Bio Chem Lab Report 3 - Djdjdhhdjdjdjhdhdhdh hdhdhdj hdhdhdj jdhdhdh djdhhddhd eusjj PDF

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Colin Dral, Kendel Drag, & Banks Handy BioChem Lab #3 10/27/2021 Dr. Ramin Radfar Spectrophotometric methods for the determination of proteins Abstract: The experiment today was performed in order to familiarize ourselves with the two methods of protein determination, the Biuret reaction and the Lowry Method. We ran both in an effort to determine an unknown protein. Introduction: In order to determine a protein, there are two different methods, the Biuret Reaction and the Lowry Method. The Biuret Reaction is a purple color. This is due to the formation of CU2+ in alkaline solution with the peptide bonds of the proteins. With a sensitivity of about 1-10mg of protein, this method allows for determination of larger proteins. IT must take place with compounds that contain multiple peptide bonds, otherwise this Cu would not be able to react correctly. The Lowry Method, on the other hand is more suited for use with small proteins. This method contains a density of 10-200 micrograms. This reaction has 2 components which result in its blue appearance. First, the Cu2+ reacts with the peptide bonds, much like the Biuret reaction, then the reduction of a complex reagent that contains phosphomolybdate. This allows a lot of flexibility with this method as there ar multiple reagents that contain this compound such as phenol reagent, Folin reagent, Lowry reagent, and the Folin-Ciocalteau reagent. The reduction occurs by tyrosine and tryptophan residues that are present in the protein. Procedure: We first ran the Biuret Reaction, this was done with 8 test tubes. The test tubes were set up with 0,0.2,0.4,0.7,1.0,2.0,3.0 ml of BSA for the first 7 then the 8 th was filled with 1 ml of the unknown protein. These test tubes were filled 2 3 ml using water. 3 ml of the Biuret Reagent was then added to each test tube, which were they incubated at room temperature for 5 minutes. After 5 minutes, we took the absorbance of each tube at 540 nanometers.

For the Lowry method, 8 test tubes were set up again. This time the first 7 contained 0,0.1,0.2,0.3,0.4,0.5, and 0.6 ml of BSA respectively. The 8 th was filled with 1 ml of the unknown protein. Each test tube was then filled to 1 ml with water. 5 ml of reagent C was added to each tube, then the tubes were allowed to incubate at room temperature for 5 minutes. After the 5 minutes, .5 ml of Reagent E (Lowry Reagent) was added to all the tubes and they were mixed

using the vortex stirrer. The tubes were then again incubated for 5 minutes at room temperature. After the 5 minutes, each tube was measured for absorbance at 540 nm. Calculations: 1) The standard curve for each reaction is shown below

Biuret REagent 0.02 0.01 0

Absorbancy

-0.01 -0.02

0

0. 5

1

1. 5

2

2. 5

3

3. 5

f(x) = − 0.02 x − 0.01 R² = 0.61

Linear ()

-0.03 -0.04 -0.05

Using the trend line equation the

-0.06 -0.07 Concentration of Reagent

concentration of the unknown is found to be 3.18 mg/ml For the Lowry Method the

Lowry Method 0 0

Absorbancy

0

f(x) = 0.01 x − 0 R² = 0.63

0

Linear ()

0 0

0

0.1

0.2

0.3

0.4

0.5

0 0

Concentration (mg/ml)

concentration of the unknown was found to be .17 mg/ml

0.6

0.7

Conclusion: The goal of this experiment was to find the concentration of an unknown protein. We did this through two methods and found the concentration to be with the Lowry Method, and with the Biuret Method....