The protein solution may not be flowing through column due to one or more of the following:
Centrifugation speed was too low.
Check the centrifuge to ensure that it is capable of generating 3,300 x g (micro kit), or 1,000 x g (midi and kits). Sufficient centrifugal force is required to move the liquid phase through the resin. Centrifugation speeds may be increased to 6,700 x g (micro kit), or 2,000 x g (midi and maxi kits), but this speed should not be exceeded.
Inadequate spin time.
Spin additional 1 minute (micro kit), or 3 minutes (midi and maxi kits) to ensure that the liquid is able to flow completely through the column.