolve 20 g TCA infinal volume of 100 ml distilled water.
2.0.5 N NaOH–Dissolve 2 g of NaOH infinal volume of 100 ml distilled water.
3.Bovine serum albumin (BSA) standard (0.5 g/dl)–Dissolve 0.5 g BSA in
100 ml distilled water or normal saline. Do not shake.
4.Biuret reagent–Dissolve 9 g of sodium-potassium tartrate in 500 ml of 0.2 N
NaOH solution. To this add 3 g of CuSO 4 previously dissolved in 100 ml distilled
water, gently with constant mixing. Then dissolve 5 g potassium iodide and make
final volume up to 1 litre with 0.2 N NaOH.

Procedure

1. Take a set of test tubes in duplicate and label as blank, standard (S 1 – S 5 ) and test
   (T).


2. Add BSA standard and distilled water as shown in table.


3. Then add 20% TCA to all tubes except blank.


4. Mix and incubate all the tubes at 37C for 30 min.


5. Centrifuge all the test tubes at 5000 rpm for 10–15 min and, discard the superna-
   tant completely.


6. Dissolve the pellet in 0.5 ml NaOH and add 1.5 ml distilled water to all tubes.


7. Then add 2 ml biuret reagent to all the tubes, incubate at 37C for 10 min, and
   take absorbance at 540 nm.

Reagent Blank S 1 S 2 S 3 S 4 S 5 T

BSA (ml) – 0.2 0.4 0.6 0.8 1.0 –

BSA amounta(mg) – 12345 –

Urine (ml) ––––––0.2

(continued)

40 9 To Determine the Quantity of Proteins in Urine Sample Using Biuret Reaction