TITLE: Radiolabeled lipid extraction protocol
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Radiolabeled - Lipid Extraction Protocol for </span><span style = "font-style:italic;">Enterococcus faecalis</span></div></div>

[STEPS]
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Grow a large culture of cells (suggest 35 mL per condition) in the lab as normal.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Once cells have reached mid-log, harvest 5mL of culture (no fatty acid added yet!) and place cells into a 15mL conical. This is time point zero.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Freeze the time point tubes, including time zero, as follow in liquid nitrogen.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
To a 50 mL conical, add 25 mL of liquid culture.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Add in radiolabeled + cold fatty acid mixture (amount to be determined based on target number of µCi [suggest 0.5]); then bring up fatty acid to appropriate concentration), or equivalent volume of ethanol for solvent control.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
For every time point, harvest 5 mL of the spiked culture. Use filter tip pipettes(1mL each)5x to avoid splashing of hot material.
For each time point, repeat steps 6 and 7
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Freeze the tube in liquid nitrogen; once all-time points have been collected, proceed at step 8.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Add in 5 mL of 1X PBS. Wash two times.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
After the second wash, tap out the remaining volume of liquid on the pellet onto multiple layers of paper towels; tap in dry locations on the paper towel to avoid saturation through the paper and to avoid collecting hot material on the lip of the conical.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Re-suspend pellet in 200uL of 4:1 v/v Ethanol : Water. Use filter tips to avoid aerosols.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Boil the suspension in 100*C dry bath for 15 minutes. Vortex about halfway through (at 7.5 min).
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Centrifuge the boil.  Pipet off the supernatant and place into a 2mL glass autosampler vial.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Re-suspend the pellet in 100ul of 4:1 v/v Ethanol : Water
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Boil for an additional 15 minutes.  Vortex half way through.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Centrifuge the boil.  Pipet off the supernatant and place it into the previous 2mL glass autosampler vial.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Boil for a final time for 15 minutes.  Vortex half way through
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Centrifuge the boil and pipet the supernatant into the previous 2mL glass autosampler vial.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Dry down the samples by leaving the vials on the heat block set to 45*C with the lid off.  Drying takes about 24 hours.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Re-suspend the dried lipids in 20uL of 2:1 v/v chloroform : methanol.
?. [Radiolabeled - Lipid Extraction Protocol for Enterococcus faecalis]
Run on TLC with trimethylamine solvent system.