Mon. Dec 9th, 2019

ANATRACE How to purify a membrane protein-Technical bulletin

2 min read
International center for biotechnology information

International center for biotechnology information

1- First Step!

Look up other purifications from similar sources and similar membranes.Different tissues, organisms and membrane types sometimes require differ-ent detergents and conditions. e.g. bacterial sources may require quitedistinct solubilization procedures from mammalian sources.

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Complete dispersion and lipid depletion are critical for successful chroma-tography. If the protein is not separately solubilized from other proteins andmuch of adhering phospholipid removed, the protein will not bind andchromatograph on the basis of its own characteristics. Instead, it will behaveheterogeneously on the basis of the other proteins and lipids attached.

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A detergent that is good for solubilizing intact membranes and removingexcess lipid may not be the best for continuing to purify the “naked”protein (may be too harsh). Thus, for the initial steps, you may want tochoose a relatively cheap, relatively pure detergent that can be readilyexchanged for another. For example, cholate and CHAPS are both strong,charged or zwitterionic, steroid detergents that break up membranes welland help in removal of excess lipid in chromatographic steps such ashydroxyapatite. They also have a high critical micelle which means theycan be readily removed by dialysis or exchanged for another on a column.

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A good dispersing detergent that will also stabilize the protein and is freeof contaminants that could inactivate the enzyme, is needed for chromato-graphic purification. For many, TRITON® X-100 has been very suitable,but has the problem of contaminants and 280 nm absorbance. A twelvecarbon chain length of hydrocarbon tail is often necessary for goodstabilization and dispersion, especially for larger multisubunit enzymes.Dodecyl-β-D-maltoside often works equally well or better than TRITON®,with none of the disadvantages. Shorter chain detergents and some chargeddetergents tend to dissociate multisubunit proteins. Trial and error is clearlyimportant at this stage with careful attention to maintaining a high enoughdetergent to protein ratio to make sure the protein is well dispersed (thedetergent concentration will be much higher than the CMC).

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Even if a protein is not active in a particular detergent, if you can show thatthe inactivity is reversible, by adding back lipid or another detergent, youmay still be able to use it.

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