Putting enzymes on pause
Nickel chromatography and protein isolation. A. alternata Cdc14 was concentrated using an AKTA protein purification system. Nickel buffer B (25 HEPES pH 7.5, 500mM NaCl, 250mM Imidazole, 10% (v/v) glycerol) for about 15 minutes at 1 ml/min, then Nickel Buffer A (25 HEPES pH 7.5, 500mM NaCl, 10% (v/v) glycerol) for 20 minutes. Then, the lysis solution containing the histidine-tagged protein was passed through a Ni 2+ column, leading to the separation of the protein from the rest of the supernatant. Then, the protein was eluted from the column by gradually increasing the imidazole concentration, which outcompeted the histidine tag that held the protein to the Ni 2+ column. The concentrated protein was then subjected to dialysis, resulting in further purification of the protein. Lastly, the concentration of the post- dialysis protein was calculated using a standard curve generated using known concentrations of BSA (bovine serum albumin). Gel electrophoresis. The gel plate was filled with SDS-PAGE buffer (containing 1.5 g Tris Base, 7.2 g glycine, 0.5 g SDS, and adjusted to 500 mL with H2O, pH 8.3). Then, thawed gel samples were loaded: 10 µl of a protein molecular weight ladder in the first lane, 20 µl of uninduced whole cell extract sample, 20 µl of induced whole cell extract sample, 20 µl of lysate, 20 µl of flow through, 20 µl of purified protein, and 30 µl of purified protein. Empty lanes were left between specific samples to ensure proper separation. The gel was then run. After the completion of gel electrophoresis, Coomassie Blue Stain was added to colour the gel, and the gel was left on the shaker for 2 hours. Finally, the gel was photographed using a gel documentation system to capture the results. Inhibitor assay. Three reactions were set up, one counting an inhibitor, one without an inhibitor, and one without an inhibitor or enzyme to act as a blank. To prepare the reaction mixture in a 96-well plate with a final volume of 200 µL, the following components were added to each well: 20 µL of 10x reaction buffer (resulting in a final concentration of 1x), 10 µL of 800 mM pNPP (final concentration 40 mM), 20 µL of enzyme (final concentration as per enzyme stock), and 8 µL of 50 mM sodium tungstate (final concentration 2 mM) for wells with the positive inhibitor. The remaining volume was filled with deionized water to reach a total volume of 200 µL. The reaction proceeded at room temperature for 5 minutes. The reaction was stopped by adding 100 µL 5 N NaOH, and absorbance was measured at a wavelength of 405 nm. Then, the absorbance of the blank was subtracted from the average of the absorbance value data collected. Then, the data were converted from absorbance values to concentrations of p-nitrophenol using the standard curve. Linear range determinant for steady-state enzyme assay. 5 different concentrations of Cdc14, ranging between 0.62 µM and 0.01 µM, were tested in a plate containing pNPP at 40mM, 0.1 µM Cdc14, 10x reaction buffer at 100mM reaction buffer, and ddH2O. Then, the absorbance value was monitored for 30 minutes, with one absorbance reading taken every 60 seconds at a wavelength of 405nm. The activity of the enzyme was recorded by the change in absorbance detected in relation to a blank (where no enzyme was present.) These changes in absorbance values were then converted to the concentration of product produced by subtracting the blank and dividing by the slope of the PNP standard curve. Then, the values were divided by the number of minutes the test was run to determine the initial velocity of each concentration. Then, these values were plotted to show the amount of product (PNP) against time (in minutes).
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