DNA after synthesis were PCR amplified using pFx enzyme respectively for both premature AI and

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.

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.

Bradford assay would be ideal if an accurate estimation of a newly isolated protein using BSA as a standard. With the data obtain from the Bradford assay, it shows that it was more sensitive and quicker, also it had less interference from substance that were possibly present in the protein solution, expect for the interfering substance SDS.

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.

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

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.

Gel electrophoresis method. Qualitative analysis shows protein concentrations in kidney, heart, and liver. 1-6 are kidney tissue. 7-14 are liver and 15-19 are the heart tissue.

In Figure 3, there is a collected data with the total washes and the elution fraction with the RFUs from the Ni+2 Agarose column. E3 had the highest elution fraction with 8554 RFUs. In the Bradford assay, E3 had the highest protein amount with 65.5ug. The specific activity for E3 was about 1.28E5 RFUs/mg. Overall, in the Figure 3, the E1-E6 had most of the rGFPs due to being bonded in Ni+2. W1-W6 rGFP is much smaller than the E1-E6 amounts.

Restriction Enzyme Digestion – The experiment was begun after putting on gloves to avoid any chemical contact with the skin. Four microtest tubes were obtained, and each of them was labeled to contain the different enzymes or suspect DNA. Two of the microtest tubes were used for suspect one and the two different restriction enzymes, while two other microtest tubes were labeled for suspect two and the two restriction enzymes. After labeling the tubes, the contents that were at the bottom were taken out by slightly tapping them. Then to begin setting up the enzyme reactions, a micropipette was used to obtain 10 μL of the reaction buffer which was added to each of the four test tubes. The buffer is important because it carries the electrical current from the power supply in the gel. After the reaction buffer was in each, the microtest tubes were individually filled with their specific enzymes and DNA, shown in summary through Table 1.1 below. The restriction enzymes are used to cleave the DNA at specific

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.

BoHV-1 genomic DNA was extracted using the TIANamp Genomic DNA Kit and Δ gD1 and Δ gD2 fragments were generated by PCR amplification using 2× GoTaq Master Mix under recommended setting. Each PCR product was purified using a Wizard SV Gel and PCR Clean-Up System by agarose gel electrophoresis. The purified PCR product, Δ gD1 or Δ gD2 was sequentially cloned into pET28a double digested by the corresponding restriction endonuclease, followed by transform into competent E. coli DH5α. The recombinant positive plasmid was confirmed by DNA sequencing was designated as pET28a-ΔgD1-ΔgD2.

In this paper I will refer to the immobilized Lewis base as the ligand. The combination of the metal ion with the ligand will be referred to as the affinity ligand. IMAC can be performed employing ion exchange resins as the ligand, but in the separation of proteins only chelating groups have been used to fix the metal ion to the support. Schmuckler compared the binding energy of transition metal cations with ordinary cation exchangers and with chelating groups. He found the chelating groups’ binding energy to be 15 to 25 kcal/mol whereas the ordinary ion exchangers have a binding energy of 2 to 3 kcal/mol. (Nes, 1999) This characteristic has led to chelating groups as the ligand of choice in IMAC. The chelating groups used in IMAC are multidentate chelating compounds providing the strength of the complex formed by the protein, metal ion and chelating group. Free coordination sites in the metal ion must be present in the structure formed after the metal ion is chelated by the chelating group to allow for the adsorption or binding of solvent molecules or proteins. (Sulkowski, 1985) Differences in the number of free coordination sites plays a part in the selectivity of chelating substances for a target protein.

For the second part of the experiment, one had to use the knowledge learn from viewing protein molecules in FirstGlance in Jmol to analyze the protein PDB ID: 4EEY. The analysis of this protein was done using the RSCB protein data bank (PDB) at (http://www.rcsb.org/pdb/home/home.do).2

Half of each tube’s contents are poured into a new test tube each respectively after the tubes are incubated for 1 hour. One set of tubes is tested for: