Lidstrom:Measuring 13C Incorporation Into Protein - CO2 Project

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Contents

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, 4oC
  • 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 4oC, pellet for 5 min at 13,500 g, 4oC
  • Repeat last step

Optional:

  • store pellet at -40 to -80oC

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 95oC 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 105oC 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
    • 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
  • 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)

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