Question

In this lab, you will quantitatively estimate the protein concentration in the following 3 unknown samples: 1. Milk 2. Diet drink 3. High protein health food drink. Before you do the lab, you are expected to know the following: 1. How will you make the dilutions of the provided standard protein BSA (Bovine Serum Albumin) in the microfuge (1.7 ml small) tubes? Draw the chart for the same. 3 marks 2. How will you make the dilutions for the above 3 unknown samples in the big 15 ml tubes? 1 m 3. Once you make the above dilutions of standard BSA and the 3 unknown samples, you will transfer 150 ul of each standard solution (6 tubes) and 3 unknowns (2 dilutions, total 6 tubes) and 1 blank (water) into big 15 ml assay tubes. What reagent/ volume will you add to these 13 tubes? What is the color that you see? 1m 4. What is the product that you expect to see after the reagent reacts with the protein samples? What is the color of this product? 1m 5. How will you measure the intensity of color of the product? 1m 6. How will you correlate the color intensity in your unknown samples with the protein concentration? 3m

Answer 1

1. Serial dilutions can be made from the BSA solution. The weighed amount of the BSA can be added to a known amount of DI water and from this stock solution, serial dilutions can be prepared.

1 ml solution of BSA

0.1 ml from a stock solution will be added to 0.9 ml of DI in another tube, by following the same, one can dilute as per requirement.

2. The serial dilutions of the unknown samples can be made in the same way as discussed above for serial dilution preparation.

3. The reagents are purely depended on the method by which the protein estimation is to be done. One can use biuret reagent, Bradford reagent or lowry reagent. The amount of reagent used depend upon the standard protocols.

4. Protein reagent complex will be formed when the protein fro the sample reacts with any of the reagents. When one uses the biuret reagent the colour of the product will be purple-blue

5. Colour intensity can be measured spectrophotometrically or colourimetrically, spectrophotometric methods are prefered due to its non-distructive nature.

6. The colour intensities of the unknown samples can be correlated with that of the standard BSA (standard curve can be prepared as concentration vs absorbance) and this can be further used to quantify the protein amount in unknown sample.