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

Samenvatting

Source: Maurakis, S., and Cornelissen, C. N. Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands. J. Vis. Exp. (2020)

This video demonstrates the procedure for detecting surface metal transporters in Neisseria gonorrhoeae grown under metal-limited conditions using a dot blot assay, involving ligand binding, antibody detection, and colorimetric visualization.

Protocol

1. Detection of Ligand Binding by Outer Membrane Metal Transporters

1. Back dilute the cultures with three volumes of CDM (chelex-treated defined medium).
2. Then incubate with shaking for 4 h.
3. Shortly before the 4 h mark, cut three pieces of filter paper and a piece of nitrocellulose to the approximate size needed to fit into a dot blot apparatus. Presoak the nitrocellulose in deionized water, then assemble the apparatus with filter paper below the nitrocellulose.
4. At 4 h, record the cell densities and standardize to an appropriate final density.
5. Pipette cell cultures onto the nitrocellulose and allow sufficient time for the filter paper to absorb all the liquid.
6. Disassemble the apparatus, allow the blot to dry, and block the nitrocellulose membrane for 1 h in 5% bovine serum albumin or nonfat milk (w/v) in Tris-buffered saline.
7. Reassemble the dot blot apparatus, replacing the filter paper with paraffin film to create a leak-proof seal under the nitrocellulose.
8. Dilute the metal-binding ligand of interest to 0.2 μM in blocker and probe cells for 1 h.
9. Siphon off the liquid with a vacuum, wash the blot, then follow standard immunological procedures to develop the signal. The wash steps may be done in or out of the apparatus.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
125 mL sidearm flasks	Bellco	2578-S0030	Must be custom ordered
2-Mercaptoethanol	VWR	M131	Open in fume hood
3MM Paper	GE Health	3030-6461	Called "filter paper" in text
Blotting grade blocker	Bio-Rad	170-6404	Nonfat dry milk
Bovine serum albumin	Roche	3116964001	Powder
Calprotectin	N/A	N/A	We are supplied with this by a collaborator
Chelex-100 Resin	Bio-Rad	142-2832	Wash with deionized water prior to use
Cotton-tipped sterile swab	Puritan	25-806	Cotton is better than polyester for this application
Dot blot apparatus	Schleicher & Schwell	10484138	Lock down lid as tightly as possible before sample loading
Ethanol	Koptec	V1016	Flammable liquid, store in flammables cabinet
Klett colorimeter	Manostat	37012-0000	Uses color transmission to assess culture density
Methanol	VWR	BDH1135-4LP	Flammable liquid, store in flammables cabinet
Nitrocellulose	GE Health	10600002	Keep in protective sheath until use
S100A7	N/A	N/A	We are supplied with this by a collaborator
Sodium chloride	VWR	470302	Powder
Tris	VWR	497	Powder