Drummond:Solubility

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(principle, and some questions answered)
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==Introduction==
==Introduction==
I'd like to measure the proportion of a protein in the soluble versus insoluble state.  Typical assays seem to use antibody probes against the supernatant and pellet of a standard lysis.
I'd like to measure the proportion of a protein in the soluble versus insoluble state.  Typical assays seem to use antibody probes against the supernatant and pellet of a standard lysis.
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==Principle==
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The basic method of all assays I've seen is to lyse cells into an aqueous buffer, spin down the pellet, pull off the supernatant and store it as the soluble fraction, then solubilize proteins remaining in the pellet using a solubilization buffer containing various detergents (e.g. Triton X-100), spin down the pellet again, and pull off the supernatant and store it as the insoluble fraction.
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Questions: How do you ensure that you've preserved the composition of total protein in each fraction?  Answer: Extract in the same amount of buffer in each case, and load identical amounts of each fraction.  Control: Do the lysis in solubilization buffer, and save that fraction as total protein.
==Links to protocols==
==Links to protocols==

Revision as of 11:57, 19 June 2007

Contents

Introduction

I'd like to measure the proportion of a protein in the soluble versus insoluble state. Typical assays seem to use antibody probes against the supernatant and pellet of a standard lysis.

Principle

The basic method of all assays I've seen is to lyse cells into an aqueous buffer, spin down the pellet, pull off the supernatant and store it as the soluble fraction, then solubilize proteins remaining in the pellet using a solubilization buffer containing various detergents (e.g. Triton X-100), spin down the pellet again, and pull off the supernatant and store it as the insoluble fraction.

Questions: How do you ensure that you've preserved the composition of total protein in each fraction? Answer: Extract in the same amount of buffer in each case, and load identical amounts of each fraction. Control: Do the lysis in solubilization buffer, and save that fraction as total protein.

Links to protocols

Knight:Protein solubility

References

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