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

4.Working protein standard:Dilute stock standard with normal saline to get
concentration range of 2, 4, 6, 8, and 10 g/dl.

10.5 Procedure

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


2. Add 0.1 ml protein standard of concentration 2, 4, 6, 8, and 10 gm/dl, respec-
   tively, in all tubes labelled as S 1 – S 5.


3. In tube T add 0.1 ml serum sample.


4. Then add 5 ml biuret reagent to all tubes except blank. In blank, add 5 ml biuret
   blank.


5. Mix and incubate all the tubes at 37C for 30 min and take OD at 540 nm.

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

BSA (ml) – 0.1 0.1 0.1 0.1 0.1 –

BSA concentration (g/dl) – 246810

BSA amount (mg) – 246810 –

Serum (ml) ––––––0.1

Distilled water (ml) 0.1 ––––––

Biuret reagent (ml) – 555555

Biuret blank (ml) 5 ––––––

10.6 Calculations

Plot a graph between amount of BSA at x-axis and absorbance at y-axis. Then
extrapolate quantity of protein in the given serum sample from the graph. The
amount extrapolated will denote protein amount in 0.1 ml of sample. Calculate
and express protein amount per 100 ml serum sample. The protein concentration
in serum sample can also be calculated by using the following equation:

Amount of protein in serum¼

ODof testamount of standard mgðÞ 100

ODof standardvolume of sample mlðÞ 1000 ∗

¼xgm=dl

*To convert mg to gram

10.6 Calculations 45