Gel filtration technique

Gel filtration technique

Preparation of column

  1. Use Sephadex G-100 for gel filtration. Prepare the gel slurry by dissolving 5 g Sephadex G-100 in 50 ml distilled water or 50 mM Tris buffer, pH. 7.5
  2. Prepare the equilibration buffer, 50 mM Tris buffer, pH. 7.5 and degas it.
  3. Rinse the column with 20% ethanol.
  4. Remove the bottom piece and remove the air from it by purging with 20% ethanol. While removing the air bubbles, ensure that the net surface does not have white appearance instead should be dark.
  5. Immediately close the outlet with dump nut and fix it with the column. Place the column in a pipette stand and add 5 ml of 20% ethanol or 50 mM Tris buffer, pH. 7.5, to avoid air bubble trapping.
  6. Repeat the same for the adaptor. Remove the air, close the outlet with dump nut and dip it in 20% ethanol kept in a beaker.
  7. Fix the reservoir over the column and pour the slurry with help of a glass rod and close the reservoir with the lid.
  8. Switch “ON” the instrument and the soft ware “PRIME VIEW”. It will show ‘Template”. Using the navigator button, bring it to “Manual Run”
  9. Remove the male nut valve in the pump, and purge to remove the air bubbles from the inlet tube. (A1)
  10. Set the conditions for setting the column. Screen shows “Manual run”. Press “OK” to start. Using the navigator key, go to flow rate and set 5.0 ml flow rate/min. Set pressure at 0.3 MPa. Select “LOAD” mode. Then, start “RUN” by clicking “OK”. Observe for the buffer flow from the Port 1 of the “Injection valve”.
  11. Connect the tube from the Port 1 of the Injection valve to the top of the reservoir. Remove the bottom dump nut and check the flow, and connect it into the “UV”.
  12. Then, press “PAUSE” and change the flow rate to “10 ml/min” and again continue. Wait until the column material is compressed (approx 10-20 min). After compression, press “PAUSE”.
  13. Remove the connecting tube from the reservoir and injection value. Also remove the bottom outlet tube of the column from the “UV” and place the dump nut.
  14. Remove the column from the stand and slowly open the reservoir to remove the excess buffer from the reservoir into a beaker. Remove the excess buffer from the column top using a syringe.
  15. Place the adaptor over the column at 450 angle to avoid air bubbles trapping inside. Fix the adaptor by adjusting the screws on top. Care should be taken not to press too much which will disturb the column.
  16. Then, press “PAUSE” and change the flow rate to 2 ml/min and press “Continue”.
  17. Check the flow from the Port 1 valve of the Injection value. Connect the connecting tube from the adaptor to the Port 1 of the Injection valve. Remove the dump nut from the bottom outlet, check the flow and connect it to the “UV”.
  18. Allow it run, until the conductivity becomes stable and end the program by pressing “END”

Sample injection

  1. Set the conditions. Flow rate 2ml/min, Pressure 0.3MPa, Fraction size 5ml/min, choose “LOAD” mode
  2. Start the program by pressing “OK” with the help of navigation button
  3. Inject 5 ml of filtered enzyme solution (filtration by 0.2 U syringe filter) into the Injection valve (Port no 3).
  4. Change “LOAD” mode into “INJECT” mode
  5. Separation takes place and fractions get collected
  6. Collect until all fractions of protein comes out by observing the UV
  7. End the program by pressing “END”
  8. Fractions form the PURIFIED ENZYME EXTRACT.
  9. Fractions collected are further checked for protein content and enzyme activity.
  10. For injecting next sample proceed as per step 1 of sample injection.
Last modified: Saturday, 12 November 2011, 7:28 AM