Protein purification experiments
Protein purification experiments
Protein molecules of different sizes enter the colloidal filtration column and can be separated according to their molecular weight differences; it is a widely used protein partition chromatography.
Operation method
colloidal filtration
Materials and Instruments
Sephacryl S-300 Colloid Move I. Instrumentation For more product details, please visit Aladdin Scientific website.
Buffer A-150
Chromatography columns Iron clamps Test tubes Iron racks Levels Collectors Centrifuges for concentration Centrifuge tubes for concentration
Chromatography column (Pharmacia C column, 1.6x100 cm), rack, clamps and level; fraction collector (about 100 clean tubes); centrifuge for concentration (low speed 5,000 rpm); Centriprep-30 (Amicon 4322) centrifuge tube for concentration. Please note the instructions for use.
Drugs and reagents
Colloid Sephacryl S-300 (Pharmacia): a. Pre-equilibrate the colloid with buffer A-150 and make the volume of the colloid after complete precipitation account for 70% to 80% of the total volume; the amount of colloid to be used should be estimated first. b. The temperature of the colloid should be the same as that of the operation place, otherwise the temperature change will produce bubbles. c. Sephacryl series colloid has a considerable absorption capacity. d. Sephacryl series colloid has a very large absorption capacity, and it can be used in a variety of ways. Sephacryl series colloids have considerable adsorption, so add more than 0.15 M NaCl to the buffer to remove non-specific adsorption. Buffer A-150: Be careful to use at the same temperature as the column colloid. （Contains thyroglobulin (670 kD), bovine gamma globulin (158 kD), chicken ovalbumin (44 kD), equine myoglobin (17 kD), and vitamin B12 (1,350).
Column loading
1. Rinse the glass column with pure water (rinsing up and down with pure water is sufficient, and the use of a test tube brush is strictly prohibited); please understand the structure and disassembly of the column, set up the column vertically, connect part of the collector with a hose, and use the bufferA-150 to test whether the pipeline is smooth; you can use hemostatic forceps or long-tailed paper clips to hold the outlet hose, which will control the dissociation of the dissolution process. Note that the system should be appropriately placed, and not installed in the main traffic.
2. Remove the Sephacryl colloid according to the estimated volume and note whether the temperature of the colloid and the buffer have been equilibrated; shake the colloid in the bottle up and down so that it is completely suspended but does not produce too many air bubbles.
3. Add about 10 cm of buffer to the column and slowly pour the colloid into the column along the wall of the tube until it reaches the top of the column, and the colloid will settle quickly when it begins to wash. When the liquid level above the colloid gradually decreases, the colloid can be added at the top to reach the desired height; the height of the colloid is about 90 cm.
4. After the colloid is completely settled, carefully fill the column with buffer A-150, close the outlet, install the top end cap and connect the buffer bottle, open the outlet to wash by gravity flow. Adjust the height of the buffer bottle so that the flow rate is about one drop every five to six seconds, and set the collection volume to 2.5 mL/tube.
5. After the column has been flow washed for about 100 mL, close the outlet, remove the top end cap, and use a dropper to draw out the solution above the colloid to a height of about 1 cm, taking care not to disrupt the smoothness of the colloid surface; then open the outlet and allow the liquid level to drop down to the surface of the colloid, and then close the outlet in preparation for injecting the samples.
Sample color analysis
1. Use a micropipette or dropper to draw up the sample (the sample volume should not exceed 3% of the total volume of the colloid), and add it slowly along the wall of the tube above the colloid, taking care not to damage the flat surface of the colloid! (Sample volume _______ mL)
2. Open the outlet and partially open the collector; when the sample is completely submerged in the colloid, close the outlet and slowly add an equal volume of buffer A-150, open the outlet and allow it to slowly enter the colloid, and repeat this process twice. The surface of the colloid must not be disturbed, causing depressions.
3. Temporarily close the outlet, fill the liquid level to the top of the column, and lock the top end cap; then open the outlet to start dissolution, adjust the height of the buffer bottle, so that the flow rate is 6 s a drop.
4. Observe the first few divisions to make sure that the entire system is functioning correctly, being careful of the part of the collector that is most susceptible to problems. It is expected that the column will be flow washed overnight and about 80 tubes will be collected.10) Collect the tubes for quantitative protein analysis and GUS activity determination and graph them.
5. Collect the GUS activity region, concentrate to 10 mL with Centriprep-30, dilute to 20 mL with buffer A-0, concentrate again to _______ mL (GF), and retain 100 uL.
6. Wash 100 mL of the column with buffer A-150 and set aside carefully for molecular weight determination.
V. Molecular Weight Determination
1. The day before the molecular weight determination, please rinse 100 mL with buffer A-150 and check the column for air bubbles. If there are serious air bubbles or dry cracks, the column must be reloaded.
2. 0.4 mL of standard molecular weight solution, plus 0.5 mL of pure target enzyme (AF portion by affinity chromatography) should be injected into the column as above, and colloidal filtration should begin immediately, with collection of each fraction. Please follow all the above instructions for the column and assay collector.
3. Collect the results and analyze them for protein quantification. Several protein peaks can be identified as the basis for molecular weight; in addition, the number of tubes with red peaks can be determined visually to determine the number of tubes in which vitamin B12 is dissociated. Using the above data, a straight line relationship between molecular weight and the number of tubes dissolved can be drawn, which can be used as a standard calibration line for molecular weight determination.
4. For the same batch, please perform a GUS analysis to determine the dissociation volume of the enzyme, and then compare with the above standard correction line to find out the molecular weight of the enzyme.
Dismantle the column and preserve the colloid.
1. If the column is not used for a long period of time, the colloid should be removed from the column, washed with buffer and stored in a refrigerator, but never in a freezer. If the colloid is packed too tightly, it may not be easy to remove, so be patient and wash it out slowly with buffer solution.
2. 0.01% NaN3 can be added to the colloid to prevent the growth of mold, but remember to wash it off before use; when using it again, please check the colloid for gray and black mold particles, and do not use it if there are lumps that cannot be easily broken up.