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Team:Cambridge/protocols/Norgen
From 2011.igem.org
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==Norgen Proteospin Inclusion Body Prep.== | ==Norgen Proteospin Inclusion Body Prep.== | ||
| - | A proprietary kit for isolating the non-soluble fraction of a cell's internal protein (a soluble internal | + | A [http://www.norgenbiotek.com/display-product.php?ID=8 proprietary kit] for isolating the non-soluble fraction of a cell's internal protein (a soluble internal fraction is also obtained but usually discarded). |
===Theory=== | ===Theory=== | ||
| - | Upon solublisation of the membrane fraction of the cell, inclusion bodies are the densest remaining component | + | Upon solublisation of the membrane fraction of the cell, inclusion bodies are the densest remaining component and can be pelleted by ultracentrifugation. The inclusion-body proteins are then solublised in a proprietary agent, with purification on protein columns. |
===Practice=== | ===Practice=== | ||
| - | The protocol used is identical to that given on the instructions with the kit. The 'basic' protocol is used | + | The protocol used is identical to that given on the [http://www.norgenbiotek.com/display-product.php?ID=8 instructions with the kit]. The 'basic' (as opposed to 'acidic') protocol is used since the pI of reflectin is 8.5. The pI of a protein can be calculated using [http://web.expasy.org/compute_pi/ this web tool]. |
| - | It was found that the centrifugation forces given in the protocol for the | + | It was found that the centrifugation forces given in the protocol for the bench-top centrifugation steps were insufficient. The speed needed to be raised by a factor of four. |
{{Template:Team:Cambridge/CAM_2011_PROTOCOL_FOOT}} | {{Template:Team:Cambridge/CAM_2011_PROTOCOL_FOOT}} | ||
Latest revision as of 03:25, 22 September 2011
Norgen Proteospin Inclusion Body Prep.
A [http://www.norgenbiotek.com/display-product.php?ID=8 proprietary kit] for isolating the non-soluble fraction of a cell's internal protein (a soluble internal fraction is also obtained but usually discarded).
Theory
Upon solublisation of the membrane fraction of the cell, inclusion bodies are the densest remaining component and can be pelleted by ultracentrifugation. The inclusion-body proteins are then solublised in a proprietary agent, with purification on protein columns.
Practice
The protocol used is identical to that given on the [http://www.norgenbiotek.com/display-product.php?ID=8 instructions with the kit]. The 'basic' (as opposed to 'acidic') protocol is used since the pI of reflectin is 8.5. The pI of a protein can be calculated using [http://web.expasy.org/compute_pi/ this web tool].
It was found that the centrifugation forces given in the protocol for the bench-top centrifugation steps were insufficient. The speed needed to be raised by a factor of four.
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