Assembly:

• Obtain the Flex column, a stand, a three-way stopcock, flow adapter and pump, rinse all with distilled water (exept for the pump!).
• MAKE SURE THE COLUMN IS NOT BLOCKED!!
• Shake Ni-agarose beads vigorously and add ~5ml to the column
• Attach the pump and allow it to compress the beads (take note of the direction!!)
• Fill the column with buffer or destilled water to the top, then add the flow adapter allowing no air bubbles inside the column.
• Allow the expandable tip of the flow adapter to touch the bead of the column, then expand it to lock it in place, but don't lock it to much, this will damage the column!
• Attach tube adapters from the bottom of the pump to a distilled water bottle
• Open tube compression of pump, add water using a syringe to the stopcock, if no air bubbles are observed, close the compression door, then release the syringe.
• Place the column and the pump in the cold room
• Wash the column with water (50 ml) and equilibrate with the appropriate buffer~50ml, allow it to flow from bottom to top (direction of pump)
• Make sure the pump tubes are not carrying any air bubbles
• Load the protein sample by placing the tube adapters in the protein solution and "back-load" the protein onto the column (i.e. this will cause the protein to be concentrated at the bottom of the column, thus eluting more concentrated protein, with a better defined peak)
• DO NOT CAUSE THE PROTEIN TO BE COMPLETELY DRAWN UP BY THE PUMP, if you are unsure as to what to do simply turn off the pump and ask someone to help you.
• Wash the column from the top with the appropriate buffer(s)  ~150-200ml
• Elute protein in 1ml bumps