BAF_Protocol_007a Chloroform/Methanol Precipitation
Nicholas Sherman
Abstract
This protocol extends BAF_Protocol_007 as an alternative to acetone precipitation (normal method). The chloroform/methanol precipitation is a better method if the sample is high in lipid or other hydrophobic small molecules. This method also produces a protein pellet and not the normal liquid interface and as such is easier.
Steps
Chloroform/Methanol PPT
100 uL of protein extract of sample added to Eppendorf tube.
400 uL of Methanol was added, vortexed well.
200 uL of Chloroform was added, vortexed well.
300 uL of Water was added, vortexed well.
Sample was centrifuged at 13,000 rpm for 2 minutes
The upper layer was carefully removed (not disturbing the protein interface).
300 uL of Methanol was added, vortexed well.
Samples were centrifuged at 13,000 rpm, for 2 minutes.
Supernatants were discarded and the protein pellet was air dried. Resuspend in 100 mM Ammonium Bicarbonate in volume based on protein amount.
