Thermo Scientific EZ-Link Maleimide-PEG Solid-Phase Biotinylation Kits provide an on-column approach to sulfhydryl-targeted antibody biotinylation that provides greater control over the labeling reaction and clean-up process.
This innovative antibody-labeling system uses nickel-chelated agarose to temporarily immobilize antibody molecules via their histidine-rich Fc regions. Once held in place on the resin, the antibody can be gently reduced at the hinge-region to produce free sulfhydryls that are then biotinylated with the Maleimide-PEG2-Biotin reagent. Excess labeling reagent and byproducts are then washed away before recovering the labeled and purified antibody from the resin using a mild imidazole solution. No gel filtration or dialysis is needed. Kits for small-scale reactions (0.1 to 1mg antibody) and larger scales (1 to 10mg antibody) are offered.
- Fast labeling and purification – the entire procedure takes only about two hours
- Easy removal of spent and excess labeling reagent – simply wash away the reaction byproducts – no need for dialysis or gel filtration
- No dilution effects – solid-phase method allows initially dilute antibodies to be recovered in a smaller volume after labeling
- Optimized protocols – specific protocols for antibody ensure gentle disulfide reduction and appropriate level of labeling to minimize the possibility of inactivation
- High-performance biotin reagent – Malemide-PEG2-Biotin uses efficient, sulfhydryl-specific maleimide chemistry and includes hydrophilic polyethylene glycol (PEG) spacer that stabilizes long-term solubility of labeled antibodies
- Eight biotin-labeling experiments per kit – Thermo Scientific No-Weigh Packaging ensures that the biotin reagent is fully active for eight separate experiments
EZ-Link Solid-Phase Biotinylation Kits use an innovative strategy for antibody biotinylation that simplifies the process in many respects, making successful biotinylation possible for the novice and expert. The strategy involves immobilizing the antibody to a metal-chelated affinity support and performing the biotinylation in the solid phase.
Antibodies bind readily to metal-chelated supports, such as nickel or cobalt, binding primarily through the histidine-rich Fc region of IgG-class antibodies. This feature allows the derivatization chemistry to be performed easily while the antibody is bound to the support. This strategy facilitates reagent delivery and removal of spent reagent. The resulting modified antibody is then selectively removed from the support by elution with a buffered imidazole solution.
The solid-phase approach to antibody biotinylation provides more control over the reaction. For example: Reagent and antibody ratios and reaction times can be adjusted to achieve the proper degree of biotinylation for the intended application.
The kits contain an aqueous soluble sulfhydryl group-specific reagent (Maleimide-PEG2-Biotin) that targets accessible sulfhydryl (-SH) groups on the surface of the antibody. The format can be applied at the multi-milligram antibody level in the 1mL column kit (Part No. 21920) or to low microgram antibody levels for biotinylations using the mini-spin column kit (Part No. 21930).
|Protocol strategy and reaction scheme for biotin labeling with the Thermo Scientific EZ-Link Maleimide-PEG Solid Phase Biotinylation Kits.
Sulfhydryl-specific Biotinylation Reagents
NHS Solid Phase Biotinylation Kits (amine-specific)