Sino Biologicals 40143-MM08 was first buffer exchanged into PBS to remove any interfering substances using Amicon filters (Sigma, St Louis, MO, 50 kDa MWCO, UFC5050). Specifically, the antibody was concentrated 20-fold and brought back to the original volume with PBS. This was done three times to remove sodium azide. The antibody was biotinylated at 1.5 mg/mL with 50 molar excess NHS-dPEG12-biotin (10198, Quanta Biodesign, Plain City, OH, USA). Excess biotin was removed using Amicon filters again, this time with five total concentration and resuspension cycles. The concentration of the biotinylated antibody was determined using the A280. The final biotinylation molar ratio was determined to be 20 moles of biotin per mole of antibody using the QuantTag Biotin Quantification Kit (Vector Laboratories, UK, BDK-2000).
Quanttag biotin quantification kit
Manufactured by Vector Laboratories
Sourced in United Kingdom
The QuantTag Biotin Quantification Kit is a laboratory tool designed for the quantitative analysis of biotin. It provides a straightforward method to determine the concentration of biotin in various sample types.
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2 protocols using quanttag biotin quantification kit
1
Antibody Biotinylation Protocol
2
Biotinylated Uricase Multimer Assay
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Uricase was biotinylated using EZ-link NHS-biotin (Thermo Scientific). Unreacted NHS-biotin was removed using Zeba spin desalting columns, 7kDa MWCO (Thermo Scientific). The extent of biotinylation was measured using the QuantTag Biotin Quantification kit (Vector Laboratories) to ensure 1:1 M ratio of uricase and biotin. Biotinylated uricase was reacted for 20 min on ice with 4:1 M ratio of biotin to streptavidin-conjugated PE or streptavidin-conjugated APC (Biolegend). Streptavidin-conjugated FITC (BioLegend) was reacted with excess free biotin to form a non-antigen-specific streptavidin probe as a control. Multimer formation was confirmed using SDS-PAGE gel. Cells were stained for flow cytometry with all three streptavidin probes at the same time as other fluorescent surface markers at a volumetric ratio of 1:200. The antigen-specific staining of B cells with multimer was verified by staining with and without 5M uricase on splenocytes from vaccinated and antigen-naive mice.
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