Apr 02, 2024
BAF_Protocol_007a Chloroform/Methanol Precipitation
- 1University of Virginia Biomolecular Analysis Facility Core
Protocol Citation: Nicholas Sherman 2024. BAF_Protocol_007a Chloroform/Methanol Precipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld83q9v5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 02, 2024
Last Modified: April 02, 2024
Protocol Integer ID: 97659
Keywords: protein precipitation, mass spectrometry, sample cleanup, lipds
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.
Guidelines
This precipitation would be substituted in for the acetone precipitation normally used in BAF_Protocol_007.
Materials
Pre-cleaned microtubes 1.5 mL - SEAL-RITE‱ 1.5 ML MICROCENTRIFUGE TUBES color: natural, USA Scientific
Pipette tips - Fisher Brand, yellow, part number: 02-681-151
ABC - Fluka analytical Ammonium Bicarbonate, Sigma Aldrich, 09830-500G
VWR Analog Vortex mixer - CAT No: 58816-121
Centrifuge 5427 R, Eppendorf
Water - Fisher Chemical, W6-4, Optima LC/MS
Methanol - A456-212, Methanol, optima LC/MS
Chloroform - AC423550010, ACS Grade 99.8+%
Chloroform/Methanol PPT
Chloroform/Methanol PPT
28m
100 uL of protein extract of sample added to Eppendorf tube.
1m
400 uL of Methanol was added, vortexed well.
2m
200 uL of Chloroform was added, vortexed well.
2m
300 uL of Water was added, vortexed well.
2m
Sample was centrifuged at 13,000 rpm for 2 minutes
4m
The upper layer was carefully removed (not disturbing the protein interface).
1m
300 uL of Methanol was added, vortexed well.
2m
Samples were centrifuged at 13,000 rpm, for 2 minutes.
4m
Supernatants were discarded and the protein pellet was air dried. Resuspend in 100 mM Ammonium Bicarbonate in volume based on protein amount.
10m