Protein Concentration Determination using Spectrophotometer

PURPOSE :

1. To describe the basic principles of spectrophotometer and know how to use it to determine Protein Concentration.
2. To describe the principal and method of measuring protein concentration by biuret method.
3. To describe method of drawing standard curve.

Protein Concentration Determination PRINCIPLE :

When a monochromatic light passes through a transparent solution medium, a portion of the light energy is absorbed. The amount of absorbed light energy is directly proportional to the thickness of the solution medium and the concentration of the solution.

LAMBERT BEER LAW : A = KCL

A : Absorbance

K : Coefficient of Absorbance

C : Concentration

L : The length of liquid layer through which light passes.

MATERIALS :

Equipment :

1. Visible Spectrophotometer

2. Micropipette and Tip

Reagent:

1. 0.9% Nacl ( Normal saline )
2. Serum ( Diluted 100 times )
3. Biuret Reagent

PROCEDURE

1. The standard curve is prepared by using five known concentration with biuret Reagent. The absorbance is observed using spectrophotometer and graph is plotted absorbance ( Y-AXIS ) vs concentration ( X-AXIS ).

1. After that two set of testube is taken with 3mL serum + 2 ml biuret Reagent and another with 3 mL (0.9% Nacl) + 2mL biuret Reagent.
2. Both testube is mixed thoroughly.
3. Absorbance is measured using the blank for zero adjustment by the photospectrometer.
4. Standard Curve is used to find the concentration of the protein in the sample.

RESULT

From the Spectrophotometer the absorbance of “U” tube I.E tube with serum was found out to be 0.065. By Ploting this on the graph the concentration was found out to be 45 g/L.

To watch my Lab Experiment on YouTube click here