Inner Membrane Permeability Assay (ONPG Assay)

The b-galactosidase activity of E. coli ML-35 measured with o-nitrophenyl-b-D-galactopyranoside (ONPG) as substrate. E. coli ML-35 is a lactose, permease-deficient strain with constitutive cytoplasmic b-galactosidase activity.

References:

1. Skerlavaj, B., R. Domenico and R. Gennaro. 1990. Rapid permeabilization and inhibition of vital functions of gram-negative bacteria by bactenicins. Infect. Immun. 58: 3724-3730.
2. Lehrer, R.I., A. Barton, K.A. Daher, S.S.L. Harwig, T. Ganz, and M.E. Selsted. 1989. Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity. J. Clinical Invest. 84: 553-561.

Materials:

• ONPG stock 30 mM = 9 mg/ml in dH20
• 10 mM sodium phosphate buffer (pH 7.5) containing 100 mM NaCl. (remember: to make Na phosphate buffer…make 10 mM Na2HPO4 and 10 mM NaH2PO4 and combine until the pH is 7.5)
• E.coli strain ML-35
• Positive control; Gramicidin S, 1-5 mg/ml
• Dual beam spectrophotometer; a dual beam model is required to subtract the background activity of cells and ONPG alone.

Method:

1. Inoculate 100 mls of media ( LB or Mueller Hinton broth ) with overnight culture of ML-35.
2. Grow up cells to mid log phase ( OD 600 = 0.4-0.6 ). Spin down and resuspend in above Na phosphate buffer to OD 600 = 0.5. Don’t wash cells with extra spins. Note: cells grown to higher OD i.e. 0.9 are still OK, but use logarithmic phase cells. Spin cells at room temperature and don’t store on ice. You can use the “Silencer” centrifuge set at “5” (approx. 4000 rpm) for 10 -15 minutes to spin down cells or the Sorvall floor model .
3. Set the spectrophotometer wavelength to 405 nm.
4. Zero the spec with water or buffer.
5. Turn chart recorder on, set speed to 15 mm/min, zero chart recorder. Full scale = OD 1.
6. In order to “subtract” the background during the assay, the reference cuvette in the back will have the same amount of cells and ONPG , but using an additional amount of buffer in place of the peptide added to the other cuvette.
7. To TWO cuvettes: Add 1/20 of ONPG stock ( 30 mM = 9 mg/ml) to cell suspension to give a final concentration of ONPG of 1.5 mM. i.e. 760 ml of cells and 40 ml of ONPG.
8. To one cuvette add buffer. i.e. 8 ml. Mix and place in the reference cuvette position.(Back)
9. To the second cuvette add peptide i.e. 8 ml of (100X) stock, mix and place in the front position.
10. Monitor change in OD, usually until the OD = 1.0. Some compounds/ peptides have a long “lag” phase, so measure for 10-15 minutes if there is little or no change in OD.
11. For each sample, a new set of two cuvettes must be used; the back reference cuvette cannot just be left in the back. Do not add the ONPG to the cells until you are ready to test each sample.