From 2011.igem.org
(Difference between revisions)
|
|
| Line 1: |
Line 1: |
| - | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}
| |
| | | | |
| - | =His-Trap Protein Purification - Second Attempt=
| |
| - | Reflectin was harvested from bacteria very similarly to the [https://2011.igem.org/Team:Cambridge/Experiments/Protein_Purification first attempt], this time attempting both variants of the [https://2011.igem.org/Team:Cambridge/Protocols/Protein_Purification 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 [[Team:Cambridge/Protocols/Protein_Purification | 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.
| |
| - |
| |
| - | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}
| |
Latest revision as of 09:16, 16 September 2011
"
