Cells were uninfected or infected with SARS-CoV-2 for indicated times. Starvation of cells was done for 3 h by incubating with RPMI Medium 1640 without l -methionine (Thermo Fisher). Cells were then incubated with the same medium supplemented with the Click-IT-AHA (L-Azidohomoalanine) reagent (Invitrogen) for 2 h. Cells were lysed in a solution containing 1% SDS in 50 mM Tris-HCl, pH 8.0. Click-chemistry reaction for protein detection was performed using biotin alkyne (PEG4 carboxamide-propargyl biotin) according to manufacturer’s instructions using the Click-iT Protein Reaction Buffer kit (Invitrogen). Labeled proteins were separated by SDS-PAGE. The membrane containing the biotin alkyne labeled proteins was incubated in a solution containing 1% BSA with streptavadin-HRP (Invitrogen) for subsequent visualization.
Streptavadin hrp
Manufactured by Thermo Fisher Scientific
Streptavidin-HRP is a conjugate of streptavidin and horseradish peroxidase (HRP). Streptavidin is a tetrameric protein that binds strongly to the vitamin biotin. HRP is an enzyme that catalyzes the oxidation of various substrates in the presence of hydrogen peroxide. This conjugate is commonly used in various bioanalytical techniques, such as enzyme-linked immunosorbent assays (ELISA), Western blotting, and immunohistochemistry, where the high affinity of streptavidin for biotin is leveraged to detect and quantify target analytes.
Automatically generated - may contain errors
2 protocols using streptavadin hrp
1
SARS-CoV-2 Proteome Dynamics Monitoring
2
SARS-CoV-2 Proteome Dynamics Monitoring
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Cells were uninfected or infected with SARS-CoV-2 for indicated times. Starvation of cells was done for 3 h by incubating with RPMI Medium 1640 without l -methionine (Thermo Fisher). Cells were then incubated with the same medium supplemented with the Click-IT-AHA (L-Azidohomoalanine) reagent (Invitrogen) for 2 h. Cells were lysed in a solution containing 1% SDS in 50 mM Tris-HCl, pH 8.0. Click-chemistry reaction for protein detection was performed using biotin alkyne (PEG4 carboxamide-propargyl biotin) according to manufacturer’s instructions using the Click-iT Protein Reaction Buffer kit (Invitrogen). Labeled proteins were separated by SDS-PAGE. The membrane containing the biotin alkyne labeled proteins was incubated in a solution containing 1% BSA with streptavadin-HRP (Invitrogen) for subsequent visualization.
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