His-Trap Protein Purification - Second Attempt

Reflectin was harvested from bacteria very similarly to the first attempt, this time attempting both variants of the purification protocol and both nickel and ABT cobalt resin (kindly donated by Carole Augustus, Thistle Scientific). The culture that provided the higher yield of reflectin last time was harvested in this attempt.

Practice

The methods described in the first attempt at his-trap purification were repeated with two new columns, one containing nickel resin and the other containing cobalt resin. With the nickel column, the procedure was repeated while paying special attention to the [tips] we developed last time, while in the cobalt column the [variant] was used. The cobalt was also washed through with 6 extra column volumes of sterile water and triple the binding buffer at the start of the protocol, as recommended by the manufacturer. Instead of protein precipitation with dialysis, we attempted [acetone precipitation] and [ethanol precipitation] to fully isolate the reflectin product.

Results

Photospectroscopy readings from the eluted solution indicated that we had obtained approximately 2mg of reflectin from each column, indicating a five-fold increase in our yield. We therefore favour the 'variant' his-trap protocol, since it is slightly easier to perform, and seems to be more robust (it is harder to go wrong!). Both acetone and ethanol precipitation worked well, with large pellets visible at the end of the procedures.