Isolation and analysis of Protein from Egg Whites
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| How do extraction methods begin? | They begin with a marceration technique (homogenization in a blender) which will destroy the cell and its substructure, thus releasing the molecular constituents.
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| What fact does the method of protein quantification? | It utilizes the fact that certain colored reagents will combine quantitatively with proteins, i.e. the higher the protein concentration, the more intensely colored it will be become when combined with the colored molecule.
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| What is the Biuret reaction? | It is based on the formation of a purple-colored complex between copper ions and two or more peptide bonds.
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| What does the Biuret reaction is performed in the presence of? | Bicinchoninic acid, or BCA, in order to produc a colorimetric reaction.
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| What is the first step of the biuret reaction? | First the biuret reaction results in the reduction of the cupric ion (CU 2+) to a cuprous ion (Cu 1+).
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| What is the second step of the Biuret reaction? | Chelation of BCA with the cuprous ion results in an intense purple color. The purple-colored reaction product is formed by the chelation of two molecules of BCA with one cuprous ion.
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| What is proportional the intensity of the purple color? | It is proportional to the number of peptide bonds present
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| What is the maximum absorbance of any molecule containing peptide bonds that can be measured quantitatively in the spectrophotometer? | 562 nm
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| What is the range of ug/ml of protein | This reaction is measurable over a range of 20-2000 ug/ml of protein and is nonspecific; meaning that all proteins will react approximately the same to it.
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| What is the protein standard that often protein concentration are determine? | Bovine Serum Albumin (BSA)
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| What is BSA? | It is a protein that forms a large proportion of all plasma proteins; it acts as a "taxi" to carrier molecules with low water solubility. It is stable and lacks interference within biological reactions.
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| What is the percentage that Ovalbumin makes up of the total protein found in egg? | 55%
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| What is the percentage that globulins make up of the total protein egg? | 8% and they are insoluble in pure water, but dissolve in salt solutions
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| What is the total protein content (total grams of protein) of an egg white? | According to the United States Department of Agriculture, the average large 33 g egg contains 3.6 g of protein in its egg white.
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| Why were samples run on a microplate rather than in glass cuvettes in a Spec 20? | Since making so many measurements using the spec 20 is tedious, a micro-plate allows smaller volumes to be tested as opposed to the large volumes needed in a cuvette
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| Why is necessary to dilute concentrated samples to determine the actual concentration, rather than extrapolating the standard curve? | because the stock solution is very concentrated; so, it must be diluted to a lesser concentration. It does not disrupt the results because dilution keeps the amount of solute constant as the total amount of solution increases.
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| What is the purpose of analyzing protein samples in triplicate? | Decreases the error within the experiment. Overall, sampling more than once allows for error to be minimized and ensures correct data analysis.
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| What does BSA stands for and what is its purpose? | The acronym BSA stands for Bovine Serum Albumin and the purpose of BSA is to act as a “taxi” to carrier molecules with low water solubility.
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| What does BCA stands for and what is its purpose | BCA stands for Bicinchoninic Acid Assay.The purpose of the BCA is to determine the total concentration of protein in a solution.
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