This study investigates the expression of metal transporters in Neisseria gonorrhoeae under metal-limited conditions. The methodology involves a dot blot technique to visualize the binding of metal ligands to bacterial transporters.
Begin with a Neisseria gonorrhoeae culture grown in a metal-depleted medium.
Metal scarcity triggers the expression of outer membrane transporters that initiate metal uptake when available.
Place a presoaked nitrocellulose membrane over moist filter papers and assemble the dot blot apparatus.
Spot the bacterial cells and allow the liquid to absorb.
Disassemble the apparatus, remove the membrane, and air-dry it.
Add a blocking agent to prevent nonspecific binding.
Reinsert the membrane in the apparatus, seal it with parafilm, and reassemble.
Add a host-derived metal-binding ligand and incubate with shaking.
The ligand binds to the bacterial transporters.
Remove the liquid and wash the membrane to remove any unbound ligand.
Add a ligand-specific primary antibody, followed by an enzyme-conjugated secondary antibody and a colorimetric detection reagent that forms a colored product.
A visible signal indicates the expression of bacterial metal transporters under metal-limited conditions.
After the mass doubling incubation, back dilute the cultures with three volumes of CDM. Then, incubate the cultures at 37 degrees Celsius with shaking for four hours. Shortly before the four hour mark, cut three pieces of filter paper and a piece of nitrocellulose to the approximate size needed to fit into a dot-blot apparatus.
Pre-soak the nitrocellulose in deionized water for about 10 minutes before assembling the apparatus with filter paper below the nitrocellulose. At the end of the incubation, record the cell densities and standardize the densities to an appropriate final density. Then, pipette the cell cultures onto the nitrocellulose.
When the filter paper has absorbed all of the liquid disassemble the apparatus and allow the blot to dry. Block the nitrocellulose membrane for one hour with an appropriate blocking solution and reassemble the dot-blot apparatus, replacing the filter paper with paraffin film to create a leak proof seal under the nitrocellulose. Dilute the metal-binding ligand of interest to 0.2 micromolar in blocker and probe the cells for one hour with moderate shaking.
At the end of the incubation, siphon off the liquid with a vacuum, wash the blot, and follow standard immunological procedures to develop the signal.
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