Lidstrom:Measuring 13C Incorporation Into Protein - CO2 Project

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Supplies)
(Cell Prep)
Line 7: Line 7:
* Discard supernatant
* Discard supernatant
* Wash pellet with 0.9% 1 mL of sodium chloride at 4<sup>o</sup>C, pellet for 5 min at 13,500 g, 4<sup>o</sup>C
* Wash pellet with 0.9% 1 mL of sodium chloride at 4<sup>o</sup>C, pellet for 5 min at 13,500 g, 4<sup>o</sup>C
-
* Repeat last step.4<sup>o</sup>C
+
* Repeat last step
''Optional:''  
''Optional:''  
-
*store pellet at -40 to -80<sup>o</sup>C  
+
*store pellet at -40 to -80<sup>o</sup>C
== Hydrolysis of Amino Acids ==
== Hydrolysis of Amino Acids ==

Revision as of 13:21, 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

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)

Personal tools