NHS-LC-Biotin

Method for labeling antibodies with NHS-LC-Biotin^[1]
This experimental protocol is compiled based on research content and is for reference only. Please adjust according to actual needs.
(1) Antibody dialysis: Dialyze the antibody (2–10mg/mL; 100μL to 1mL total volume) against carbonate buffer (0.1M NaHCO₃; pH=8) for 1h at room temperature, then repeat dialysis once with fresh carbonate buffer.
(2) Concentration adjustment: After dialysis, adjust the sample to 1mg/mL with carbonate buffer.
(3) Preparation of NHS-LC-Biotin solution: Prepare a 1mg/mL solution of NHS-LC-Biotin in anhydrous DMSO or DMF.
(4) Labeling reaction: Add the NHS-LC-Biotin solution dropwise to the antibody at the desired molar ratio. For example, a typical polyclonal IgG antibody can be labeled at a 10:1 molar ratio (biotin:antibody): 1mg antibody (≈6nmol) corresponds to 30μg NHS-LC-Biotin (i.e., 30μL of 1mg/mL NHS-LC-Biotin solution). Typically, molar ratios of 3–10 are suitable for most applications.
(5) Incubation: Incubate the reaction mixture on a rotator for 4h at room temperature or overnight at 4°C.
(6) Purification: Dialyze the labeled antibody solution against three changes of PBS (pH=6.5–7.5) to remove unbound biotin, after which it can be used in the desired application.
Precautions:
(1) If precipitation occurs during the labeling reaction step, add the biotin solution more slowly.
(2) If the labeled antibody shows weak signal or poor reactivity, reduce the amount of biotin to avoid over-labeling of the antigen-binding region, or choose to crosslink to other amino acid side chains or oxidized glycosylation sites.
(3) For your safety and health, please wear a lab coat and disposable gloves during operation.

References:
[1] Berg EA, Fishman JB. Labeling Antibodies with N-Hydroxysuccinimide-Long Chain (NHS-LC)-Biotin. Cold Spring Harb Protoc. 2020 Jan 2;2020(1):099259.