Drummond:ChlorMethExtraction: Difference between revisions
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* Remove as much methanol as possible. Be careful, because the pellet is delicate. You should be able to remove all but a few uL of methanol with care, which will speed drying. | * Remove as much methanol as possible. Be careful, because the pellet is delicate. You should be able to remove all but a few uL of methanol with care, which will speed drying. | ||
* Dry under vacuum. | * Dry under vacuum. | ||
Adapted from Wessel, D. and Flügge, U.I. (1984) <i>Anal. Biochem.</i> 138 141–143. | |||
</div> | </div> | ||
{{Drummond_Bottom}} | {{Drummond_Bottom}} | ||
Revision as of 07:21, 15 July 2011
Introduction
Detergents, like SDS, and salts, like NaCl, can disrupt LC-MS/MS runs. Precipitation with chloroform and methanol results in dry protein material, free of salt and detergent.
Protocol
To 100uL protein sample (~100ug protein) in a 1.5mL eppendorf tube:
- Add 400 uL methanol and vortex thoroughly.
- Add 100 uL chloroform and vortex.
- Add 300 uL H2O—mixture will become cloudy with precipitate—and vortex.
- Centrifuge 1 minute @ 14,000g. Result is three layers: a large aqueous layer on top, a circular flake of protein in the interphase, and a smaller chloroform layer at the bottom.
- Remove top aqueous layer carefully, trying not to disturb the protein flake.
- Add 400 uL methanol and vortex.
- Centrifuge 5 minutes @ 20,000g, which will slam dandruffy precipitate against the tube wall.
- Remove as much methanol as possible. Be careful, because the pellet is delicate. You should be able to remove all but a few uL of methanol with care, which will speed drying.
- Dry under vacuum.
Adapted from Wessel, D. and Flügge, U.I. (1984) Anal. Biochem. 138 141–143.
