Zenon APC-DL755 was generated by conjugating the Zenon APC Human IgG labeling reagent, from the Zenon Allophycocyanin Human IgG Labeling Kit (Z25451) to DyLight 755 NHS Ester (62279; Thermo Fisher Scientific) according to manufacturers’ instructions. Zenon PE-DL650, was generated by conjugating the Zenon PE Human IgG labeling reagent from the Zenon R-Phycoerythrin Human IgG Labeling Kit (Z25455) with DyLight 650 NHS Ester (62266; Thermo Fisher Scientific) according to the manufacturers’ instructions. To create decoy reagents, Zenon APC-DL755 and Zenon PE-DL650 were incubated with Zenon blocking reagent at a 1:1 ratio.
Dylight 755 nhs ester
Manufactured by Thermo Fisher Scientific
Sourced in United States
DyLight 755 NHS Ester is a fluorescent labeling reagent. It is used for covalent labeling of proteins and other biomolecules containing primary amines.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using dylight 755 nhs ester
1
Zenon Antibody Conjugation and Decoy Creation
2
Recombinant SARS-CoV-2 RBD Production and Tetramerization
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Recombinant SARS-CoV-2 RBD (Wuhan-1, Wu-1) was generated by standard transient transfection followed by IMAC purification as described previously (Walls et al., 2020 (link)). Recombinant SARS-CoV-2 RBD (B.1.1.351, β) was generated by transient transfection from the SARS-CoV-2-Beta-RBD-Avi (K417N-E484K-N501Y) construct synthesized by GenScript into CMVR with an N-terminal mu-phosphatase signal peptide and a C-terminal octa-histidine tag, flexible linker, and avi tag (GHHHHHHHHGGSSGLNDIFEAQKIEWHE) as described previously (Tortorici et al., 2021 (link)). For tetramer generation, RBD proteins were biotinylated with the BirA500 kit (Avidity), tetramerized with streptavidin-phycoerythrin (SA-PE) or streptavidin-allophycocyanin (SA-APC) (Agilent) and stored in 50% glycerol at -20oC as previously described (Krishnamurty et al., 2016 (link)). Decoy reagents were generated by tetramerizing an irrelevant biotinylated protein with SA-PE previously conjugated to Dylight594 NHS Ester (ThermoFisher) and Dylight650 NHS Ester (ThermoFisher) or SA-APC previously conjugated to Dylight755 NHS Ester (ThermoFisher).
3
Hybridoma Screening and Antibody Purification
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The positive hybridoma screening was performed via ELISA and FCM. Positive hybridomas were cloned twice with a limiting dilution method. For ascites production, 5 × 105 positive hybridomas were injected into a Balb/c mouse that had been pre-injected with 500 µL pristane. The ascites was extracted continuously, and the antibody was purified with an IgG Purification Kit (Beyotime, Shanghai, China), according to the manufacturer’s instructions. The Ig subclass and light-chain class of mAb were determined with a mouse immunoglobulin-isotyping ELISA kit (BD Pharmingen™, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Purified mAb was labelled using DyLight® 755 NHS Ester (Thermo Fisher, Waltham, MA, USA), as per the manufacturer’s instructions.
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