Bradford Protein Assay Essay

The protein molecules in many foods provide the amino acid building blocks required by our own cells to produce new proteins. To determine whether a sample contains protein, a reagent called Biuret solution is used. Biuret solution contains copper ions. However, the chemical state of the copper ions in Biuret solution causes them to form a chemical complex with the peptide bonds between amino acids (when present), changing the color of the solution. Biuret solution is normally blue, but changes to pink when short peptides are present and to violet when long polypeptides are present.

The purpose of this lab was to test different substances using various procedures to see what biomolecules were present and ultimately find out what restaurant Anna Lyza had eaten at before she died. For the first control test, we used vegetable oil to test for lipids. So, if the solution does not contain lipids, it does not become translucent when placed onto a paper bag square and held up to a light. So, it is a negative result. However, in the presence of lipids, the solution will become translucent when placed onto a paper bag square and held up to a light. Therefore in this case, the result is positive. On the other hand, we used albumin egg to test for proteins in another control test. If the solution does not contain proteins, it will not experience any color change and so it is a negative result. When there are proteins existing in the solution, it will turn bluish/purplish and for this reason it is a positive result. Furthermore in the third control test, we used dextrose to test for simple carbohydrates such as glucose. If the solution does not contain simple carbohydrates, it will not undergo any color change and will remain a blue color. So, it is a negative result in this circumstance. If there are simple carbohydrates present in the solution, the solution will turn reddish and so the result is positive. For the last control test, we used starch solution to test

The null hypothesis will be that the test tubes with an increase in temperature, pH values, enzyme concentrations, and substrate concentration will have a very small color change or no color change at all. The alternate hypothesis is that the test tubes containing an increase in temperature, pH values, enzyme concentrations, and substrate concentration will all have an intense color change; the more the change, the more intense the color change will be.

This Lab Report is an analysis of the results of a two-part experiment. In the first part, we used a gel filtration column to separate the components of a mixture composed of protein and non-protein molecules. By doing so we hoped to obtain fractions that contained single components of the mixture, while also gaining insight into the relative molecular weight of each component compared to each other. We would then plot these fractions onto nitrocellulose paper in order to determine which fractions had protein. In the second part, we would use the fractions which we had determined had protein to conduct an SDS-PAGE. By doing so we hoped to determine an estimate on the molecular weight of the proteins present in each fraction by comparing it to a tracker dye composed of a variety of molecules of differing molecular weight.

Protein Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from 0-2000 µg/ml was prepared from a stock solution of 2 mg/ml BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at 370C and then the absorbance was measured at 562nm.

In order to examine the variation in sensitivity of lysozyme, gelatin, and BSA, the absorbance reading was obtained using the Bradford and BCA assay

Colorimetric assay is a process of determining a concentration of a solution based on absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way to determine an unknown concentration of two samples of protein. The Bradford assay is done by measuring wavelength of light passing through a cuvette filled with Bradford dye and concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard curve based on concentration (x-axis) and wavelength (y-axis). The standard curve is then used to measure an educated guess on the concentrations of unknown protein concentrations. We hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can determine an accurate concentration of two unknown concentrations of proteins. The results of this lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to demine the concentration of an unknown concentration.

A spectrophotometer’s purpose is to use colors of the light spectrum to determine the concentration of light absorbing molecules in a solution. (p.59) In this particular lab, our mission was to determine the protein concentration and the standard curve of the unknown sample of BSA. This, by preparing five dilutions of the unknown solution of BSA together with other known concentrations, and then experimenting by observing how the concentrations were passed through the spectrophotometer. The outcome resolved in the absorption levels being decreased, and this

Analysis: In this experiment, every substance tested positive for at least 1 organic compound. Corn oil tested positive for lipids. Honey and oats tested positive for starch. Honey, apple juice, and unknown 1 tested positive for sugar. Egg white, gelatin, and unknown 2 all tested positive for proteins. So, not a single substance didn't test positive for at least 1 organic compound. All the indicators used in this experiment change color when in the presence of a compound; therefore, they chemically react with the compound. No errors were made during this experiment

The homogenates provided were made by homogenizing tissues in a sucrose phosphate buffer in a 1:20 ratio. The protein concentration in bovine cells was measured by diluting the homogenate with a 1:5 ratio; 50 microliters of homogenate and 200 microliters of water. Then 5 known protein concentration samples which were 0.4, 0.8, 1.2, 1.6, 2.0 mg/ml of bovine serum were used to determine absorbance with a spectrophotometer. Two additional samples were made; one was blank and the other was for the specific homogenate sample. Then 3 microliters of bradford assay reagent, which indicates the amount of protein present

You can tell because the protein was not digested in tube #1. There was no color change & a density of 0.__

After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.

There are two methods using which the BCA Protein Assay can be performed- Test tube method and Micro plate method.

The objective of this lab was to learn the principle of affinity chromatography by isolating a carbohydrate-binding protein from an extract of jack bean meal.

1. Thin slices of cooked egg white with same size (about 0.5 cm²) are placed in four test tubes.