VHHs (E-tagged) were tested for binding to the various recombinant BoNT preparations (ciBoNT/B1, HCB, LC/B, etc) by standard ELISAs (Mukherjee et al., 2012 (link)) using plates coated with 100 μl of 1 μg/ml of the protein, or by first coating plates with 5 μg/ml of a BoNT-binding VHH (myc tagged) followed by a blocking step and then capturing a 1 ug/ml BoNT preparation. Typically ELISAs started at 125 nM VHH and 1:5 dilutions were performed prior to detection with HRP-goat anti-E-tag (Bethyl). BoNT/B neutralization studies were done similar to those previously reported for VHHs binding to BoNT/A (Mukherjee et al., 2012 (link)), but employing 1 nM BoNT/B treatments of primary neurons and detecting VAMP cleavage on western blots as a ratio of VAMP to SNAP25 (1:25,000 rabbit anti-SNAP25 combined with 1:1000 rabbit anti-VAMP (Millipore)) and detected with 1:10,000 HRP-goat anti-rabbit IgG (GE Healthcare). BoNT cleaved VAMP is not recognized by the anti-VAMP antibody, so the assay relies on the ratio of intact VAMP:SNAP25 as comparted to controls.
Rabbit anti snap25
Manufactured by Merck Group
Rabbit anti-SNAP25 is a laboratory reagent used in research applications. It is a polyclonal antibody raised in rabbits that specifically binds to the SNAP25 protein, which is involved in the regulation of synaptic vesicle exocytosis. This product can be used in various laboratory techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation, to detect and analyze the SNAP25 protein.
Automatically generated - may contain errors
2 protocols using rabbit anti snap25
1
Binding and Neutralization Assays for BoNT/B
2
Binding and Neutralization Assays for BoNT/B
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VHHs (E-tagged) were tested for binding to the various recombinant BoNT preparations (ciBoNT/B1, HCB, LC/B, etc) by standard ELISAs (Mukherjee et al., 2012 (link)) using plates coated with 100 μl of 1 μg/ml of the protein, or by first coating plates with 5 μg/ml of a BoNT-binding VHH (myc tagged) followed by a blocking step and then capturing a 1 ug/ml BoNT preparation. Typically ELISAs started at 125 nM VHH and 1:5 dilutions were performed prior to detection with HRP-goat anti-E-tag (Bethyl). BoNT/B neutralization studies were done similar to those previously reported for VHHs binding to BoNT/A (Mukherjee et al., 2012 (link)), but employing 1 nM BoNT/B treatments of primary neurons and detecting VAMP cleavage on western blots as a ratio of VAMP to SNAP25 (1:25,000 rabbit anti-SNAP25 combined with 1:1000 rabbit anti-VAMP (Millipore)) and detected with 1:10,000 HRP-goat anti-rabbit IgG (GE Healthcare). BoNT cleaved VAMP is not recognized by the anti-VAMP antibody, so the assay relies on the ratio of intact VAMP:SNAP25 as comparted to controls.
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