This Water-Soluble Long-Arm Biotin Labeling Kit uses Sulfo-NHS-LC-LC-Biotin for labeling. Sulfo-NHS-LC-LC-Biotin reacts with primary amines (N-terminal and the side chain of lysine residues) in antibodies and other proteins, forming a stable amide bond between the antibody/protein and biotin.  The long-arm (spacer arm) reduces steric hindrance when several biotinylated molecules bind with avidin/streptavidin, effectively optimizing labeling and detection results.

Due to the charged sulfonate group, Sulfo-NHS-LC-LC-Biotin will not permeate the cell membrane and can be used to label proteins that are exposed on the surface of the cells.

384846A: Sulfo-NHS-LC-LC-Biotin, 3 x 0.2mg 384846B: Labeling Buffer, 1 x 10ml 384846C: Ultrapure Water, 1 x 500ul 384846D: Filtration tube (6 tubes with 0.5ml capacity per tube)

Adjustable high-precision transfer pipets (10-1000 uL) Incubator (37°C) Centrifuge (capable of speed up to 12,000 x g).

Store Sulfo-NHS-LC-LC-Biotin (384846A) with dessicant at -20°C. Store the rest of the kit components at 4°C. Unopened kit can be stored for one year before opening and should be used before this expiration date. For maximum recovery of product, centrifuge the original vials prior to removing the cap.

Molecular Weight: 670 Molecular Formula: C26H40O10N5S2Na Spacer Arm Length: 30.5 A

Coupling Procedure:   The following procedure is based on labeling of 1mg of antibody/protein. Adjust volume of Sulfo-NHS-LC-LC-Biotin Solution according to the actual amount of antibody/protein to be labeled.

1. Mix 1mg of protein sample and corresponding volume of labeling buffer to filtration tube to make the total volume equal to 0.5ml. Centrifuge at 12,000 x g for 10 minutes. Note: The maximum volume capacity of the filtration tube is 0.5ml. If the protein concentration in the sample is low, the sample can be concentrated first using centrifugal ultrafiltration (use filters with appropriate molecular weight cut-off).

2. Dissolve Sulfo-NHS-LC-LC-Biotin by adding 30ul Ultrapure Water to the vial of Sulfo-NHS-LC-LC-Biotin. Let stand for 10 minutes to allow complete dissolution. The final concentration of Sulfo-NHS-LC-LC-Biotin is 10mM.

3.  Add 13.3ul of Sulfo-NHS-LC-LC-Biotin Solution and appropriate volume of Labeling Buffer to the filtration tube to make the final concentration of the protein solution equal to 2mg/ml. Mix thoroughly using a pipet and incubate the tube for 30 minutes at 37°C.

4. Centrifuge at 12,000 x g for 10 minutes.

5. Add appropriate volume of Labeling Buffer to the filtration tube to make the total volume equal to 0.5ml. Mix thoroughly using a pipet and centrifuge at 12,000 x g for 10 minutes. 

6. Repeat step 5.

7. Add 0.2ml of Labeling Buffer to the filtration tube and mix thoroughly using a pipet. Invert the filtration tube into another centrifugal tube. Centrifuge at 6,000 x g for 10 minutes.

8. Collect the solution in the centrifugal tube; this liquid will contain the biotinylated antibody/protein.