Glutathione S-Transferase Purification Lab Report

The five reagents that were used in this lab were Ninhydrin, Biuret, Orcinol, I2KI, and Nile blue. Ninhydrin in solution will react with amino acids producing a blue product. In the absence of amino acids, nope color develops. this test is both qualitative and quantitative. the development of a blue color indicates the presence or absence of amino acids and the intensity of that color reflects the concentration of amino acids present in the sample being tested. the next reagent that was used was the biuret reagent. this region is used to test for the presence of proteins. when an aqueous sample (using water as the solvent) is mixed with this reagent, the appearance of a purple color indicates that protein is present in the sample. in the absence of proteins, the simple commands light

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.

MstII digestion of 15µl amplified reaction, 1µl of MstII was added to the reaction and then incubated at 37°C for 1-2 hours and this incubation period the reaction was terminated by heating the to 70°C for 5 minutes followed by rapidly cooling on ice. This was then stored at 4°C.

One sample of grey milk was obtained and 6 serial 10-fold dilutions were conducted. 0.1 ml of these dilutions were spread onto Brain Heart Infusion (BHI) agar and incubated for 48 hours at 32°C. After incubation, 100 µl of PrepMan Ultra Reagent was aseptically pipetted into a microcentrifuge tube and an isolated colony was transferred into the tube. This tube was vortexed for approximately 15 seconds and was placed in a 100°C heat block for 10 minutes. After the 10 minutes, the sample was removed from the heat block and allowed to cool for 2 minutes. The tube was then spun in a Microcentrifuge at the highest speed for 2 minutes. Using a sterile pipette tip, 50 µl of the supernatant was transferred into an Eppendorf tube, taking precaution

Once lab began, the IPTG was added to the time 0 samples cultures, a final concentration of 1mM was added to both cultures, and the timer was started. Every 30 minutes over a time period of 90 minutes 2ml was removed from each culture and the initial flask was returned to the 37° environment to continue growing. The 2ml was equally divided into two different test tubes. The tubes were labeled A and B. There was four tubes at each time point; 2 for lacZ+ and 2 for lacZ-. Tube A was the control tube, which contained .2ml of water. Tube B was the experimental tube, which contained .2ml of water and .013M ONPG. The tubes were then incubated in 37° bath water for 20 minutes. After 20 minutes elapsed the tubes were removed and 2.7ml of 1M Na2CO3 was added to each tube. Next 1ml was transferred from each tube into a cuvette, which was placed in the spectrophotometer at 420nm. A spectrophotometer, also known as a spec, reads the intensity of light (absorbance level of liquids). Tube A was inserted first in order to blank the spec, and tube B was second, so we could record the absorbance of the solution. The absorbance was recorded for each cuvette containing a solution from tube B. The A420 recordings we were needed to determine ONP produced in mmol and the activity. The formula for activity is ONP produced divided

In each tube, 10 drops of the eluted sample were collected for a total volume of 0.5 mL of sample per tube. Drops were being continuously collecting in the tubes until we observed the blue color of Blue Dextran and then clear samples, then the red eluted solution of Vitamin B-12, meaning that samples were collected until the standard solution had flown through the entire column, while continuously adding saline. We kept record of how much volume eluted was corresponding to the presence or absence of color and or to change in intensity of color. Our lab group has followed these step to determine the volume eluted for the standards whereas the other lab teams had followed the same steps but used the two unknown proteins as their samples: cytochrome c and hemoglobin. For that, the exact same steps were followed where each protein sample was run through the column while keeping the gel from drying by slowly adding saline. The eluted samples were collected and data was recorded keeping track of the color and

The gel slice was weighed in a colorless tube. 3 volumes of buffer QG to 1 volume gel were added. The tube was incubated at 50 celsius for 10 minutes. This dissolved the gel slice completely. To help it dissolve the gel, the tube was vortexed every 2 or 3 minute. Once the gel was completely melted, if the gel was orange or violet, 10 µl 3M sodium acetate had to be added. Once it became yellow, 1 gel volume of isopropanol was added to the sample. The QIAquick spin column was then placed in the 2ml collection tube. To bind the DNA, the sampled was applied to the column and centrifuged for 1 minute. The flow through was discarded and the column was placed back into the same tube. 500 µl Buffer QG were added to the QIAquick and the mixture was centrifuged for a minute. As before, the flow-through was discarded. Then, the QIAquick column was placed into a clean 1.5 ml microcentrifuge tube. To elute DNA, 50 µl buffer EB(10 mM Tris CI, pH 8.5) was added. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to 4 minutes increased the yield of purified DNA. After the DNA had been loaded onto the gel, the DNA could be analyzed for banding

The sulfating activity of the recombinant human SULT2B1b allozymes will be assayed by using PAP [35S] as a sulfate donor. The standard assay mixture final volume is 20µL and consist of 50 mM HEPES buffer at pH 7.0, 1mM DTT, 14 mM PAP [35S], and the substrate. The essay will be performed on different exogenous and endogenous substrates (see table 6). The controls for this experiment will be prepared with either water or dimethyl sulfoxide (DMSO) replacing substrate. The reaction will be initiated by adding 2µl of the enzyme. The reaction will be allowed to continue for 10 minutes in water bath at 37°C then the reaction will be terminated by placing the reaction tube on the heating block source for three minutes. Then to remove the participate,

An equal amount of solution is to be placed into three test tubes containing Lugol’s iodine. Three different enzyme concentrations were prepared

In this experiment, it was attempted to purify acid phosphatase from wheat germ in its native confirmation with retention of enzymatic activity. The fraction that would contain the purified acid phosphatase (Supernatant IV), had 4.0mg of total protein, but no enzymatic activity and 0% recovery. The experiment was not successful. Acid phosphatase was not purified in its native confirmation with retention of enzymatic activity. In part 2c of the experiment, an error was made in the amount of saturated (NH4)2SO4 and EDTA added to diluted Supernatant IV. An excess of 1.05 mL of (NH4)2SO4 was added to Supernatant IV, and 1.05 mL of EDTA was deficient. EDTA was added to chelate metal ions so that proteases would not denature the acid phosphatase. (NH4)2SO4 was added to precipitate out proteins other than acid phosphatase that were still present in the supernatant. The conclusion can be made that either (1) not enough EDTA was added to prevent proteases from denaturing the acid phosphatase; or (2) the excess (NH4)2SO4 added caused the acid phosphatase to precipitate prematurely, and it was lost.

In order to isolate and purify Lactate Dehydrogenase (LDH), we first extracted LDH from bovine muscle tissue. We minced 8 grams of meat and homogenized using blender. The homogenate was centrifuged and the pellet consisting of membranes, organelles, cytoskeletal components and structural fragments was discarded. The crude extract was subjected to 40% (NH4)2SO4 fractionation. The purpose of this is to remove molecules of lesser solubility than LDH like lipids, fats and low soluble proteins. For this, 9.24 g of was slowly added to crude extract while stirring reaching final solution saturation of 40%. The mixture was then centrifuged and the pellet consisting the contaminants was discarded. From table I, the data indicate that the yield for this was 88% with purification factor of 1.5. The total protein in 40% supernatant was 121 mg which decreased from 210 mg present in crude extract while retaining 88% of LDH suggesting some contaminant proteins were removed. Due to some discrepancies in protein data for crude extract and 40% supernatant, we re-assayed these. Even though week 3 data may not be very reliable, the crude extract assay from week 2 and 40% supernatant assay from week 3 gave the most appropriate data with least error.

First, six different buffers were used and 5 mL of each buffer was added to six different test tubes. The six buffers were as follows: pH 4.00 was Potassium Biphthalate (Item # 280-4.00), pH 5.00 was Potassium Biphthalate/ Sodium Phosphate (Item # 280-5.00), pH 6.00 was Potassium Phosphate/ Sodium Phosphate (Item # 280-6.00), pH 7.00 was Sodium Phosphate/ Potassium Phosphate (Item # 280-7.00), pH 8.00 was Sodium Phosphate/ Potassium Phosphate (Item # 280-8.00), pH 9.00 was Sodium Bicarbonate/ Sodium Carbonate (Item # 280-9.00). All of the aforementioned hydrion buffer salts were obtained from Micro Essential Laboratories in Brooklyn, NY. Secondly, 1.5 mL of a 1% w/v α-Amylase solution from porcine pancreas, Type VI-B, ≥10 units/mg solid, from Sigma-Aldrich in St. Louis, MO, (Item # A3176-500KU) was added to each test tube, and the solutions in the test tubes were mixed. Next, 100 μL of 2% Iodine Solution, I2KI, from Carolina Biological Supply Company in Burlington, NC (item # 869093) was put into wells in several rows of seven different

Preparation of PBS/BSA: 10mL of 1% bovine serum albumin (BSA) in PBS was prepared (0.1mg BSA in 10mL PBS) and the solution

For the determination of the kind of inhibition and the enzyme-inhibitor dissociation constant (Ki), the substrate concentration was varied: 0.25, 0.50, 0.75 and 1.0 mmol/L while the inhibitor (5, 6 DHT) was kept at constant concentration for each experiment: 140, 280, 420 or 560 µmol/L.

In order to produce crude extract, bovine tissue was obtained, precisely minced to exclude extra fat, and then blended with pH 7.2 phosphate buffer. The purpose of blending the tissue with the buffer was to pulverize the cells, causing them to release their contents evenly—most importantly lactate dehydrogenase (LDH)—into the solution. Since other cell components such as proteases which reduce LDH were also released from the lysed cells, the slurry was kept on ice to minimize their kinetic activity. The solution was then centrifuged, creating a pellet made of cellular debris and leaving behind the crude product in the supernatant. Using data from the enzyme assay, the crude extract revealed 155±7 mg total protein, 4980±80 units of total activity, 32±2 units/mg of specific activity, a 100% yield, and a purification factor of 1 fold (Table 1).