Team:Cambridge/Protocols/Protein Purification

Protein Purification

A his-trap mechanism was used to isolate reflectin from the lysate produced after performing an [inclusion body prep]. The protocol below was adapted from [http://tools.invitrogen.com/content/sfs/manuals/xprpur_man.pdf here].

Theory

Practice

Preparing the column

1. When taking his-trap resin out of storage, the resin is likely to have precipitated from its storage buffer. Resuspend the resin in its bottle by inverting and gently

tapping the bottle repeatedly.

1. Pipette 2 ml of the resin into a 10-ml Purification Column. Allow the resin to settle completely by gravity (5-10 minutes) before carefully aspirating the supernatant.
2. Add 6 ml of sterile, distilled water and resuspend the resin by alternately inverting and gently tapping the column.

Allow the resin to settle using gravity or centrifugation as described in

Step 2, and gently aspirate the supernatant. 5. For purification under Denaturing Conditions, add 6 ml of Denaturing Binding Buffer. 6. Resuspend the resin by alternately inverting and gently tapping the column. 7. Allow the resin to settle using gravity or centrifugation as described in Step 2, and gently aspirate the supernatant. Repeat Steps 5 through 7.

Purification procedure

Safety

All safety measures relating to guanidine hydrochloride in solution from the buffer protocol apply here when dealing with GLB. In addition, all consumables and liquid waste that may have come into contact with the bacteria must be autoclaved before disposal.