page 74 column chromatography rev 2012

							                               Anion Exchange Cellulose Preparation of
                                    Preswollen Whatman DE-52

Material Preparation

STOCK/ELUTION BUFFER. Prepare 50-100 mL of “10 X stock buffer”, 0.1 M phosphate, pH 7.6.
This is 10-fold more concentrated than the concentration of the buffer you are using to dialyze your
protein (0.0IM sodium phosphate buffer pH 7.6). Store this buffer in the refrigerator. It can be used to
make up 0.01 M buffer when or you need; it is also the elution buffer for your column.

LOADING BUFFER. Make up 500 mL per person of 0.01 M phosphate, pH 7.6. This will be used to
swell and equilibrate the column and is the loading buffer.

Weigh out 4 g of Whatman DE52 beads.


DE 52 Whatman protocol

NOTE: pouring columns is an exacting procedure. The column must be poured CONTINUOUSLY.
Breaks in the material, especially any that introduce air bubbles will essentially destroy the columns
ability to purify samples.

1.      In a 50 mL beaker, combine the DE52 beads with 10 mL of buffer of loading buffer (0.01 M
        phosphate) per gram of beads while stirring slowly.

NOTE: The beads are FRAGILE! Rapid stirring while break them and hurt your column.

2.      While stirring slowly with a glass rod, adjust to pH 7.6 with 1M HC1 or 1M NaOH. Let stir a
        minute and adjust pH again if necessary.

3.      Then allow the slurry of DE 52 to settle on ice. (Go to step 6 while you wait)

4.      Decant the solution by carefully pouring off the liquid and any “fines”. Fines are very small
        (usually damaged) bead particles that require a long time to settle.

        What volume of hydrated beads do you have? _______

5.      Resuspend the DE 52 in an equal volume of starting buffer, 0.01M PB pH 7.6. to make a
        thin suspension or “slurry”. Store on ice.

6.      Set up the small chromatography column on a ring stand. Measure the useable internal volume of
        the column. This is the total internal volume up to 1 cm below the top. Write down this volume.
        _______

7.      Fill the column with cold buffer and open up the valve. Solution should drip out. If it does not
        begin flowing, then the valve is probably blocked by an air bubble. This can be cleared by using a
       syringe and a connector to suction a bit of the buffer through the tubing.

8.     Leave about 1 inch of buffer in the column and close off the column valve.

9.     Attach a funnel to the top of the column.

10.    Mix your bead slurry WELL and pour about half of the DE 52 along the edge of the funnel
       such that it does not trap air.

11.    Open the column valve and allow the buffer to flow SLOWLY. It should not flow so fast that
       you can't count the drops. Tap the column gently with a pencil or similar weight to “shake” the
       beads to the bottom.

12.    As the column packs, continue to add slurry continuously until the gel packs to within 1 cm of the
       top (between 0.2 and 2 cm is acceptable). Shut off the column and remove the funnel.

13.    Rinse the funnel free of any remaining gel and replace the funnel on the column.

14.    Add some cold starting buffer, 0.01M PB pH 7.6 to the top of the column. Add the buffer down
       the inside wall of the funnel slowly. Try not to disturb the gel in the column. Open the valve and
       let the buffer begin to flow; not too fast.

15.    Wash the gel with 5 mL of starting buffer.

16.    The column must never dry out, or it will be ruined. Store it with buffer on top. Seal both the top
       and the bottom, carefully, with parafilm before storing it for any time. It should be stored
       UPRIGHT. Put it in the refrigerator for overnight storage.




Lone Star College Montgomery Biotechnology Program.
Original Protocol: William Geoghegan; revised 2009, x2 L Loomis-Price
                                  Purification of Rabbit IgG

Once your rabbit serum precipitate has been dissolved and dialyzed against several changes of 0.01 M PB
pH 7.6 and your column has been equilibrated you are ready to begin purification of the rabbit IgG. Here’s
an animation of what’s happening: http://www.youtube.com/watch?v=9GiLjH9Oym8

1. Set up a beaker for your slow-through IgG and a rack of 10 numbered test tubes to hold your
   fractions. Measure 2 mL of water into one and mark the level. Discard the water. Get a beaker of ice to
   store your protein fractions in.

2. Definition: “running a solution INTO the column” means to let the solution flow until the meniscus
   actually touches the surface of the gel. This helps avoid mixing of old and new material.

3. Run your phosphate buffer into the DE 52. Discard the eluate (the buffer that runs out the bottom).
   This is the last buffer you will discard. Save EVERYTHING else that comes off the column!!

4. Place a small beaker under the column.

5. Next carefully add a few drops of the dialyzed rabbit serum protein pellet to the top of the gel,
   dropwise with a Pasteur or transfer pipette. Try not to disturb the top of the gel when you add the
   protein. Run this into the gel. The carefully add the rest of the protein – up to 10 mL total.

6. Run the last of the protein into the column. Collect the “flow-through” into your beaker. Store on ice.

7. Switch to your rack of tubes before starting the next step.

8. Run about 0.5 mL of STARTING (0.01 M PB) buffer into the column and then add 5.5 mL more of
   the same buffer to rinse the gel. Immediately begin to collect 2 mL fractions (measure to your mark
   and try to collect equal volumes in each tube). Store the tubes on ice after you collect them.

9. What do you note about the color of the proteins going on the column, sticking on the column and
   flowing through? ___________________________________________

10. Now switch over to elution buffer (0.1 M phosphate), 0.5 mL (run into gel) and then as much as
    needed until you’ve collected all your fractions. Collect 6 more 2 mL fractions.

11. What do you note about the colors of the fractions? _______________________________

12. Cover the tubes and refrigerate until you can measure the Abs (280), next lab.

Lone Star College Montgomery Biotechnology Program.
Original Protocol: William Geoghegan;       revised 2009, 2012 L Loomis-Price