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How to Calibrate Gel Filtration Columns

Gel filtration columns are used to separate proteins from complex mixtures. Since each protein has a unique molecular weight, they can be induced to separate into distinct layers by migrating them through a filtration column containing an evenly distributed gel matrix. To correctly identify each protein that is separated out, you will need to calibrate the columns using several proteins of known molecular weight.

Things You'll Need

Carbonic anhydrase
Cytochrome C
Aprotinin
Basal salt
Centrifuge tubes
Ferritin
Centrifuge
Pipet pump
250 ml graduated cylinder
Buffer liquid
Scientific cleaning wipes
Syringe barrel

Instructions

- 1
  Remove the vials of carbonic anhydrase, cytochrome C and aprotinin from the refrigerator or freezer and allow them to reach room temperature. Add 4 mg of each protein to a centrifuge tube per milliliter of gel in the filtration column. Add 961 mcl of basal salt per milligram of protein to each centrifuge tube to dissolve the proteins. Add 39.2 mcl of ferritin per milliliter of gel to each tube and then mix in a centrifuge.
- 2
  Turn off the filtration column pump and close the stopcock at the column entrance. Unscrew, but do not remove, the top of the filtration column. Set the pump to the maximum flow rate for the column and restart the pump until the buffer material has been drawn off. Stop the pump.
- 3
  Add 1 ml of each protein to the filtration column using a pipet pump. Drip the proteins down the inside of the column from a height of 1 cm to 2 cm, working around the inner edge of the column. Allow the protein mixture to settle on top of the gel matrix.
- 4
  Place the outlet tubing of the pump in a clean, dry 250 ml graduated cylinder. Restart the pump at a rate of 0.30 ml per minute and run the proteins into the gel matrix. While the pump is running, rinse the sides of the tube using the pipet pump and a small amount of buffer liquid. Once the proteins have fully entered the gel matrix, reduce the pump speed to 0.02 ml per minute and pump in a 5 cm to 7 cm layer of buffer liquid on top of the matrix.
- 5
  Disconnect the column cap from the stopcock. Clean the column cap and the inside of the column using scientific cleaning wipes. Reinstall the column cap. Fill the buffer reservoir with buffer liquid. Attach a syringe barrel to the stopcock and open it. Use the syringe to draw a small amount of buffer through the tubing and into the syringe. Close the stopcock and reattach it to the column top. Open the stopcock and check for leaks.
- 6
  Run the column at 0.02 ml per minute for several days, until the ferritin band nears the end of the column. Collect all the filtration gel that is pumped out in the graduated cylinder.

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