DEAE Anion Exchange Protein Purification Protocol

Biochemistry, Molecular Biology, and Cell Biology Protocols  >> DEAE Anion Exchange Protein Purification Protocol

Running a DEAE column

Solutions required
~100mL 40mM Tris, pH 7.8

~60mL 40mM Tris, pH 7.8 +0.6M NaCl

Depending on how strongly your protein is bound, you may want to use a higher molarity of NaCl for your second solution

Dialyze samples
Stir in cold room
Change the outside buffer before leave (should have stirred at least one hour)
Change outside buffer again the next day and stir at least one hour
Spin down dialyzed samples to make sure there’s no pellet
If there is a pellet, resuspend in high-salt solution and run some on gel
Pour the rest into the column

Prepare column
Pour 8 mLs of beads into a tube
Wash 3x with water, 1x with Tris only, 1x with Tris + high salt
Put enough of the “Tris only” solution to resuspend the beads again (~16mL if 8 mL of beads)
pour into column and let run 
Pour some “Tris only” solution and let column run a bit in the same tube that samples ran into
Run some of that sample on gel
Run “Tris only” solution through column ‘til OD280 is decreased to ~0.02 to 0.01.
Start the gradient (high-salt solution on left, low-salt solution on right if the gradient apparatus starts from the right side; vice versa if the liquid flows from the left first)
--push to get rid of the air bubbles
Keep the chamber on the right stirring
Column should empty at the same rate that solution enters

Read the OD280 of each of the 40 tubes
Pour back into correct tube after each reading

Copyright 2006-2012 by Biologicalworld.com  Disclaimer   Privacy Policy