Lidstrom:Measuring 13C Incorporation Into Protein - CO2 Project

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Cell Prep

• Grow 1.5 mL of OD₅₅₀ 0.8 - 1.5 (approximately 0.3 - 0.8 mg) of cell culture
• Centrifuge 5 min at 13,500 g, 4^oC
• Swirl 30-40 seconds in ethanol dry ice bath or liquid N₂ (don't freeze sample with liquid N₂)
• Discard supernatant
• Wash pellet with 0.9% 1 mL of sodium chloride at 4^oC, pellet for 5 min at 13,500 g, 4^oC
• Repeat last step

Optional:

• store pellet at -40 to -80^oC

Hydrolysis of Amino Acids

All steps in this section should be done in the fume hood across from the GC:

• Suspend cell pellets in 1 mL of 6N HCl
• Transfer resuspended cells into 2 mL GC-MS autosampler vials
• Seal tubes with screw caps to prevent evaporation of HCl
• Bake the well-sealed 2 mL tubes for 12-24 hours in a heating block set to 105 - 110 (not hotter)^oC
  • Hotter temps will may destroy some amino acids
• Dry the hydrolysate at 95^oC with constant air flow (or N₂) gas flow in the fume hood until the sample is completely dry.
  • Use the "nitrogen tree"
• Bake the dried samples at 105^oC for another 10 minutes to ensure no moisture is left.

Derivitization of Amino Acids

• Add 200 uL nanopure water to reconstitute the dried sample
  • If you have a lot of biomass, add 100 uL instead
• Centrifuge at 13,500 g for another 1 - 2 minutes to remove ash
  • Use a filter centrifuge tube if sampling a large fraction of this volume
  • If using a very small volume (~ 10 uL) you can pellet without a filter and sample from the top of the supernatant. Centrifuge longer (10 min) if using this approach.
• Transfer into clean GC-MS vial with insert, and repeat drying step with nitrogen tree as done above or speed-vac
  • Yanfen uses 10 uL; we should use 20 uL
  • We can prepare 40 uL + samples to use if the signal from 20 uL is too low
  • If using speed-vac: 35^oC for 1-2 hours until dry. Hold vials in 15 mL tubes.
• Prepare pyridine:
  • Add one layer of molecular sieve to scintillation vial
  • Add 1 mL pyridine per sample: found in Hackett lab cabinet under fume hood next to FPLC fridge.
  • Let sit for 5 min. No agitation.

For each sample:

• Add 20 uL of molecular-sieve treated pyridine to the dried sample using a syringe (would dissolve pipette tip)
  • The solvent may turn slightly brown
• Add another 20 uL of Trifluoromethanesulfonic acid tert-butyldimethylsilyl ester (TBDMS) and seal well
  • use the same blue screw-cap but replace the septum
• Rinse syringe in leftover pyridine

Repeat for all samples

Supplies

EMD Molecular Sieve Type 3A 8-12 (MX1583D-1)

Pyridine Sigma ACS Reagent (360570-500mL)

Agilent Screw Caps with Red PTFE/white silicone Septa 100 pk (5182-723)

Agilent Extra Septa Red PTFE/white silicone 500 pk (5182-730)

Agilent Vial Glass Small Volume Inserts (5183-2085)

Agilent Glass Vial 2 mL with write on spot (5182-0715)