Estimation of Protein Concentration

UNIVERSITI PERUBATAN ANTARABANGSA

INTERNATIONAL MEDICAL UNIVERSITY

MALAYSIA

Biological Science

Practical 2 Report:

Title: Estimation of protein concentration

Introduction:

It is important to know the concentration of protein in a biological material in biochemical analyses. There are several method of determining the concentration of protein, but basically is based on the binding of a chromophore to specific amino acids in the protein. The resulting colour development can be detected at some wavelength of visible light by spectrophotometer. Normally a standard curve of absorbance versus protein concentration is used to estimate the protein concentration of an unknown solution. One of the method to estimate the protein concentration is Bradford protein assay which based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is converted into its bluer form to bind to the protein being assayed.

Objective:

1. To prepare a standard curve of absorbance versus protein concentration by using Bovine Serum Albumin (BSA).

2. To determine BSA concentration in two sample solutions.

3. To determine protein concentration in apple juice.

Materials:

Bradford Reagent, bovine serum albumin, apple juice sample, test tubes, cuvette, micropipette, tips.

Procedure:

Part 1: Preparation of standard curve of BSA

7 test tubes is prepared and labeled from 1 to 7. Appropriate amount of BSA and distilled water is pipetted into respective test tubes according to Table 3.1. Next, 500 l of Bradford Reagent is added into all the 7 test tubes. Then, the mixture is vortexed for proper mixing and then leave for 2 minutes. The absorbance at wavelength 595 nm is measured.

Results:

Concentration of BSA (µg/ml) in the test tube 1-7

Ma = Initial concentration Mb= Final concentration where b=1,2,3...

Va = Initial volume Vb = Final...