Nov 07, 2022

Public workspaceCyanobacteria Total Lipid Extraction from Cell Pellets

 Forked from Cyanobacteria Total Lipid Extraction from Polycarbonate Filters
DOI
dx.doi.org/10.17504/protocols.io.4r3l2oe3jv1y/v1
  • 1University of Tennessee, Knoxville
Robbie Martin
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DOI: dx.doi.org/10.17504/protocols.io.4r3l2oe3jv1y/v1
Protocol Citation: Robbie Martin, Steven W Wilhelm, Katarina A. Jones, Shawn Campagna 2022. Cyanobacteria Total Lipid Extraction from Cell Pellets. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2oe3jv1y/v1
Manuscript citation:
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.
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: May 12, 2022
Last Modified: November 07, 2022
Protocol Integer ID: 62491
Keywords: cyanobacteria, lipids, extraction
Abstract
This protocol is designed/used for extraction of total cellular lipids from cyanobacteria samples (either lab cultures or field samples) collected via centrifugation 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.
Protocol materials
ReagentButanolFischer ScientificCatalog #71-36-3
Step 20
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)
Concentrate cells via centrifugation using methods appropriate for your available lab equipment and for your species of interest. Decant supernatant. Transfer pelleted cells to a 2-mL centrifuge tube. Cells may need to be re-pelleted and residual supernatant removed after transfer to final 2-mL centrifuge tube.

Note: Appropriate volume of lab culture or field samples to concentrate and extract depends on cell concentration. As a guideline, we have been successful concentrating 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.