Sulfo-NHS-LC-Biotin 

Sulfo-NHS-LC-Biotin is an amine-reactive ester derivative of biotin and cannot pass the intact blood-retinal barrier. It can be used to assess vascular permeability of the brain and retinal vasculature.

Guide (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
Calculations:
For best results, use ≥ 12-fold molar excess of biotin for a 10 mg/mL protein solution or ≥ 20-fold molar excess of biotin for a 2 mg/mL protein solution.
1.Calculate millimoles of biotin reagent to add to the reaction for a 20-fold molar excess:
mL protein × (mg protein/mL protein) × (mmoL protein/mg protein) × (20 mmoL protein/mmoL protein) = mmoL Biotin.
* 20 = Recommended molar fold excess of biotin for 2 mg/mL protein sample
2. Calculate microliters of 10 mM biotin reagent solution (prepared in Step B.3) to add to the reaction:
mmoL Biotin × (1000000 μL/L) × (L/10 mmoL) =μL Biotin
Biotin Labeling Reaction:
1. Dissolve 1-10 mg protein in 0.5-2.0 mL PBS according to the calculation made in Calculations.
2. Before use, prepare a 10 mM solution of the biotin reagent in an organic solvent such as DMSO.
3. Add the appropriate volume (μL) of 10 mM biotin reagent solution to the protein solution.
4. PBS or other amine-free buffer having pH 7-8 for use as reaction buffer.
5. Incubate reaction on ice for two hours or at room temperature for 30 minutes.
6. To tests the labeled protein by ELISA or Western blot.

6. Wash cells three times with ice-cold PBS (pH 8.0) to remove amine-containing media and proteins from the cells.
7. Suspend cells at a concentration of ~25 × 10⁶ cells/ml in PBS (pH 8.0).
8. Add 1.0 mg of Sulfo-NHS-LC-Biotin reagent per mL of cell suspension (results in ~2 mM biotin reagent).
9. Incubate reaction mixture at room temperature for 30 minutes.
Note: Performing this incubation at 4°C may reduce active internalization of the biotin reagent.
10. Wash cells three times with PBS + 100 mM Glycine to quench and remove excess biotin reagent and byproducts.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

534.60

C₂₀H₃₀N₄O₉S₂

190598-57-3

O=C(C(CC1=O)S(=O)(O)=O)N1OC(CCCCCNC(CCCC[C@H]2[C@]3([H])[C@](NC(N3)=O)([H])CS2)=O)=O

Room temperature in continental US; may vary elsewhere.

Please store the product under the recommended conditions in the Certificate of Analysis.

• [1]. Wan E, Ogawa M, Okajima T. A Blood-retina Barrier Permeability Assay in Young Mice Using Sulfo-NHS-LC-biotin Perfusion[J]. Bio-protocol, 2018: e3064-e3064.