Purification of PPO crude enzyme solution
Purification of PPO crude enzyme solution
The purification of PPO can be applied to (1) the study of PPO nutritional mechanisms and (2) PPO physiological mechanisms and regulation.
Operation method
gel chromatography
Principle
Dextran Gel Sephadex G-200 Gel Column Chromatography utilizes the fact that large molecules cannot enter the interior of the gel particles and are the first to flow out of the column, while small molecules enter the interior of the gel particles with a slow flow rate and are the last to flow out of the column, to separate substances of different molecular sizes. (G-200 can separate proteins with molecular weight of 800-80000D).
Materials and Instruments
PPO crude enzyme solution Move Dextran gel filtration chromatography 1.Dextran gel treatment, column loading and equilibrium (1) column material processing weighing a certain amount of SephadexG-200 dry powder, plus non-ionized water or distilled water soaked and dissolved for 48h, to remove the suspended fine particles, in order to prevent the presence of air in the gel particles, affecting the effect of chromatography, the gel must be loaded into the column before the gel in the liquid to get rid of the gas, which is the method is to be put into the filtering bottle of the gel liquid, vacuum processing, until no bubbles in the gel liquid until no bubbles appear in the gel solution. (2) loading column will be cleaned vertical column fixed to the chromatography rack, add 1/3 of the volume of non-ionized water, open the lower outlet, the water flow is smooth, immediately will be a small beaker containing the appropriate concentration of gel solution (gel concentration is too high will produce bubbles, affecting the speed of separation of proteins, the concentration is too low easy to produce the gel delamination, affecting the effect of separation of proteins) gently poured into the dialysis column, the gel naturally and slowly settles at the bottom of the column, the gel settling in the Gently pour into the chromatographic column, the gel will slowly settle at the bottom of the column, and the gel will be deposited until 1.5-2cm from the upper end of the column, then stop loading the column, and cut a piece of filter paper equal to the inner diameter of the column and cover it on the gel. The inlet port of the upper end of the chromatographic column is connected to a constant flow pump, and the lower outlet port is connected to a protein monitor to be equilibrated in the chromatographic column. (3) equilibrium in the column chromatography on the sample before the column must be equilibrated, the so-called equilibrium is the solution in the column with the buffer of the chromatographic process (eluent) replacement, so that the buffer system in the chromatographic column and the column chromatography process in the same system. The method is as follows: use the constant flow pump at the upper end of the column to pump the equilibrium buffer into the column, open the outlet at the lower end of the column, the flow rate of equilibrium solution is at 0.5-1 ml/min, and the column reaches equilibrium when the PH value of the effluent from the lower outlet is consistent with that of the equilibrium buffer. In this experiment, the SephadexG-200 gel was equilibrated with 0.002MTris-HCL buffer PH7.4 (containing 0.0001MEDTA). 2. Sampling: the crude enzyme solution was loaded onto the column. 3. Elution: elute with 0.02MTris-HCL buffer Ph7.4 (containing 0.001MEDTA), collect the eluate for enzyme activity, protein concentration, polyacrylamide gel electrophoresis molecular weight of the basic properties of the analysis and determination. 4. Determination of enzyme activity and protein concentration. Caveat 0.02MTris-HCL buffer Ph7.4 (containing 0.001MEDTA) was prepared by dispensing x 10-fold concentrate 1000ml. Common Problems Regulating PPO expression is a hot spot for future research . On the one hand because injury or senescence leads to melanin formation, negatively regulating PPO can massively improve crop quality. On the other hand overexpression of PPO reduces crop attack by pests, either by affecting plant proteins to form antinutritional mechanisms or by initiating polymerization through it in trichome exudates to capture insects. Therefore negatively regulating PP0 expression or regulating PPO so that it is overexpressed has good prospects and important practical significance in production and application. For more product details, please visit Aladdin Scientific website.
Tris-HCL buffer NaCL polyethylene glycol DEAE-cellulose DE52
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