Biochemistry of Proteins. Isolation of Ovalbumin: Characterisation of Thiol Groups and Separation by Gel Filtration

3ml of sample was taken first flask at 4 minutes and added to the appropriate tube of sodium hydroxide, from the second flask at 4.5 minute and so on, each flask was sampled at 30 second intervals. The sampling was then repeated starting at 8,12,16 minutes. The final sample from the last flask was taken at 18.5 minutes. Once the sampling was completed, measurements of absorbance were obtained for solution in each tube at 405 nm.

concentration was plotted in order to estimate the concentration of the unknown solution (Figure 3).

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.

In doing the lab, one was able to determine the characteristics of the given solutions, containing different macromolecules, whilst doing the multiple tests. The tests performed were,

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.

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.

Amino Acids are essential nutrients that are the primary building blocks of proteins found in meat, dairy products, and legumes. Proteins make up 20 percent of the human body, and the amino acids that make up these proteins play a critical

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.

The absorbance is measured using a Plate reader and a Standard curve is generated. Also, the different types of pipetting techniques are assessed in this Assay.

Methionine has a thioether side chain, and cysteine's contains a thiol group. These side chains exist as free thiols inside the cell, and are oxidised causing them to pair up and form disulphide bonds in an extracellular environment.

By using acid-base titration, we determined the suitability of phenolphthalein and methyl red as acid base indicators. We found that the equivalence point of the titration of hydrochloric acid with sodium hydroxide was not within the ph range of phenolphthalein's color range. The titration of acetic acid with sodium hydroxide resulted in an equivalence point out of the range of methyl red. And the titration of ammonia with hydrochloric acid had an equivalence point that was also out of the range of phenolphthalein.. The methyl red indicator and the phenolphthalein indicator were unsuitable because their pH ranges for their color changes did not cover the equivalence points of the trials in which they were used. However, the

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.

Bettelheim, Brown, Campbell and Farrell assert that polypeptide chains do not extend in straight lines but rather they fold in various ways and give rise to a large number of three-dimensional structures (594). This folding or conformation of amino acids in the localized regions of the polypeptide chains defines the secondary structure of proteins. The main force responsible for the secondary structure is the non-covalent

An acid-base titration is the determination of the concentration of an acid or base by exactly neutralizing the acid/base with an acid or base of known concentration. This allows for quantitative analysis of the concentration of an unknown acid

For this experiment, a pH meter was used so this part of the experiment began with the calibration of the pH meter with specified buffers. The buret was then filled with the standard HCl solution and a set-up for titration was prepared. 200g of the carbonate-bicarbonate solid sample was weighed and dissolved in 100 mL of distilled water. The sample solution was then transferred into a 250-ml volumetric flask and was diluted to the 250-mL mark. The flask was inverted several times for uniform mixing. A 50-mL aliquot of the sample solution was measured and placed unto a beaker. 3 drops of the phenolphthalein indicator was added to the solution in the beaker. The electrode of the pH meter was then immersed in the beaker and the solution containing the carbonate-bicarbonate mixture was titrated with the standard HCl solution to the phenolphthalein endpoint. Readings of the pH were taken at an interval of 0.5 mL addition of the titrant. After the first endpoint is obtained, 3 drops of the methyl orange was added to the same solution and was titrated with the standard acid until the formation of an orange-colored solution. Readings of the pH were also taken at 0.5 mL addition of the titrant.