ReadiView™ biotin succinimidyl ester

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.

Note: The pH of the protein solution (Solution A) 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.

Note: 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.

Note: 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.

Note: The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.

Add anhydrous DMSO into the vial of ReadiView™ biotin succinimidyl ester to make a 10 mM stock solution. Mix well by pipetting or vortex.

Note: Prepare the ReadiView™ biotin succinimidyl ester (Solution B) before starting the conjugation. Use promptly. Extended storage of the ReadiView™ biotin succinimidyl ester stock solution may reduce its activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with ReadiView™ biotin succinimidyl ester. You might need further optimization for your particular proteins.

Note: Each protein requires distinct ReadiView™ biotin succinimidyl ester/protein ratio, which also depends on the properties of ReadiView™ biotin succinimidyl ester. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low ReadiView™ biotin succinimidyl ester/protein ratio gives reduced sensitivity.

1. Use 10:1 molar ratio of Solution B (ReadiView™ biotin succinimidyl ester)/Solution A (protein) as the starting point:  Add 5 µL of the ReadiView™ biotin succinimidyl ester stock solution (Solution B, assuming the ReadiView™ biotin succinimidyl ester 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.

   Note: We recommend to use 10:1 molar ratio of Solution B (ReadiView™ biotin succinimidyl ester)/Solution A (protein). If it is too less or too high, determine the optimal ReadiView™ biotin succinimidyl ester/protein ratio at 5:1, 15:1 and 20:1 respectively.

2. Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.

The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.

1. Prepare Sephadex G-25 column according to the manufacture instruction.
2. Load the reaction mixture (From "Run conjugation reaction") 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 ReadiView™ biotin-protein conjugate.

   Note: For immediate use, the ReadiView™ biotin-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses.

   Note: For longer term storage, ReadiView™ biotin-protein conjugate solution need be concentrated or freeze dried.

The Degree of Substitution (DOS) is the most important factor for characterizing Biotin-labeled protein. The Biotin DOS depends on the number of thiol groups on the protein. For effective labeling, the degree of substitution should be controlled to have 4-8 moles of Biotin to one mole of antibody.

To measure the absorption spectrum of a Biotin-protein conjugate, it is recommended to keep the sample concentration in the range of 1- 10 µM depending on the extinction coefficient of the Biotin. 

For most spectrophotometers, the sample (from the column fractions) needs to be diluted with de-ionized water so that the O.D. values are in the range of 0.1 to 0.9. The O.D. (absorbance) at 280 nm is the maximum absorption of protein, while 385 nm is the maximum absorption of biotin. To obtain accurate DOS, ensure the conjugate is free of the non-conjugated biotin.

You can calculate DOS using our tool by following this link: https://www.aatbio.com/tools/degree-of-labeling-calculator