Archives: Methods

1. Rinse blot with 2X SSC and air dry
2. Place blot into hybridization tube with ~100 ml 0.5% SDS, 0.1 SSC. Prehybridize for 1 hour at 65C.
3. Discard prehybridization solution and add hybridization buffer; use approximately 7-10 ml per large hybridization tube.
   

   25 ml 1.0 M NaHPO4

   200 µl 0.5 M EDTA

   50 ml formamide

   7 g SDS

   H2O to 100 ml

4. Prehybridize for ~5 – 10 minutes at 37-42ºC.
5. Add denatured and labeled probe.
6. Hybridize 16 – 24 hours at 37-42ºC.
7. Discard hybridization solution to radioactive waste. Wash the membrane briefly in 2X SSC, 0.1% SDS at 23ºC (room temperature).
8. Wash the membrane in ~ 100 ml 0.1X SSC , 0.1% SDS for 15-30 minutes at 37-65ºC.
9. Repeat step 8.
10. Blot the membrane onto filter paper, wrap in plastic and expose to film.

Strip nylon membrane for reuse:

1. Wash the blot twice in 100 ml 0.1X SSC, 0.1% SDS at 95ºC for 30 minutes.
2. Wash the blot as in step 1 but at 23ºC.

Reference:

Molecular Cloning: A Laboratory Manual, 2nd ed., pp.1.25-1.28.

Solutions:

1. TE buffer 50 mM glucose, 25 mM Tris-Cl (pH 8.0), 10mM EDTA (pH 8.0); filter sterilized and stored at 4ºC.
2. 0.2N NaOH (freshly diluted from 10N stock), 1% SDS; This solution should be made up fresh on the day of use.
3. 3 M potassium and 5 M acetate. Store at 4ºC.

Method:

1. Grow up a 5 ml culture overnight in the presence of the appropriate antibiotic.
2. Harvest 1.5 ml of culture by centrifugation in an Eppendorf tube.
3. Resuspend the pellet in 100 µl of ice-cold solution 1.
4. Store for 5 minutes at room temperature.
5. Add 200 µl of solution 2 and mix the contents by inverting the tube rapidly two or three times. Do not vortex.
6. Store for 5 minutes on ice.
7. Add 150 ul of ice-cold solution 3 and mix by vortexing in an inverted position.
8. Store for 5 minutes on ice.
9. Centrifuge for 5 minutes in a Eppendorf centrifuge at 4ºC.
10. Transfer supernatant to a fresh tube and add an equal volume of phenol/chloroform. Mix by vortexing the tube and centrifuge for 2 minutes in an eppendorf centrifuge at room temperature.
11. Transfer the top layer to a fresh tube and add two volumes of ethanol. Vortex gently and let stand at room temperature for 2 minutes.
12. Centrifuge for 5 minutes in an Eppendorf centrifuge at 4ºC.
13. Remove supernatant and add 1 ml of 70% ethanol. Vortex briefly and recentrifuge.
14. Remove supernatant and dry the pellet briefly in a vacuum dessicator.
15. Add 50 µl of TE (pH 8.0) containing DNase-free pancreatic RNase (20 µl/ml).

Biotinylation of peptides at cysteine with Nalpha-(3-maleimidylpropionyl)biocytin.

Author:

Annett Rozek (Postdoctoral fellow, Hancock Lab); July 2005.
(Revised/updated by Susan Farmer, Research Associate, Hancock Lab, October 2012).

References:

1. Lau, Y.E., A. Rozek, M.G. Scott, D.L. Goosney, D.J. Davidson and R.E.W. Hancock. 2005. Interaction and cellular localization of the human host defence peptide, LL-37, with lung epithelial cells. Infect. Immun.73:583-591.
2. Bowdish, D.M.E., D.J. Davidson, Y.E. Lau, K. Lee, M.G. Scott, and R.E.W. Hancock. 2005. Impact of LL-37 on anti-infective immunity. J. Leukocyte Biol. 77:451-459.

The Nalpha-(3-maleimidylpropionyl)biocytin is from Molecular Probes/Invitrogen, cat# M-1602.

LL-37 needs to have a cysteine to link to the biocytin. At the time we had decided to add it at the C-terminus (“LL37C”).

Procedure for LL37C:

1. Dissolve 12 mg of Nalpha-(3-maleimidylpropionyl)biocytin (MW=524 g/mol) in 20 ml of Tris buffer (50 mM, pH 7) at room temperature in 50 ml Falcon polypropylene tube. Put on shaker until completely dissolved (about 10-15 min).
2. Dissolve 20 mg LL37C (MW=4596 g/mol) in 10 ml of same buffer. It should dissolve immediately.
3. Combine reagents and let react for 2h on shaker at room temperature.
4. Wrap tube with aluminum foil to protect from light.
5. Stop reaction and scavenge excess Nalpha-(3-maleimidylpropionyl)biocytin by adding 10 ul 2-mercaptoethanol.
6. Separate on reverse phase column (Resource RPC-3, Pharmacia) using a water (0.1% TFA)/acetonitrile (0.1% TFA) gradient. Biotinylated LL37 elutes around 40% acetonitrile, scavenged biocytin elutes around 20 %).

Yield is ~50%, 4.8 mg (quantified by LOWRY, BCA), 12.8 mg (by weight).

Membrane prep:

1. Wash cells with 10 mM Tris pH 8
2. French press
3. Slow speed spin
4. High speed spin
5. Resuspend in 10 mM Tris
6. Sonicate 2 x 15 sec to remove ß-lacatamases
7. Wash in 10 mM Tris
8. Resuspend in 10 mM Tris
9. Store at –70ºC until needed.

Assay:

1. Incubate membranes with ~5 µCi ³H-penicillin at 25ºC for 10 minutes
2. Add 1000X cold penicillin
3. SDS-PAGE
4. Incubate in 1 M sodium salicylate for 30 minutes
5. Dry at 80ºC for 2 hours under vacuum
6. Expose to photographic film for 3-30 days.

This method is based on the classical microtitre broth dilution recommended by the National Committee of Laboratory Safety and Standards (NCLSS) as published in Amsterdam, D. 1996. Susceptibility testing of Antimicrobials in liquid media. In “Antibiotics in Laboratory Medicine”, Lorian, V., ed. Fourth Edition, pp.52-111. Williams and Wilkins, Baltimore. The modifications were first introduced by Intrabiotics Inc at the 1996 ICAAC, and recommended for general use by Bob Hancock at the First Gordon Conference on Antimicrobial Peptides. They are included here in the hope that they will be used standardly by workers in the field for antimicrobial susceptibility testing to permit comparisons to be made between the data of different labs. We have found this method to be reliable and reproducible.

Below we have indicated some results from our comparisons of different methods.

Materials:

It is important that you use the materials mentioned below. For example, do not substitute polystyrene for polypropylene microtitre plates. Cationic peptides bind polystyrene (especially “tissue culture treated” polystyrene).

1. Sterile tubes (16 X 125 mm) containing 5 ml of Mueller Hinton Broth (MHB, Cat. No. 0757-17-6, DIFCO)
2. Mueller Hinton agar plates (MHA)
3. Sterile 96-well polypropylene microtitre plates (COSTAR Cat. No. 3790, distrubuted as FISHER Cat. No. CS003790 or SIGMA Cat. No. M4404)
4. Polypropylene microcentrifuge tubes (Cat. No. 000-MICR-150, Elkay Products, Inc.) or glass tubes coated with Sigmacote
5. Sterile petri dishes
6. Test peptides quantated by amino acid analysis
7. 0.01% acetic acid (Cat. No. A-38-4, FISHER) containing 0.2% bovine serum albumin (BSA, Cat. No. 735086, Boehringer Mannheim GmbH)
8. 0.02% acetic acid containing 0.4% BSA

Methods:

1. Inoculate 5 ml MHB in tubes with test strains from MHA plates and grow overnight at 37^oC on a shaker (180 rpm).
2. Make serial dilutions of test peptides (at 10 times the required test concentrations) in 0.01% acetic acid, 0.2% BSA in polypropylene or coated glass tubes:
   1. dissolve test peptide in dH₂O at 20 times the required maximal concentration (enough final volume for all tests to be performed on a given day);
   2. dilute it into an equal volume of 0.02% acetic acid, 0.4% BSA to get 10 times the required maximal concentration;
   3. then do serial doubling dilutions in 0.01% acetic acid, 0.2% BSA to get serial dilutions of peptides at 10 times the required test concentrations, eg., 640, 320, 160, …2.5 µg/ml.
3. Dilute overnight bacterial cultures in MHB to give 2 – 7 x 10⁵ colony forming units/ml.
4. Dispense 100 µl of bacterial suspension in each well from column 1 to column 11. Do not add bacteria to column 12, and instead dispense 100 µl of MHB (sterility control and blank for the plate scanner).
5. Add 11 µl of 10x test peptide each well from column 1 to column 10 (column 11 is a control for bacteria alone, with no peptide).
6. Incubate the plates at 37^oC for 18-24 hours. Check again at 40-48 hours.
7. MIC can be taken as the lowest concentration of drug that reduces growth by more than 50%.
8. Plate l0 µl 10^-6 dilution of overnight cultures on MHA plates to determine a viable count. The MBC (Minimal bactericidal concentration) can be determined by plating out the contents of the first 3 wells showing no visible growth of bacteria onto MHA plates and incubating at 37^oC for 18 hr. The lowest concentration of peptide that prevents any residual colony formation is the MBC.

Effect of MIC determination method on the MIC value recorded. Methods 1 and 2 are the methods given above and method 6 is the broth microdilution method of Amsterdam (see above reference).

* “tissue culture treated”

Materials:

• liquid cultures of bacteria at suitable growth phase
• sterile petri dishes
• sterile 96-well microtitre plates (round-bottom wells are best)
• filter sterilized antibiotics
• sterile diluents
• test tubes.

Method:

1. Grow the test strains in the chosen medium to the right A₆₀₀. Have antibiotic solutions and plates ready before the cultures reach the desired growth phase.
2. Thaw and weigh the antibiotics. Take a note of the purity at this stage, e.g. gentamicin, 577ug/mg solid. Dissolve the antibiotics (solvent depends on the compound), then dilute in the test medium to 2x the top concentration desired in the test, e.g. if highest desired concentration is 128ug/ml, dilute to 256ug/ml. Keep on ice until use.
3. Using the multipipettor, dispense 100ul of medium into all wells of a microtitre plate. Label the plate and lid.
4. Pipette 100ul of appropriate 2x antibiotic solutions into the wells in column 1 (far left of plate).
5. Using the multipipettor set at 100ul, mix the antibiotics into the wells in column 1 by sucking up and down 6-8 times. Do not splash.
6. Withdraw 100ul from column 1 and add this to column 2. This makes column 2 a twofold dilution of column 1, e.g. for the example above this would be 64ug/ml. Mix up and down 6-8 times. Transfer 100ul to column 3. Repeat the procedure down to column 10 only. The same set of tips can be used for the entire dilution series.
7. Discard 100ul from column 10 rather than putting it in column 11.
8. Pour bacteria of the right A₆₀₀ into a sterile petri dish. The bacteria may be diluted first depending on the desired inoculum. The appropriate inoculum size for standard MIC is 10⁴ to 10⁵ CFU/ml.
9. With the smaller multipipettor set to 5ul, dispense bacteria into wells in columns 11 to 1 in that order. Do not add bacteria to column 12 (sterility control and blank for the plate scanner).
10. Incubate the plates at 37^oC or other desired temperature.
11. Streak the bacterial cultures on plates to check their purity.
12. When satisfactory growth is obtained (18-36 hours) scan the plates with an ELISA reader (e.g. Levy’s lab). Use column 12 as the blank (this means putting the plate in back-to-front).
13. MIC can be taken as the lowest concentration of drug that reduces, by more than 50% or 90% for MIC₅₀ or MIC₉₀ respectively.

Note:

Put different drugs on different rows of the same plate, but try to avoid putting bacteria together, to prevent cross-contamination.

Reference:

Gootz & Sanders. A.A.C.

Method:

1. Inoculate regular growth medium with overnight culture of bacteria. (1/20 – 1/50)
2. Grow to OD₆₀₀ = 0.3 under usual conditions (37^oC, shaking).
3. Add sterile antibiotics so the final concentration is 1/2 the MIC of that antibiotic for that bacteria. Include a control of no antibiotic for each strain.
4. Grow for another 3 hours. Check final OD₆₀₀. (Cells should still grow.)
5. Add 1mM NaAz (final concentration) to stop cell metabolism.
6. Centrifuge cells in Sorvall 7K, 15′ in G3 or 10K, 5′ in SS34.
7. Wash 2X in 5mM Hepes pH 7.2.
8. Resuspend each pellet in small volume of buffer (approx. 2 mls) (or 1/100 of original growth volume).
9. Place each sample in small plastic test tube. Keeping samples on ice, sonicate each one 3X for 1 minute bursts, using small probe at 35%. Alternatively: add polymyxin B to a final concentration of 1mg/ml or French Press in the small cell.
10. Spin down in ultracentrifuge 70.1 Ti, 23K, 30′, 5^oC.
11. Carefully pipet off supernatant. This is the crude B-lactamase extract. Store: short term: 4^oC (loses activity with time) long term: -20^oC (loses activity with freeze-thawing)
12. By doing protein assays and nitrocefin assays on each sample, you can then determine the specific activity. See “permeability Calculations: 10 easy steps” for calculations of specific activity.