Lidstrom:Measuring 13C Incorporation Into Protein - CO2 Project

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== Supplies ==
== Supplies ==
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[ http://www.emdmillipore.com/chemicals/molecular-sieve-type-3a/EMD_CHEM-MX1583D/p_uuid;sid=eXGkpC4I2H2YpH5U0s7J84bI_vK2VESvQV-68p2lNt7cOKCGVZHHDig2biopGYXUy7O0uC3RhEmNo6fB2_UUuvUcWIW94OLYSuschZYRSHZISA_8YDUqKgLo EMD Molecular Sieve Type 3A 8-12 (MX1583D-1)]
+
[http://www.emdmillipore.com/chemicals/molecular-sieve-type-3a/EMD_CHEM-MX1583D/p_uuid;sid=eXGkpC4I2H2YpH5U0s7J84bI_vK2VESvQV-68p2lNt7cOKCGVZHHDig2biopGYXUy7O0uC3RhEmNo6fB2_UUuvUcWIW94OLYSuschZYRSHZISA_8YDUqKgLo EMD Molecular Sieve Type 3A 8-12 (MX1583D-1)]
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[ http://www.sigmaaldrich.com/catalog/product/sial/360570?lang=en&region=US Pyridine Sigma ACS Reagent (360570-500mL)]
+
[http://www.sigmaaldrich.com/catalog/product/sial/360570?lang=en&region=US Pyridine Sigma ACS Reagent (360570-500mL)]
-
[ https://us.vwr.com/store/catalog/product.jsp?catalog_number=89040-590 Agilent Screw Caps with Red PTFE/white silicone Septa 100 pk (5182-723)]
+
[https://us.vwr.com/store/catalog/product.jsp?catalog_number=89040-590 Agilent Screw Caps with Red PTFE/white silicone Septa 100 pk (5182-723)]
-
[ https://us.vwr.com/store/catalog/product.jsp?catalog_number=89040-590 Agilent Extra Septa Red PTFE/white silicone 500 pk (5182-730)]
+
[https://us.vwr.com/store/catalog/product.jsp?catalog_number=89040-590 Agilent Extra Septa Red PTFE/white silicone 500 pk (5182-730)]
-
[ https://us.vwr.com/store/catalog/product.jsp?catalog_number=89203-498 Agilent Vial Glass Small Volume Inserts (5183-2085)]
+
[https://us.vwr.com/store/catalog/product.jsp?catalog_number=89203-498 Agilent Vial Glass Small Volume Inserts (5183-2085)]
-
[ https://us.vwr.com/store/catalog/product.jsp?catalog_number=89203-492  Agilent Glass Vial 2 mL with write on spot (5182-0715)]
+
[https://us.vwr.com/store/catalog/product.jsp?catalog_number=89203-492  Agilent Glass Vial 2 mL with write on spot (5182-0715)]

Revision as of 13:20, 21 September 2012

Back to Protocols

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.4oC

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)

Agilent Vial Glass Small Volume Inserts (5183-2085)

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

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