For immunoblot analysis, total membrane proteins (25 μg) resolved by SDS-PAGE under nonreducing (in the absence of β-mercaptoethanol) conditions (37 (link)) as described above were electrotransferred onto an Immun-Blot polyvinylidene difluoride (PVDF) membrane (Bio-Rad) and probed for LptD or LptE using anti-LptD (1:20,000) and anti-LptE (1:30,000) polyclonal rabbit antibodies (GenScript) and goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma-Aldrich). Anti-LptD and anti-LptE antibodies were generated at GenScript using keyhole limpet hemocyanin (KLH)-conjugated peptides as the antigens (LptD epitope, QLHQKEAPGQPEPVC , and LptE epitope, ELLDKETTRKDVPSC ). The epitopes were selected using the OptimumAntigen design tool (GenScript). The transferred membrane was developed with Clarity Western ECL substrate (Bio-Rad) and visualized in a FluorChem Q imager (ProteinSimple).
Goat anti rabbit igg horseradish peroxidase hrp conjugated secondary antibody
Manufactured by Merck Group
Goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody is a laboratory reagent used to detect the presence of rabbit primary antibodies in immunoassays. The antibody is conjugated with the enzyme horseradish peroxidase, which can be used to catalyze a colorimetric or chemiluminescent reaction for the visualization and quantification of target proteins.
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2 protocols using goat anti rabbit igg horseradish peroxidase hrp conjugated secondary antibody
1
Immunoblot Analysis of LptD and LptE
2
Immunoblot Analysis of LptD and LptE
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For immunoblot analysis, total membrane proteins (25 μg) resolved by SDS-PAGE under nonreducing (in the absence of β-mercaptoethanol) conditions (37 (link)) as described above were electrotransferred onto an Immun-Blot polyvinylidene difluoride (PVDF) membrane (Bio-Rad) and probed for LptD or LptE using anti-LptD (1:20,000) and anti-LptE (1:30,000) polyclonal rabbit antibodies (GenScript) and goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma-Aldrich). Anti-LptD and anti-LptE antibodies were generated at GenScript using keyhole limpet hemocyanin (KLH)-conjugated peptides as the antigens (LptD epitope, QLHQKEAPGQPEPVC , and LptE epitope, ELLDKETTRKDVPSC ). The epitopes were selected using the OptimumAntigen design tool (GenScript). The transferred membrane was developed with Clarity Western ECL substrate (Bio-Rad) and visualized in a FluorChem Q imager (ProteinSimple).
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