Bradford Assay On Unknown Concentrations Of Proteins

In Exercise 1, diaminofluorene is used to determine the hemoglobin concentration in the daphnids. A higher hemoglobin concentration is indicated by a darker blue color. A spectrophotometer was used to determine the absorbance at 610nm. When measuring the absorbance levels a blank is necessary to have a zero reference, the blank is the “starting point” for the measurements of the sample (re-word). The blank consists of 10µL of diaminofluorene(DAF), 50µL of hydrogen peroxide, and 0.5mL of PBS. The PBS acts as a buffer in this experiment. The cuvette with the sample of daphnids consisted of 10µL of DAF, 50µL of hydrogen peroxide, and 0.5mL of the sample of Daphnia. In Exercise 2, the Pasteur pipette was used to obtain the sample of Daphnia. The depression slide used in this experiment isolated the daphnid, cotton was used to keep the daphnid still while the heartbeat was counted. The ocular micrometer on the microscope allows the tail spine length to be measured accurately, as well as using an ocular magnification table.

Figure 1 contains gel electrophoresis for protein samples. The lanes were labeled from 1 to 10 from the right to the left. Lane 1 contained the ladder fragment. Lane 2 contained the filtrate. Lane 3 contained the S1 sample. Lane 4 contained the P1 sample. Lane 5 contained the P1 medium salt sample. Lane 6 contained the P1 high salt sample. Lane 7 contained the S2 sample. Lane 8 contained the P2 sample. Lane 9 contained the P2 medium salt sample. Lane 10 contained the P2 high salt sample.

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 experiment is to prepare Allura Red standard solutions, use a colorimeter to measure the absorbance value of each standard solution, find the relationship between absorbance and concentration of a solution and use the results of this experiment to determine the concentration of Allura Red

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

10 microliters of the sample is then added and the assay absorption is measured at 340nm. If absorbance was above 1.5, samples were diluted.

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.

Incorporation of assay controls included setting up a spectrophotomer and running the chart recorder with a full-scale deflection before the start of the assay. The set recorder had a corresponding value of 1 for the change in the absorbance. Therefore, prior testing was done to observe whether a change occurred in the readings. This helped to indicate that the results were valid, as they could have been affected by a fault during the setting up of the spectrophotometer. On the other hand this was considered as one of the controls for the experiment. Nevertheless, a new cuvette had to be used for each assay.

The shape and magnitude of the UV spectra depends on the composition of amino acid in each protein. Due to the aromatic amino acid residues in the protein, the observed UV absorbance was mainly in the 240 nm to 340 nm region. In Figure 1 to 3, the maximal absorbance of each protein was approximately at 280 nm. The difference in magnitudes of the peak observed was linked to the differences in the amino acid contents in each of the proteins. The peak of lysozyme was greater than those of BSA and gelatin, because lysozyme has a greater number of tryptophan residues. Lysozyme has six tryptophan residues, whereas BSA and gelatin has two and zero, respectively (Department of Chemistry, 2014). Lysozyme has three times more tryptophan residues

The enzyme-linked immunosorbent assay (ELISA) is a common laboratory technique used to measure the concentration of an analyte (usually antibodies or antigens) in solution. In the practical anti-BSA antibodies that had undergone serial dilutions were added to a BSA solution in an ELISA plate with goal of seeing how the concentration of anti-BSA antibodies would affect the colour change of the BSA solution. The results clearly showed a direct correlation as the more diluted the anti-BSA antibody solutions became the lower the Wavelength readings at 405nm, which showed that there was less of a colour change.

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.

Spectrophotometer; the finding of protein concentration of an unknown sample of BSA, and by using the standard curve.

3. The tubes are allowed to incubate in a 37˚C water bath for 1 hour. The final pH of the solutions is tested and the amount of protein digestion is estimated using a scale of (+++), (++), (+), and (-) by

The table shows that the Biuret reagent had an effect on the Albumin and Gelatin solution compared to the Glucose, Water and the Unknown Solution. The data shows that protein was present in the Albumin and Gelatin solution only. I hypothesized that the solution would change purple after the Biuret reagent was added to the solution; resulting in protein present in the solution. The data of this experiment supports my hypothesis. I also made an earlier prediction that the amount of protein present in the solution would have an effect on the color’s vibrancy. This was proven to be correct. The albumin solution was much darker in color than the Gelatin solution, which was a lighter shade of purple.

Name ____________________________ I) Introduction All cells contain four major types of macromolecules: carbohydrates, lipids, nucleic acids, and proteins. In today’s lab, we will be studying three of the four-proteins, carbohydrates and lipids. Various chemical tests can be used to detect the presence of each of these molecules. Most of the tests involve a color change visible to the eye. If a color change is observed, the test is considered positive. If the color change is not observed, the test is negative, indicating that a particular molecule is not present. In all the chemical tests we will be performing, we will also be using a control. In most cases, the control will be a sample of