Assessment of HALO-cIVP882 cleavage was carried out in V. parahaemolyticus 882 strains as described [8 (link)] with modifications. Specifically, overnight cultures carrying the plasmid harboring HALO-cIVP882 were diluted 1:200 in medium and grown for 2.5 h with shaking. Cultures were divided in half and administered ciprofloxacin or water as specified. The treated cultures were incubated without shaking for an additional 1.5 h. Cells were collected by centrifugation (16,100 X g for 1 min), resuspended in a lysis buffer containing 1x BugBuster, benzonase, 300 mM NaCl, and 1 μM HALO-TMR (excitation/emission: 555/585 nm). Clarified lysates were loaded onto NEBExpress Ni spin columns (NEB), washed once with lysis buffer containing 10 mM imidazole, and eluted in lysis buffer containing 500 mM imidazole. Eluted samples were subjected to electrophoresis on 4–20% SDS-PAGE stain-free gels. Gels were imaged using an ImageQuant LAS 4000 imager under the Cy3 setting prior to being exposed to UV-light for 7 min and re-imaged under the EtBr setting. Exposure times never exceeded 30 sec.
Ni spin columns
Manufactured by New England Biolabs
Ni spin columns are a tool used for the purification of proteins with a histidine tag. The columns contain a nickel-charged resin that binds to the histidine tag, allowing the target protein to be separated from other components in the sample. The columns can be used in a centrifuge to facilitate the purification process.
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2 protocols using ni spin columns
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HALO-cIVP882 Cleavage Assay
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HALO-cIVP882 Cleavage Assay in V. parahaemolyticus
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Assessment of HALO-cIVP882 cleavage was carried out in V. parahaemolyticus 882 strains as described [8 (link)] with modifications. Specifically, overnight cultures carrying the plasmid harboring HALO-cIVP882 were diluted 1:200 in medium and grown for 2.5 h with shaking. Cultures were divided in half and administered ciprofloxacin or water as specified. The treated cultures were incubated without shaking for an additional 1.5 h. Cells were collected by centrifugation (16,100 X g for 1 min), resuspended in a lysis buffer containing 1x BugBuster, benzonase, 300 mM NaCl, and 1 μM HALO-TMR (excitation/emission: 555/585 nm). Clarified lysates were loaded onto NEBExpress Ni spin columns (NEB), washed once with lysis buffer containing 10 mM imidazole, and eluted in lysis buffer containing 500 mM imidazole. Eluted samples were subjected to electrophoresis on 4–20% SDS-PAGE stain-free gels. Gels were imaged using an ImageQuant LAS 4000 imager under the Cy3 setting prior to being exposed to UV-light for 7 min and re-imaged under the EtBr setting. Exposure times never exceeded 30 sec.
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