Detection of the Surface Metal Transporters in Neisseria gonorrhoeae Using Dot Blot

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 pie