To assess human IgM binding to the surface of bacterial cells, 1 × 109 colony-forming units (CFUs) of the indicated bacterial strains were incubated with 500 μL of 20% NHS (heat inactivated) or PBS for 1 hr at room temperature. After washing the bacteria with PBS four times, bound serum proteins were eluted by resuspending the samples in 100 μL of 0.1 M glycine-HCl followed by centrifugation. A total of 80 μL supernatants were collected and subsequently neutralized with 20 μL of 1 M (tris(hydroxymethyl)aminomethane) (Tris)-HCl buffer (pH 8.0). Neutralized samples and bacterial pellets were precipitated with methanol-chloroform and resuspended with Laemmli sample buffer. Samples were subjected to 10% SDS-PAGE gel. After electrotransfer, the polyvinylidene fluoride (PVDF) membranes (Merck Millipore) were probed with HRP-conjugated anti-human IgM antibody (Jackson ImmunoResearch). As an internal control, bacterial pellets were separated by SDS-PAGE, blotted onto PVDF, and probed with anti-GroEL (Sigma). The blots were developed using ECL (PerkinElmer), and images were obtained on an imager (BioSpectrum Imaging System, UVP).
Hrp conjugated anti human igm antibody
Manufactured by Jackson ImmunoResearch
The HRP-conjugated anti-human IgM antibody is a laboratory reagent used to detect and quantify human immunoglobulin M (IgM) in various immunoassays. The antibody is conjugated with horseradish peroxidase (HRP), which serves as a reporter enzyme for colorimetric or chemiluminescent detection. This product can be used in techniques such as ELISA, Western blotting, and other immunochemical applications to identify and measure the presence of human IgM in biological samples.
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2 protocols using hrp conjugated anti human igm antibody
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Evaluating IgM Binding to Bacterial Cells
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Evaluating IgM Binding to Bacterial Cells
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To assess human IgM binding to the surface of bacterial cells, 1 × 109 colony-forming units (CFUs) of the indicated bacterial strains were incubated with 500 μL of 20% NHS (heat inactivated) or PBS for 1 hr at room temperature. After washing the bacteria with PBS four times, bound serum proteins were eluted by resuspending the samples in 100 μL of 0.1 M glycine-HCl followed by centrifugation. A total of 80 μL supernatants were collected and subsequently neutralized with 20 μL of 1 M (tris(hydroxymethyl)aminomethane) (Tris)-HCl buffer (pH 8.0). Neutralized samples and bacterial pellets were precipitated with methanol-chloroform and resuspended with Laemmli sample buffer. Samples were subjected to 10% SDS-PAGE gel. After electrotransfer, the polyvinylidene fluoride (PVDF) membranes (Merck Millipore) were probed with HRP-conjugated anti-human IgM antibody (Jackson ImmunoResearch). As an internal control, bacterial pellets were separated by SDS-PAGE, blotted onto PVDF, and probed with anti-GroEL (Sigma). The blots were developed using ECL (PerkinElmer), and images were obtained on an imager (BioSpectrum Imaging System, UVP).
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