ICG-Sulfo-OSu

Molecular Weight: 930.07
Solvent: DMSO
Extinction Coefficient: 230,000 cm-1M-1
Excitation/Emission: 780/800 nm
CF at 260/280 nm: 0.113/0.073

Extinction coefficient are at their maximum absorption wavelength. CF at 260 nm is the correction factor used for eliminating the dye contribution to the absorbance at 260 nm (for oligo and nucleic acid labeling). CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptide and protein labeling). Fluorescence intensity is significantly increased upon coupled to proteins. This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with ICG. You might need further optimization for your particular proteins.

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Mix 100 µL of a reaction buffer (e.g., 1 M sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration ≥2 mg/ml if possible) to give 1 mL protein labeling stock solution. The pH of the protein solution should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0 - 9.0 using 1 M sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer. The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2 - 7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2 - 7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation. Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results. For optimal labeling efficiency, the final protein concentration range of 2 - 10 mg/mL is recommended, with the conjugation efficiency significantly reduced if less than 2 mg/mL.

Add anhydrous DMSO into the vial of ICG dyes to make a 10 - 20 mM stock solution. Mix well by pipetting or vortex. Prepare the dye stock solution before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce dye activity.

1. Use 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point: Add 5 µl of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µl of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD. The concentration of the DMSO in the protein solution should be <10%.
2. Run conjugation reaction.
3. Repeat Step 2 with the molar ratios of Solution B/Solution A at 5:1; 15:1 and 20:1 respectively.
4. Purify the desired conjugates using premade spin columns.
5. Calculate the dye/protein ratio (DOS) for the above 4 conjugates (if step 3 is done).
6. Run your functional tests of the above 4 conjugates to determine the best dye/protein ratio to scale up your labeling reaction.

1. Add the appropriate amount of dye stock solution (Solution B) into the vial of the protein solution (Solution A) with effective shaking. The best molar ratio of Solution B/Solution is determined above. If skipped, we recommend using 10:1 molar ratio of Solution B (dye)/Solution A (protein).
2. Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.

1. Prepare Sephadex G-25 column according to the manufacture instruction.
2. Load the reaction mixture to the top of the Sephadex G-25 column.
3. Add PBS (pH 7.2 - 7.4) as soon as the sample runs just below the top resin surface.
4. Add more PBS (pH 7.2 - 7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate. For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses. For longer term storage, dye-protein conjugate solution need be concentrated or freeze dried.