Western blot was performed using an anti-SARS-COV-2 S antibody following a protocol described previously (Chen et al., 2015 (link)). Briefly, full-length S protein samples were prepared from cell pellets and resolved in 4-15% Mini-Protean TGX gel (Bio-Rad, Hercules, CA) and transferred onto PVDF membranes. Membranes were blocked with 5% skimmed milk in PBS for 1 hour and incubated a SARS-CoV-2 (2019-nCoV) Spike RBD Antibody (Sino Biological Inc., Beijing, China, Cat: 40592-T62) for another hour at room temperature. Alkaline phosphatase conjugated anti-Rabbit IgG (1:5000) (Sigma-Aldrich, St. Louis, MO) was used as a secondary antibody. Proteins were visualized using one-step NBT/BCIP substrates (Promega, Madison, WI).
One step nbt bcip substrates
Manufactured by Promega
One-step NBT/BCIP substrates are a colorimetric detection system used to visualize enzymatic activity, particularly in western blotting and immunohistochemistry applications. The substrates contain nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3'-indolyphosphate (BCIP), which react with enzyme-labeled probes to generate a dark purple/blue insoluble precipitate.
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Lab products found in correlation
Alkaline phosphatase conjugated anti rabbit igg, by Merck Group (2 mentions)
Mini protean tgx gel, by Bio-Rad (2 mentions)
Spike rbd antibody, by Sino Biological (1 mentions)
Iblot2, by Thermo Fisher Scientific (1 mentions)
2019 ncov spike rbd antibody, by Sino Biological (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
2 protocols using one step nbt bcip substrates
1
SARS-CoV-2 Spike Protein Detection by Western Blot
2
SARS-CoV-2 Spike Protein Western Blot
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Full-length S protein samples were resolved in 4-15% Mini-Protean TGX gel (Bio-Rad) and transferred onto PVDF membranes (Millipore) by an Iblot2 (Invitrogen by Thermo Fisher Scientific). Membranes were blocked with 5% skimmed milk in PBS for 1 h and incubated with a SARS-CoV-2 (2019-nCoV) Spike RBD Antibody (Sino Biological, 40592-T62) at a concentration of 1 μg ml -1 for another hour at room temperature. Alkaline-phosphatase-conjugated anti-rabbit IgG (1:5,000) (Sigma-Aldrich) was used as a secondary antibody. Proteins were visualized using one-step NBT/BCIP substrates (Promega).
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