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, 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:
- Suspend cell pellets in 1 mL of 6N HCl
- Transfer resuspended cells into 2 mL tubes
- Seal tubes 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 100 uL nanopure water to reconstitute the dried sample
- Transfer into filter centrifuge tubes
- Centrifuge at 13,500 g for another 1 - 2 minutes to remove ash
- Transfer into clean GC-MS vial with insert, and repeat drying step with nitrogen tree as done above
- Add 20 uL of pyridine to the dried sample
- The solvent may turn slightly brown
- Add another 20 uL of Trifluoromethanesulfonic acid tert-butyldimethylsilyl ester (TBDMS) and seal well
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)