2. Prepare at least 20 bed volumes (10Vt) of buffer and filter it through 0.22 μm filter in a filtration unit (Figure 22.1A).
Figure 22.1: A buffer filtration unit (panel A) and an Erlenmeyer flask for degassing (panel B)
3. Calculate the amount of dry matrix required for packing the column:
a. The swelling factor of the gel matrix is provided by the manufacturer.
b. The amount of dry gel required in grams
4. Weigh slightly more than the calculated amount of the gel matrix.
5. Take 2 bed volumes (2Vt) of buffer in an Erlenmeyer flask with a thick side-arm for applying vacuum (Figure 22.1B).
6. Transfer the dry gel matrix into the buffer present in the Erlenmeyer flask.
7. Stir the suspension gently using a glass rod (Important : Do not use a magnetic stirrer as it can disrupt the soft gel particles).
8. Cover the flask with a rubber stopper that fits well in the flask mouth; seal the side arm with parafilm.
9. Allow the gel to swell overnight at room temperature.
10. During swelling, the gel settles down in the beaker. The upper buffer region may contain broken beads and looks hazy. Remove the hazy buffer portion by decanting.
11. Suspend the settled gel in 2 – 4 fold excess of buffer and allow ~95% gel to settle down. Decant the buffer that contains non-settled gel particles. Repeat this process until the gel matrix settles as a sharp zone (usually 4 – 5 times is sufficient).
Packing of the column
- 12. Dilute the gel slurry two-fold by adding approximately the same volume of buffer as that of the settled gel.
13. Degas the gel by applying vacuum on the side-arm of the Erlenmeyer flask for 15 min (Figure 22.1B). Swirl the flask occasionally to release the air bubbles formed.
14. If more than 50% of the column tube needs to be packed, attach an extension on top of the column tube that can, together with the column, hold the entire volume of the gel slurry.
15. Mount the column tube on a stable laboratory stand. The column along with its attachments is shown in figure 22.3.
16. Remove air from the bottom adapter tubing of the column by attaching a buffer-filled syringe and forcing the sufficient buffer volume up though the bed support-net.
17. Insert the bottom adapter to the desired level in the column tube, remove the syringe, tighten the adapter, and attach the stop plug.
18. Ensure that the column is vertical using a carpenter's level or a plumb-line.
19. Swirl the gel slurry and pour the entire slurry into the column along a glass rod that is in contact with the inner wall of the column or column extension.
20. Fill the remaining space completely with the buffer by pouring it carefully along the glass rod so that the gel layer is not disturbed.