Purification Of Purification And Purification

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.

Salting out is the first isolation technique carried out for this lab. This isolation technique separates the Rubisco based on solubility. The method of salting out requires an addition of salt such as ammonium sulfate to the solution containing protein Rubisco to allow precipitation (Duong-Ly & Gabelli, 2014). The additional of ammonium sulfate to the Rubisco

Experiment 55 consists of devising a separation and purification scheme for a three component mixture. The overall objective is to isolate in pure form two of the three compounds. This was done using extraction, solubility, crystallization and vacuum filtration. The experiment was carried out two times, both of which were successful.

The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of techniques including homogenization, ammonium sulfate precipitation, dialysis, and affinity chromatography. Activity and Protein assay were used to track the overall amount of LDH present in the samples.

P1 and P2 centrifuged for three minutes at 1000rpm. Supernatant transferred to Eppendorf tubes, 1ml of each saved and set aside. P1 diluted by a factor of 100 and loaded in a column with 5mL. 5mL undiluted P2 loaded into a separate column. 10mL Buffer A used to wash the column. A 10mL of low-salt buffer loaded into each column, 1-2mL collected into each cuvette. Cuvettes scanned with a spectrophotometer, blanked with low salt buffer. Fraction contained the most protein identified and isolated into an Eppendorf tube and placed on ice. The same procedure followed for medium salt and high salt, the blank correlated with loaded buffer. The beads cleaned with a 10mL resin cleaning buffer.

This experiment was conducted as per the BCHM 310 Laboratory Manual [3]. The first objective of this experiment was to analyze the purity of the invertase fractions collected during experiment 6, and to determine the molecular weight of LDH-H4, LDH-M4 and invertase subunits. This was accomplished using sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE). In this procedure, SDS, a negatively charged amphipathic molecule, was used to denature the proteins and to give each protein a similar charge-to-mass ratio [4]. As a result, most oligomeric proteins separated into individual subunits, and each subunit assumed a rod-like shape [4]. The distance travelled by each subunit, along the polyacrylamide gel, was a function of its molecular weight; where proteins with a greater molecular weight moved a smaller distance than proteins with lower weights [5]. Since SDS is not a reducing agent, and no other reducing agent was added, oligomers with disulfide bonds between subunits would have remained intact [4]. However, this was not expected to be problematic for analyzing invertase or LDH isozymes, as these proteins lack disulfide interactions between their subunits [2,6]. In addition, since invertase and LDH are homo-oligomers, each protein’s subunits were expected to migrate the same distance [2,6].

Next, in figure 2, the 96-well plate shows a change of color after the incubation of BCA reagent and both dilutions. Wells 9 A-B contained the 1:5 dilution, while wells 9 E-G contained the 1:10 dilution. The BSA standards are found in wells 1, 3, and 5 A-G. Wells 1, 3, and 5 H were used as blanks; they only contained 40 μl of IP lysis. The results obtained from the spectrophotometer were used to create the graph and obtain the equation found in figure 3. The equation was used to find the concentration of the protein, which is 3.45 μg/μl.

The prepared samples were heated with 12.5% (v/v) β-mercaptoethanol for 5 minutes in a boiling water bath to denature the proteins. The SDS-polyacrylamide gel (Biorad, Any kD, Mini-PROTEAN TGX Gel, 10 well, 30 µl) was loaded with heated protein samples of crude extract, flow through, pre-dialysis, post dialysis, and molecular weight standard (Biorad). The gel was run at 24 mA until the tracking dye reached the bottom of the gel. The gel was removed and placed within a stain (0.4 mg/mL Coomassie Blue R250, 40% (v/v) methanol, 5% (v/v) acetic acid) until bands were

RNA Isolation: First we will extract the total RNA from CML and MLL cell lines using 10 mLTrizol reagent (Invitrogen) according to the manufacturer's recommendations.

For run four, the volume of PBS in each cuvete was reduced by 10 ul to allow room in the original total volume for the addition of 10ul of the inhibitor, clavulanate. The cuvetes were prepared otherwise as normal. The same enzyme from run three was used in run four, but it was not added until after the cuvetes were already in the spectrophotometer. After all four runs were complete, the station was cleaned and the data analysis began.

An appropriate step has been accordance to kit instructions to determine the concentration range, intra-assay precision and cross-reactivity with other related proteins.

Materials: Graduated cylinder, flasks, distilled water, Lugol’s iodine, test tube, pipette, vortexer, spectrophotometer, cuvette, Starch Solution, ph buffer, enzyme extract, ice water bath, hot water bath Methods: Alternating Enzyme Concentration to Determine Its Effect on a Constant Concentration of Substrate The enzyme is to be placed in flask one and distilled water in flasks two and three. A dilution of 1:3 is created by taking some of the enzyme from flask one into two. A dilution of 1:9 is created by taking and mixing some of the enzyme from flask two into three. Some from flask three is removed.

This experiment was carried out with many precautions to minimise error margins. These measures included using the same sample for all experiments, using the same apparatus for the repeated steps of the experiments, having the same person record the results, add substrate and enzyme. However there were still some results that were indicative of error which led to results that were substantially apart from the other

The physicochemical and organoleptic qualities of proteins may be refined by controlled enzymatic hydrolysis, which generates free amino acids and abundant short peptides with less salt and carcinogenic compounds (Weir, 1992). Significantly milder conditions are employed: The pH is typically maintained at pH 5–7 corresponding to optimum enzyme activity and the hydrolytic process occurs at 50–60°C for 10–24 h, which minimizes unwanted side reactions (Clemente, 2000). Proteins are only partially hydrolysed due to the inability of most proteases to cleave glycoproteins, phosphoproteins and protein domains containing numerous covalent-linked disulfide bridges (Gibbs et al., 2004).

As a result of this finding, the Head of Quality Assurance has requested full validation to be performed on the method to assure the quality of the assay results generated