Term Paper Touching On Biomolecule Analysis (Term Paper Sample)

Protein analysis.
 
Introduction
Discuss the importance of protein determinations in the laboratory biochemistry setting.
Protein determination aims at determining the concentration of protein by comparing the unknown sample with the known sample. The known sample is the standard. Samples that show a similar value of absorbance is assumed to have the same concentration in Bradford assay. Protein determination is essential since it enables efficient purification of the protein of interest. The protein will be quickly identified and separated from other unwanted biomolecules. Protein determination is also needed for the decision of the cost of production of the desired yield. Identifying the protein concentration of the unknown sample will enable one to be able to determine the value of production of a given mass of the desired protein.
Discuss Beer's Law in detail and the appropriateness of this law for concentration measurements in biological samples.
Beer Lambert’s law refers to the directly proportional relationship between absorbance and the concentration of the absorbing molecule. The higher the absorbance of a species the more its solution will be concentrated in water or any other medium. Beer’s law is significant for the measurement of minimal units of proteins and other biomolecules since it’s difficult to measure quantities of milligram and microgram units. It’s also impossible to get rid of all the water in biomolecules like protein; therefore, Beer Lambert’s law will be the most appropriate method to measure the concentration of proteins without including the mass of water ("Beer-Lambert Law," 2017).
Describe methods for protein determination and their advantages and disadvantages three different.
Kjeldahl is a method used for protein analysis to determine protein concentration. The food sample to be analyzed is treated with a strong acid. The reaction between the substrate and the acid generates nitrogen which is detected via titration. Nitrogen’s concentration is then used to calculate the amount of protein in that sample. Kjeldahl is universally accepted and highly accurate. The analysis can also be duplicated. This technique also has its limitations.It’s time-consuming, the strong acid used to digest the food sample is denatured at high temperatures stopping the whole process (People. umass.edu, 2017).
Enhanced Dumas method is another protein analysis technique that rivals Kjeldahl procedure. It’s an automated method that is so fast and efficient. The sample to be analyzed is heated to very high temperatures in the presence of oxygen. Carbon ( IV) oxide, water, and nitrogen are released. Carbon dioxide and water are selectively absorbed in the special column, and nitrogen’s concentration is therefore determined by passing the carbon dioxide and water free nitrogen via a calibrated instrument. This method is more rapid than Kjeldahl. It’s independent of catalysts or chemicals. It’s also automated. Its demerit is that it’s so expensive.
UV-visible spectroscopy is also used to measure protein concentration. This technique depends on the ability of proteins to absorb or repel light. Chemical or physical modification of proteins either enables them to absorb or resist light. The turbidity of the solution being analyzed is quantified, and its protein concentration is computed from the calibration graph. Biuret is an example of a UV-visible method. In biuret method dilute copper sulfate solution in an alkaline medium interacts with peptide bonds forming a violet-purplish color. Turbidity is then measured after 15-30 minutes at 540 nm. The merits of this method are that it’s fast. It’s able to detect even low concentrations.It’s basic and uncomplicated hence can easily be used. Limitations of this method are that it’s only applicable to contaminant-free solutions since contaminants will compete with the protein of interest to scatter or absorb light.
Discuss the theory behind the spectrophotometer
Spectrophotometry refers to the interaction between matter and light which depends on the structure of the test substance or molecule and the wavelength of light. Matter either absorbs or transmits light over a definite range of wavelength. This theory is used in the quantitative analysis of biomolecules clinically or industrially whereby a spectrophotometer detects the intensity of light absorbed by the sample solution, and it eventually determines the concentration of the test sample by computing the intensity of light. During the interaction between light and the test substance, longer wavelengths either stretches or bends the bonds while shorter wavelengths eject electrons from the molecules to higher energy levels. A decrease in the amount of light passing through the test sample will signify an increase the concentration("Spectrophotometry," 2017).
Methods
Describe the methodology described for protein determination via the Bradford assay.
This method uses a dye known as Coomassie Brilliant Blue G-250.The dye is negatively charged. Once a protein is dipped into a solution containing this dye, the red color of the dye changes to blue. Interaction takes place between the dye and the protein. The dye is negatively charged, and it’s attracted to the positive chains of the protein. A blue complex is formed. The intensity of the blue color signifies the concentration of the protein.
 
Data
...