Team:Cambridge/protocols/Norgen

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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