Experiment 3: Detection of Proteins Procedure: 1. Obtain tube: distilled water,

Question

Experiment 3: Detection of Proteins Procedure: 1. Obtain tube: distilled water, albumin, test sample 1, test sample 2. (12) Propose a Hypothesis about which samples will give a positive result with Biuret reagentc n four tests tubes and label them 1-4. Assign the following samples to one of a. Ho: b. Ha: 2. Add Iml of assigned solutions to tubes 1-4. Note the initial color of each sample. 3. Add 5 drops of Biuret reagent to tubes 1-4. 4. Swirl gently to mix and record any color changes Table 4: Results of Detection of Protein with Biuret Sample nitial ColorInitial Color+Final Color Conclusion (+/-) (13) What color change indicates a+ result for protein with Biuret reagent? (14) What was the positive control for this experiment? (15) What was the negative control for this experiment? (16) Explain why these test results are considered qualitative. (17) How could the detection of proteins be made quantitative rather than qualitative? In other words, what tool could be used to quantitatively asses the amount of color change?

Explanation / Answer

12) a)Ho - The given sample is not a protein sample.

b) Ha - The given sample is a protein sample..

13) Emergence of a violet colour indicates a positive reaction.

This is because the biuret reagent has copper ions which on chelating with the nitrogen of peptide bonds of proteins gives a purple colour.

14) The albumin containing tube is the positive control.

Positive control is one in which is sure to give a positive reaction. Since albumin is a protein, it has peptide bonds and hence will definitely give a purple colour with the biuret reagent.

15) The water containing tube is the negative control.

Since water does not have peptide bonds, no colour change will be observed even after adding the reagent.

16) The above test results are qualitative since they do not give anyinformation about the quantity of protein present. The test only gives an idea about the presence or absence of the protein. It does not tell how much of the protein is present.

17) A standard curve can be plotted by carrying out the test with known concentrations of a protein (e.g.Bovine Serum Albumin). The plot will have the optical density versus the concentration of the protein (which is known). From the standard curve, the cocentration of the test sample can be calculated by measuring its optical density. Optical density or absorbance and hence colour change can be measured using a spectrophotometer. The intensity of the colour is directly proprtional to the amount of protein. For Biuret reagent the absorbance can be measured at a wavelength of 540nm.

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