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Methods:Carbonate extraction

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Contents

Purpose

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Materials Needed

Protocol

Protocol adapted from Molloy et al. (2000) [1]

  1. 50mL culture at A550 of 0.2. Induce expression for 30 mins. Harvest.
  2. Resuspend pellet in 1mL FPB (100mM Na2HPO4, 5mM EDTA, 100mM KCl; pH 8.0) and add 2μL 0.5M DTT, 5μL protease inhibitor (commercially available), 10μL 0.1M PMSF (optional), 20μL 1M MgCl2, 5μL 10mg/mL DNase.
  3. Pass sample through the French press. Centrifuge at 14 000 rpm for 2 mins to remove cell debris and unlysed cells.
  4. Divide the supernatant into 2 samples & centrifuge in a TLA 100.3 rotor for 1 hr at 4oC at 50 000 rpm.
  5. To one of the pellets, add 500μL 1% Triton X-100. To the other pellet, add 500μL 0.1M Na2CO3 (MADE FRESH!).
  6. Put a tiny stir bar in each and mix until the pellet is completely dissolved (about 3h).
  7. Centrifuge again in TLA 100.3 at 4oC for 1 h at 50 000 rpm.
  8. Remove supernatant and resuspend pellets in 500μL sample loading buffer and boil.

Usage Examples

User notes

Related GO Terms

References

  1. Molloy, MP et al. (2000) Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem. 267 2871-81 PubMed EcoliWiki page