Aim: estimation of protein.

Method: blank standard test.

Principle:

Protein + Cu^{2+        alkaline medium}         Cu-Protein complex

Requirements: three test tubes, micropipette-20 micro liter and 100 micro liter, calorimeter, beaker, micro pipette tips,

Reagents:

Burette reagent, protein, standard, serum, distilled water.

The reagents contain:

1.        Copper sulphate solution (12 mmol)

2.        Potassium iodide ( 20 mmol)

3.        NaOH (600 mmol)

4.        Sodium potassium tartarate (32 mmol)

Procedure:

a.        Calorimeter was switched ON and left ON for 1 hour before the experiment.

b.        Three test tubes were taken and marked as B (blank), T(test tube), S (standard).

c.        Calibration of calorimeter: calorimeter was set to zero i.e. 100% transmittance by the blank solution. Blank solution is prepared by taking 1000 micro liter burette reagent and 20 micro liter of distilled water in test tube.

d.        Now 1000 micro liter of burette reagent was taken in test tube S and T.

e.        Then 20 micro liter of protein standard was added to s marked test tube and 20 micro liter of serum was added in T marked test tube by the help of micropipette.

f.         After mixing the contents of test tube properly, the test tubes were incubated at room temperature for five minutes.

g.        Now the absorbance of T and S test tube solution was measured.

	B	S	T
BIURETE READING	1000 µl	1000 µl	1000 µl
PROTEIN STANDARD	------	20 µl	------
SERUM	------	------	20 µl
DISTILLED WATER	20 µl	------	------

Observation:

Serial number	Absorbance of S	Absorbance of T
1	0.26	0.22
2	0.29	0.27
3	0.12	0.09
4	0.21	0.19
5	0.23	0.21

Calculations:

                                                =

                                                =

                                                =4.4 mg/dl