Lidstrom:Measuring 13C Incorporation Into Protein - CO2 Project: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(New page: Back to Protocols == Cell Prep == * Grow 1.5 mL of OD<sub>550</sub> 0.8 - 1.5 (approximately 0.3 - 0.8 mg) of cell culture * Centrifuge 5 min at 13,500 g, 4<sup>o</...) |
No edit summary |
||
| Line 8: | Line 8: | ||
* 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.4<sup>o</sup>C | ||
''Optional:'' | |||
*store pellet at -40 to -80<sup>o</sup>C | |||
== Hydrolysis of Amino Acids == | == 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 | * Suspend cell pellets in 1 mL of 6N HCl | ||
* Transfer resuspended cells into 2 mL tubes | * Transfer resuspended cells into 2 mL tubes | ||
* Seal tubes to prevent evaporation of HCl | * 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)<sup>o</sup>C | |||
** Hotter temps will may destroy some amino acids | |||
* Dry the hydrolysate at 95<sup>o</sup>C with constant air flow (or N<sub>2</sub>) gas flow in the fume hood until the sample is completely dry. | |||
** Use the "nitrogen tree" | |||
* Bake the dried samples at 105<sup>o</sup>C for another 10 minutes to ensure no moisture is left. | |||
== Derivitization of Amino Acids == | == Derivitization of Amino Acids == | ||
Revision as of 11:10, 21 September 2012
Back to Protocols
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.
