IEX Chromatography Instructions 2014

IEX Chromatography Instructions 2014 - Bovine LDH...

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Bovine LDH Purification Experimental Procedures – Ion Exchange Chromatography Materials: 1.5 x 20 cm Biorad Econo column 50 ml Sigma Q Sepharose ion exchange media equilibrated in 0.3 M Bicine pH 8.5 Biorad Biologic LP chromatography computer and Biorad 80 tube 2110 fraction collector Column set up: 1. Degas 50 ml of Q-sepharose for 15 minutes. 2. Attach the Econo column to the Biologic LP plumbing. 3. Purge the system with 50 ml 0.3 M Bicine buffer. Make sure the mesh netting in the end of the Econo column is wet and there are no air bubbles in the stopcock. Leave 5-10 ml of Bicine in the column and close the stopcock. 4. Carefully pour the slurry of Q-sepharose and bicine down the sides of the column until the fluid level reaches ½ inch from the top of the column. 5. Replace the top of the column and use a syringe filled with Bicine to remove the air in the top of the column. 6. Reattach the inlet tube and pack the column at a flow of 6.5 ml/min. 7. You may need to repeat steps 5 and six until the bed of packed beads is within 1 inch of the top of the column. The remainder of the column should be filled with Bicine buffer.
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