Identification And Quantification Of Amino Acids

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.

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.

2. Four unknown samples were included in the lab kit: flax seed meal, potato starch, egg whites, and dried milk. Using the results of the biochemical testing, determine which number corresponds to the correct unknown. (8 points)

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.

(TCO 6) Which part of an individual amino acid distinguishes it from other amino acids?

Chemistry 102 is the study of kinetics – equilibrium constant. When it comes to the study of acid-base, equilibrium constant plays an important role that tells how much of the H+ ion will be released into the solution. In this lab, the method of titrimetry was performed to determine the equivalent mass and dissociation constant of an unknown weak monoprotic acid. For a monoprotic acid, it is known that pH = pKa + log (Base/Acid). When a solution has the same amount of conjugate base and bronsted lowry acid, log (Base/Acid) = 0 and pH = pKa. By recording the pH value throughout the titration process and determining the pH at half- equivalence point, the value of Ka can be easily calculated. In this experiment, the standardized NaOH solution has a concentration of 0.09834 M. The satisfactory sample size of known B was 0.2117 g. The average equivalent mass of the unknown sample was found to be 85.01 g, pKa was found to be 4.69, which was also its pH at half-equivalence point and Ka was found to be 2.0439×〖10〗^(-5). The error was 1.255% for equivalent mass and 0.11% for Ka. In other word, the experiment was very precise and accurate; the identity of the unknown sample was determined to be trans-crotonic by the method of titrimetry.

Eisenthal, R. & M.J. Danson (Eds.) (2002). _Enzyme Assays: A Practical Approach_. United Kingdom: Oxford University Press

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

In this experiment, a 50 mL buret, buret clamp, magnetic stirrer, stir bar, pH meter, pH electrode, clamp, and a 50 mL beaker was obtained. In order to prepare the solution, approximately 0.3 g of the amino acid (exact mass was recorded) hydrochloride was measured out into the beaker. Then, 50 mL of deionized water and a stir bar was added to it. The beaker was placed onto the stirrer, and the stirrer was turned on. The 50 mL buret was rinsed with deionized water, and filled with 0.3170 M NaOH until the meniscus was at/below 0.00 mL. The buret was held by the clamp with the tip positioned slightly above the beaker. The pH electrode was clamped so that the tip was under the solution, but not touching the beaker or the stir bar. The initial pH

The titration curve of the unknown exhibited many characteristics, such as equivalence points, pKa of ionizable groups, isoelectric point, and buffer regions, that are particularly distinct to lysine. For unclear reasons, the pH during the titration did not reach the pH for pure 0.2 M NaOH nor 0.2 M HCl and normal equivalence points expected at two extreme ends of the titration curves for all amino acids were not observed. The titration of a phosphate buffer showed that the buffer capacity is directly proportional to the molarity of the buffer. However, our results showed that although the initial pH of the phosphate buffer was less than the pKa value, the measured buffer capacity was higher towards acid than base. The accuracy of the pH meter and calibration process was questioned under assumptions that the pH of the prepared phosphate buffer was actually above pKa.

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

Purpose the purpose of this experiment was to perform test to detect the presence of carbohydrates, proteins, lipids, and nucleic acids. Explain the importance of a positive and a negative control in biochemical test. Use biochemical test to identify an unknown compound.

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

Different techniques and principles for protein extraction and characterization were demonstrated in this experiment. Various proteins were extracted from different sources: 1.67 g yeast invertase, 1.03 g egg white albumin, and 5.15 g of milk casein. Activity assay for invertase was performed using Benedict’s test and the enzymes inverting action on sucrose was confirmed. Warburg-Christian Method and Bradford Assay were also employed to determine the protein concentration in the albumin and the casein samples. The concentrations for the albumin and casein samples were found to be 0.519 and 0.327 mg/mL, respectively based on Warburg-Christian Assay; and 6.5x10-3¬ and 1.9x10-2 mg/mL