At time points indicated in the text cells were washed once with PBS and resuspended in 100 μl Laemmli buffer containing 5% β-mercaptoethanol. Proteins were separated on 8% or 10% poly-acrylamide gels. Membranes were probed with antibodies recognizing IE1/2, pp28, pp65, UL44, UL84, (Virusys, 1:1000 dilution), IE2 proteins (clone 5A8.2, Millipore, 1:1000 dilution) and β-actin (SIGMA, 1:5000 dilution). All primary antibodies were detected using anti-mouse- or anti-rabbit-horseradish peroxidase (HRP) conjugated antibodies (Millipore and Cell Signaling Technology, respectively). Chemiluminescence solution (GE Healthcare) was used in each case to detect secondary antibodies using film. Relative band intensity (band intensity relative to β-actin signal in the same lane) was analyzed using ImageJ software, obtained from the National Institutes of Health (USA).
Anti mouse or anti rabbit horseradish peroxidase hrp conjugated antibodies
Manufactured by Merck Group
Anti-mouse- or anti-rabbit-horseradish peroxidase (HRP) conjugated antibodies are laboratory reagents used to detect and quantify target proteins in various immunoassay techniques. They consist of antibodies specific to mouse or rabbit immunoglobulins that are covalently linked to the enzyme horseradish peroxidase. These conjugated antibodies can be used to amplify and visualize the signal of antigen-antibody interactions in Western blotting, ELISA, and immunohistochemistry applications.
Automatically generated - may contain errors
4 protocols using anti mouse or anti rabbit horseradish peroxidase hrp conjugated antibodies
1
Western Blot Analysis of HCMV Proteins
2
Immunoblot Analysis of HCMV Proteins
3
Quantitative Immunoblotting of HCMV Proteins
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At time points indicated in the text cells were washed once with PBS and resuspended in 100 μl Laemmli buffer containing 5% β-mercaptoethanol. Proteins were separated on 8% or 10% polyacrylamide gels. Membranes were probed with antibodies recognizing IE1/2, pp28, pp65, UL44, UL84, (Virusys, 1:1000 dilution), IE2 proteins (clone 5A8.2, Millipore, 1:1000 dilution) and β-actin (SIGMA, 1:5000 dilution). All primary antibodies were detected using anti-mouse- or anti-rabbit-horseradish peroxidase (HRP) conjugated antibodies (Millipore and Cell Signaling Technology, respectively). Chemiluminescence solution (GE Healthcare) was used in each case to detect secondary antibodies using film. Relative band intensity (band intensity relative to β-actin signal in the same lane) was analyzed using ImageJ software, obtained from the National Institutes of Health (USA).
4
Western Blot Analysis of Viral Proteins
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HFF cells were infected at the MOI indicated in each Figure or prepared for analysis at the time of infection. After washing with PBS, cells were resuspended in Laemmli buffer containing 5% β -mercaptoethanol. Proteins were separated on 8% polyacrylamide gels. Membranes were probed with antibodies recognizing IE1/2, (Virusys, 1:1000 dilution), IE2 proteins (clone 5A8.2, Millipore, 1:1000 dilution), MAP4K4 (ab155583, Abcam, 1:500 dilution) and β -actin (SIGMA, 1:5000 dilution). All primary antibodies were detected using anti-mouse- or anti-rabbit-horseradish peroxidase (HRP) conjugated antibodies (Millipore and Cell Signaling Technology, respectively). Chemiluminescence solution (GE Healthcare) was used to detect secondary antibodies on film. Where necessary blots were striped and re-probed. Relative band intensity (band intensity relative to β -actin signal in the same lane) was analyzed using ImageJ software obtained from the National Institutes of Health (USA). Thusly, in lanes where relative band intensity was analyzed, densitometry was used to calculate the percentage difference in band intensity between β -actin bands in those lanes. The percentage difference in band intensity for specific proteins in those lanes was then calculated. Specific protein band intensity was divided by β -actin intensity to calculate relative band intensity.
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