All protein samples were run under reduced conditions by dilution in 6× SDS containing dithiothreitol (DTT) and 5% beta-mercaptoethanol (Fisher Scientific; ICN19483425). The protein was subsequently incubated in a heating block at 95°C for 15 min, run on a 4 to 15% SDS-PAGE gel, and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blocked with phosphate-buffered saline blocking buffer (LI-COR; 927-700001) and then probed with the following antibodies. Antibodies against SARS-CoV2 (2B3E5 from Thomas Moran and GTX632604 from GeneTex), ACE2 (66699-1-Ig from Proteintech and Rb ab108252 from Abcam), VSV-G (A00199 from GenScript), VSV-M (EB0011 from Kerafast), anti-HA (NB600-363 from Novus), and CoX IV (926-42214 from LI-COR) were used. For secondary staining, membranes were washed and incubated with the appropriate Alexa Fluor 647-conjugated anti-mouse antibody or Alexa Fluor 647-conjugated anti-rabbit antibody. Alexa Fluor 647 was detected using the ChemiDoc MP imaging system (Bio-Rad). Relative ACE2 or TMPRSS2 abundance was calculated by first normalizing abundance relative to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression and then normalization to wild-type expression.
Rb ab108252
Manufactured by Abcam
Rb ab108252 is a recombinant monoclonal antibody targeting Rabbit IgG (H+L). It is designed for use in various immunoassay applications.
Automatically generated - may contain errors
2 protocols using rb ab108252
1
SARS-CoV-2 Spike Protein and Host Receptor Analysis
2
SARS-CoV-2 Protein Immunoblot Analysis
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All protein samples were run under reduced conditions by dilution in 6X SDS containing dithiothreitol (DTT) and 5% beta-mercaptoethanol (Fisher Scientific; ICN19483425). The protein was subsequently incubated in a heating block at 95°C for 15mins, run on a 4–15% SDS-PAGE gel, and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blocked with phosphate-buffered saline blocking buffer (LI-COR, 927–700001), and then probed with the indicated antibodies. Antibodies against SARS-CoV2 (2B3E5 from Dr. Thomas Moran and GTX632604 from GeneTex), ACE2 (66699–1-Ig from Proteintech and Rb ab108252 from abcam), VSV-G (A00199 from Genescript), VSV-M (EB0011 from Kerafast), anti-HA (NB600–363 from Novus), and CoX IV (926–42214 from LI-COR) were used. For secondary staining, membranes were washed and incubated with the appropriate Alexa Fluor 647-conjugated anti-mouse antibody or Alexa Fluor 647-conjugated anti-rabbit antibody. Alexa Fluor 647 was detected using the ChemiDoc MP imaging system (Bio-Rad). Relative ACE2 or TMPRSS2 abundance was calculated by first normalizing abundance relative to GAPDH expression, then normalizing to wild type expression.
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