Hancock Lab

Inner Membrane Permeabilization: DiSC35 Assay Methods

Categorized: Peptides

References:

Christensen, B., Fink, J., Merrifield, R. B., and Mauzerall, D. (1988) Proc. Natl. Acad Sci. U.S.A. 85, 5072-5076

Sims, P. J., Waggoner, A.S., Wang, C.H., and Hoffman, J. F. (1974) Biochemistry 13, 3315-3329

Buffers and Media:

  • MH Broth tubes (5ml) (or LB no salt)
  • MH Plates (or LB no salt)
  • 5mM HEPES, 5mM glucose (E.coli), pH 7.4
  • 5mM HEPES, 20mM glucose (S. aureus), pH 7.4
  • 400uM DiSC35 stock solution in ethanol
  • 4M KCI stock solution

Preparation of Cells:

  1. Inoculate 5Ml of LB no salt or MH broth for an overnight culture
  2. Use this overnight to prepare a 1 in 100 dilution (50ul in 5ml broth)
  3. Grow to an OD600 of 0.5 to 0.6
    1. coli C500, S. aureus C622 = 2-3 hours (check at 1.5 hours)
  4. Pellet cells at 3000rpm for 10 min
  5. Wash pellet with appropriate buffer
  6. Resuspend cells in same buffer to OD600 of 0.05 (usually 40 ml)

E.coli Specific:

  1. add DiSC35 to a final concentration of 0.8uM and incubate at room temperature with shaking for 1 hour (1 hour 30 minutes may work better)
  2. add KCI to a final concentration of 200mM and incubate at room temperature with shaking for 15 minutes (30 minutes may work better)

S.aureus Specific:

  1. add KCI to a final concentration of 200mM or 2mL and incubate at room temperature with shaking for 30 minutes
  2. add DiSC35 to a final concentration of 0.8uM or 80uL and incubate at room temperature with shaking for 40 minutes (cover with tin foil)

Assay procedure:

  1. settings: Excitation = 622nm, Emission = 670mm, slit width = 10nm, ensure auto cut-off filter is off and stir bar is set to low
  2. Add 2ml of cell solution to cuvette and record background fluorescence for 100 seconds
  3. Add desired concentration of peptide and continue to measure fluorescence until plateau is reached

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