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Team:Cambridge/Experiments/Protein Purification
From 2011.igem.org
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==Practice== | ==Practice== | ||
| - | *After high copy expression plasmids for his-tagged reflectin were successfully [https://2011.igem.org/Team:Cambridge/Experiments/Assembly_of_Reflectin_Constructs assembled] and [https://2011.igem.org/Team:Cambridge/Protocols/Transformation_of_E.Coli transformed] in E. coli, [https://2011.igem.org/Team:Cambridge/Protocols/Overnight_Culture cell cultures] were incubated overnight with | + | *After high copy expression plasmids for his-tagged reflectin were successfully [https://2011.igem.org/Team:Cambridge/Experiments/Assembly_of_Reflectin_Constructs assembled] and [https://2011.igem.org/Team:Cambridge/Protocols/Transformation_of_E.Coli transformed] in E. coli, [https://2011.igem.org/Team:Cambridge/Protocols/Overnight_Culture cell cultures] were incubated overnight with 1mM arabinose to induce reflectin expression. |
*[https://2011.igem.org/Team:Cambridge/Protocols/Buffers Buffers] were prepared, and their pH checked and readjusted if necessary on the day of purification. | *[https://2011.igem.org/Team:Cambridge/Protocols/Buffers Buffers] were prepared, and their pH checked and readjusted if necessary on the day of purification. | ||
*An [https://2011.igem.org/Team:Cambridge/Protocols/Inclusion_Body_Prep inclusion body prep] was performed with 50ml of overnight culture. Reflectin was [https://2011.igem.org/Team:Cambridge/Protocols/Protein_Purification purified] from the resulting lysate using a his-trap column. | *An [https://2011.igem.org/Team:Cambridge/Protocols/Inclusion_Body_Prep inclusion body prep] was performed with 50ml of overnight culture. Reflectin was [https://2011.igem.org/Team:Cambridge/Protocols/Protein_Purification purified] from the resulting lysate using a his-trap column. | ||
Revision as of 18:30, 21 August 2011
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Protein Purification
Bacteria expressing his-tagged reflectin were lysed, and the protein was purified using a his-trap column and a denaturing protocol in order to solubilise reflectin.
Practice
- After high copy expression plasmids for his-tagged reflectin were successfully assembled and transformed in E. coli, cell cultures were incubated overnight with 1mM arabinose to induce reflectin expression.
- Buffers were prepared, and their pH checked and readjusted if necessary on the day of purification.
- An inclusion body prep was performed with 50ml of overnight culture. Reflectin was purified from the resulting lysate using a his-trap column.
- This procedure was repeated using a culture of the same bacteria from a different flask.
Results
Photospectroscopy readings from the eluted solution indicated that we had obtained approximately 0.6mg of reflectin from the column. After dialysis...
