Biotin NHS

Biotin NHS is an amino reactive biotin reagent used in the preparation of biotinylated surfaces or polypeptides. In Vitro:Guide (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
1. Buffer Exchange: Ensure the protein is in an amine-free buffer (e.g., PBS, 100 mM sodium phosphate, 150 mM NaCl) at pH 7.2-7.5. Avoid Tris or glycine.
2. Reagent Prep: Equilibrate the NHS-Biotin vial to room temperature before opening to avoid condensation. Dissolve the reagent in high-quality anhydrous DMSO or DMF immediately before use.
3. Reaction Ratio: Use a 12-20 fold molar excess of biotin reagent for protein concentrations of 2-10 mg/mL.
4. Incubation: Incubate for 30-60 minutes at room temperature, or 2 hours at 4°C.
5. Purification: Remove unreacted biotin using desalting columns (e.g., Sephadex G-25) or dialysis.
6. Verification: Estimate incorporation using the HABA assay, where biotin displaces HABA from avidin, decreasing absorbance at 500nm.
Note:
1. Sulfo-NHS vs. NHS: Sulfo-NHS-Biotin is water-soluble and ideal for aqueous reactions, while NHS-Biotin requires organic solvent dissolution first.
2. Moisture Sensitivity: NHS reagents are highly moisture-sensitive; store desiccated at ?20°C (or 4°C for some LC-NHS) and protect from humidity.