The aim of this experiment was to further purify sucrase protein by column chromatography, using DEAE cellulose resin as an ion-exchange agent for hierarchical separation by column chromatography.
Operation method
ion exchange chromatography
Principle
Column chromatographic hierarchical separation using DEAE cellulose resin as an ion exchanger.
Materials and Instruments
Enzyme Protein Move 1. Ion exchanger treatment For more product details, please visit Aladdin Scientific website.
DEAE cellulose NaOH HCL Tris-HCl buffer NaCl
Chromatography Column Collector Magnetic Stirrer Stirrer Beaker Glass Sand Funnel Water Pump Filter Bottle pH Test Paper pH Meter Tee Tube Stopper Clip Suction Ear Ball UV Colorimetric Cup Urine Sugar Test Paper Dotting Plate Conductivity Meter
Weigh 1.5 g of DEAE cellulose (DE-23) dry powder, add 0.5 mol/ LNaOH solution (about 50 ml), stir gently, soak for at least 0.5 h (not more than 1 h), filter with a glass sand funnel. 5 mol/ LNaOH solution (about 50 ml), stir gently, soak for at least 0.5 hours (not more than 1 hour), filter with a glass sand funnel, and wash with deionized water until nearly neutral, then drain, put into a small beaker, add 50 ml of 0.5 mol/ L HCl, stir well, soak for 0.5 hours, rinse with deionized water until nearly neutral, and then repeat with 0.5 mol/ L NaOH. Repeat the process with 0.5 mol/ L NaOH, wash with deionized water until nearly neutral, and then pump it dry and set it aside (because DEAE cellulose is expensive, it must be recycled after use). In practice, the cellulose is usually soaked and recycled, and can be washed in the order of "alkali → acid", because it is easier to wash with water to neutral after acid washing. When washing with alkali, because of the difficulty of filtration, you can first remove fine particles by flotation, pumped dry and treated with 0.5 mol/ L NaOH-0.5 mol/ L NaCl solution, and then washed to neutral.
2. Column loading and equilibrium
The column should be mounted vertically, and the cellulose should be washed several times with 0.02 mol/ L, pH 7.3 Tris-HCl buffer in a beaker, and the large particles of cellulose at the bottom of the beaker should be loaded into the column with a dropper, and then the column should be washed with this buffer to the point that the conductivity of effluent is the same as that of the buffer, and then it can be loaded on the sample.
3. Sampling and elution
Prepare the gradient eluent before sampling, 20 ml of 0.02 mol/ L, pH 7.3 Tris-HCl buffer and 20 ml of 0.02 mol/ L, pH 7.3 Tris-HCl buffer containing 0.2 mol/ L concentration of NaCl were used for linear gradient elution. Take two small 50 ml beakers of the same diameter, one containing 20 ml of high ionic strength solution containing NaCl and the other 20 ml of low ionic strength solution, place them on a magnetic stirrer, and put a small stirrer (a small piece of wire inside a thin plastic tube with both ends sealed by heating with an alcohol lamp) into the beaker of low ionic strength solution, and place this beaker on top of the stirrer's rotating magnet. Insert a glass tee into both beakers, connect a piece of latex tubing to the top end, clamp on a stopcock, carefully draw the solution into the tee with a suction ear ball (gently loosen the stopcock), immediately clamp the latex tubing so that the two beakers of solution form a continuum, and take care that the two beakers are properly placed and that one cup does not rise above and one cup does not fall below.
Dissolve alcohol grade II with 5 ml of 0.02 mol/ L, pH 7.3 Tris-HCl buffer (note that the tip of the glass stirrer must be rounded, and the centrifuge tube must not be scratched when stirring to dissolve), and if the solution is turbid, remove the insoluble material by centrifugation with a small test tube at 4,000 r/min. Take 1.5 ml of the supernatant (1.5 ml). Take 1.5 ml of supernatant (i.e., alcohol-grade fraction Ⅱ sample, reserved for the next experiment to measure enzyme activity and protein content), the remaining 3.5 ml of supernatant carefully. Add the remaining 3.5 ml of supernatant to the column carefully without disturbing the column bed, and take care to use a partial collector to collect the sample from the beginning of the sample, 2.5~3.0 ml per tube/10 min. Wash the sample twice with buffer, and then use about 20 ml buffer to wash out the unadsorbed proteins in the column until the A280 drops to below 0.1, clamp the exit of the column, and put the thin plastic tubing from the inlet of the constant-flow pump into a low ionization chamber with no NaCl. NaCl-free low ionic strength solution into a small beaker, fix the plastic tube with adhesive tape, connect the chromatography column, turn on the magnetic stirrer, let go of the chromatography column outlet, start the gradient elution, collect the eluate continuously, after the eluate in the two small beakers is exhausted, in order to elute adequately, you can also be prepared by the remaining 30 ml of high ionic strength eluent poured into a small beaker to continue to elute, control the flow rate of 2.5 ~ 3.0 ml / The flow rate was controlled to be 2.5-3.0 ml for 10 min.
The A280 absorbance of each tube was measured.
4. Qualitative determination of enzyme activity in each tube of eluent.
Add one drop of 0.2 mol/ L acetic acid buffer, pH 4.9, one drop of 0.5 mol/ L sucrose and one drop of eluent into each well of the plate, and react for 5 min. Insert a small strip of uroglucose test paper into each well at the same time, and observe the color change of the test paper after 10～20 min. The color change of the paper was observed after 10-20 min. The number of + sign indicates the color shade, i.e. the enzyme activity of each tube. Combine the 2-3 tubes with the highest activity, measure the total volume and divide it into 10 parts, pour them into 10 small test tubes, seal them with plastic wrap, store them in the freezer, and take out one tube when using, which is the column-level fraction III.
Note: There may be two active peaks from the beginning of the sample collection, the first peak before the start of gradient elution is not adsorbed, the experiment takes the active peak washed down after the start of gradient elution.
Plot the enzyme activity, NaCl concentration (can be replaced by conductivity) and absorbance value A280 for all tubes on the same graph as the elution gradient line.