CH184 cultures were grown overnight anaerobically at 37°C in 2-mL tubes full to the brim. Cells were harvested, and the pellets were suspended in Laemmli buffer (2% SDS, 10% glycerol, 60 mM Tris-HCl [pH 7.4], 0.01% bromophenol blue). The amount of protein loaded onto the gel was standardized for each culture based on its A600 value. Samples were then heated for 10 min at 95°C and separated by SDS-PAGE. Immunoblot analysis was performed according to standard procedures: the primary antibody was a guinea pig anti-MsrP antibody (kindly provided by Jean-François Collet, De Duve Institute, Belgium). The secondary antibody was an anti-guinea pig IgG conjugated to horseradish peroxidase (HRP) (Promega). For loading controls, a rabbit polyclonal anti-RecA antibody was used (Abcam 63797). Chemiluminescence of immunoblots was measured with an ImageQuant LAS4000 camera (GE Healthcare Life Sciences).
Horseradish peroxidase hrp
Manufactured by Promega
Horseradish peroxidase (HRP) is an enzyme commonly used in various laboratory applications. It catalyzes the oxidation of substrates in the presence of hydrogen peroxide. HRP has a wide range of uses in biochemical and immunochemical techniques, such as Western blotting, ELISA, and immunohistochemistry.
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2 protocols using horseradish peroxidase hrp
1
Immunoblot Analysis of MsrP Protein
2
MsrP Production Quantification in Bacterial Strains
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To assess MsrP production by CASA (0.04%) or chlorate (1, 10, 100, and 200 μM), cells from overnight anaerobically cultures of the CH184, ∆hprR, and ∆hprS strains were harvested. The pellets were suspended in Laemmli buffer (2% SDS, 10% glycerol, 60 mM Tris–HCl [pH 7.4], 0.01% bromophenol blue). The amount of protein loaded onto the gel was standardized for each culture based on its A600 value. Samples were then heated for 10 min at 95°C and loaded on SDS‐PAGE. Immunoblot analysis was performed according to standard procedures: the primary antibody was a guinea pig anti‐MsrP antibody (provided by Jean‐François Collet Lab, De Duve Institute, Belgium). The secondary antibody was an anti‐guinea pig IgG conjugated to horseradish peroxidase (HRP) (Promega). An ImageQuant LAS4000 camera (GE Healthcare Life Sciences) was used for image chemiluminescence.
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