Protein Concentration - Lab Report Example

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Proteins are large organic compounds built from 20 different amino acids. A protein will contain in the range of 50-2000 amino acid residues joined together by a chemical reaction that results in the formation of a peptide link and the release of a molecule of water…
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Protein Concentration
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Download file to see previous pages The two most common examples are -helix and -sheet (fig. 1)
Proteins are not completely rigid systems and they shift between several related structures while they perform their biological functions or roles. Such changes are often induced by the binding of a substrate to the protein's active site.
The Biuret reagent is composed of potassium hydroxide (KOH), copper (II) sulphate (CuSO4) and potassium sodium tartrate (KNaC4H4O64H2O). This reagent is used in a protein assay, a colorimetric test to determine protein concentration by detecting the Cu2+ ion with the help of spectrometric methods. In the presence of proteins, this blue reagent turns purple, while when there are short-chain polypeptides in solution it turns pink [MadSci Network].
A spectrometer is an optical instrument used to measure properties of light (intensity, polarisation, etc) over a specific range of the electromagnetic spectrum (in this experiment UV-Vis at a 560nm). The independent variable is generally the wavelength of the light expressed as nanometers. These instruments are used in spectroscopy to measure the interaction between radiation and matter.
In this experiment, UV/Vis spectroscopy is used to determine the concentration of a solution of proteins with the aforementioned Biuret agent. The Beer-Lambert law (A=lc, where A is the absorbance, is the absorption coefficient, l is the distance light travels through the material, c is the concentration of the species that absorbs the light) establishes that the absorbance of any solution is directly proportional to the concentration of the solution [Sheffield Hallam University]. To establish the concentration of a solution, it is necessary to know first how the absorbance changes with the concentration. In order to achieve this, the calibration curve must be determined.
AIMS
Protein solutions are colourless so cannot be easily determined by simple colorimetric methods. Biuret reagent (copper sulphate, sodium hydroxide, and sodium potassium tartrate) reacts with compounds containing two or more peptide bonds to give a purple-violet colour, so this reaction can be used for a colorimetric method for estimating the concentration of protein in solution.
This experiment involves treating a series of proteins of known concentration with Biuret reagent. The data obtained may be used to draw a standard curve. This curve can then be used to estimate the concentration of 2 protein solutions for which the concentrations are not known.
METHODS
You have been provided with:
(a) protein standard solution (albumin) 10 mg/ml
(b) albumin solutions X and Y of unknown concentration
(c) Biuret reagent
(d) de-ionised water
Prepare the following in labelled test tubes, mix well and leave for 20-30 minutes at room temperature:
Tube
A
B
C
D
E
F
G
Protein standard (ml)
4
3
2
1
0
-
-
De-ionised water (ml)
0
1
2
3
4
-
-
Unknown albumin X (ml)
-
-
-
-
-
4
-
Unknown albumin Y (ml)
-
-
-
-
-
-
4
Biuret reagent (ml)
6
6
6
6
6
6
6
Read the absorbance at 540nm for solutions A - G (first using tube E as the blank to zero the ...Download file to see next pagesRead More
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