Cyanobacteria Total Lipid Extraction from Polycarbonate Filters
Steven W Wilhelm, Robbie Martin, Katarina A. Jones, Shawn Campagna, Hector Castro
Abstract
This protocol is designed/used for extraction of total cellular lipids from cyanobacteria samples (either lab cultures or field samples) collected on polycarbonate filters for use in lipid analysis and quantification via mass spectrometry.
Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) or Robbie M. Martin (rmarti49@vols.utk.edu) for additional information regarding this protocol.
Modified from Guan, X. L., Riezman, I., Wenk, M. R., & Riezman, H. (2010). Yeast lipid analysis and quantification by mass spectrometry. Methods in Enzymology , 470 , 369-391.
Before start
Steps
Prepare the three separate solutions needed for this extraction protocol as follows:
- lipid extraction solvent: a 15 : 15 : 5 : 1 : 0.18 ratio by volume of 95% ethanol, water, diethyl ether, pyridine, and 4.2 N ammonium hydroxide, respectively.
- water-saturated butanol: a 1:1 ratio of butanol and Milli-Q water
- purified lab water: (Milli-Q water)
Unfold polycarbonate filter and place into a 2-mL centrifuge tube with cell side of filter facing outwards.
Note: Appropriate volume of lab culture or field samples to filter and extract depends on cell concentration. As a guideline, we have been successful filtering 10-25 mL of lab cultures of Microcystis aeruginosa and ~50 mL of either raw lake water or mesocosm samples.
Add 1 mL of extraction solvent, ~100 µL of glass beads, and vortex ~5 s.
Incubate sample in 60 °C water bath for 20 min.
Centrifuge sample at 10,000 x g for 10 min.
Remove supernatant and place into a 1-dram glass vial (dram vial #1). The first two extractions from a sample will be placed in this vial (#1).
Repeat steps 3-6, except DO NOT ADD more glass beads.
Dry the collected supernatant in dram vial #1 under a stream of nitrogen.
Re-suspend dried sample in 300 µL of water-saturated butanol and 150 µL of Milli-Q water.
Vortex and transfer to a 2-mL centrifuge tube.
Centrifuge at 10,000 x g for 2 min.
Remove top butanol phase and place into a NEW 1-dram glass vial (dram vial #2).
Wash original dram vial (#1) with 300 µL saturated butanol and transfer to residual aqueous phase in 2-mL centrifuge tube from step 10. Vortex.
Centrifuge at 10,000 x g for 2 min. Remove top butanol phase and place into dram vial #2.
Dry the collected butanol phase in dram vial #2 under a stream of nitrogen.
Re-suspend dried sample in 300 µL of 9:1 methanol:chloroform.
The samples are now ready for analysis via LC/MS.
Prepare the three separate solutions needed for this extraction protocol as listed below.
lipid extraction solvent (step 2) with a 1:1 ratio of butanol and lab purified water (water-saturated butanol, and lab purified water
