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 vamp
Manufactured by Merck Group
Rabbit anti-VAMP is a laboratory reagent used for the detection and analysis of VAMP (Vesicle-Associated Membrane Protein) in various biological samples. It is a polyclonal antibody raised in rabbits against the VAMP protein.
Automatically generated - may contain errors
2 protocols using rabbit anti vamp
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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