ReadiLeave™ Reversible 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

1. Prepare a 900 uL protein solution in 1X phosphate-buffered saline (PBS), pH 7.2-7.4.

   Note: If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4.

   Note: Protein solution should be free of stabilizers like bovine serum albumin (BSA) or gelatin.

   Note: The presence of sodium azide or thimerosal might also interfere with the conjugation reaction.

   Note: The protein concentration range of 2-10 mg/mL is recommended for optimal labeling efficiency.

2. Add 100 µL of a reaction buffer (e.g., 1 M sodium bicarbonate solution or 1 M phosphate buffer with pH ~8.5 to 9.0) to the target protein solution to adjust pH to 8.5 ± 0.5.

1. Add anhydrous DMSO into the vial of RLR Biotin SE to make a 10 mM (6.85mg/ml) stock solution.

   Note: Prepare the dye stock solution before starting the conjugation. Use promptly.

   Note: RLR Biotin SE stock solution can be stored in the freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.

   Note: Extended storage of the dye stock solution may reduce the dye activity.

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with RLR Biotin SE.

1. Use a 10:1 molar ratio of RLR Biotin SE:Protein.

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

1. Purify the conjugate mixture to 1x PBS buffer (pH=7.2-7.4) with a ReadiUse™ Disposable PD-10 Desalting Column (Cat no. 60504) according to the manufacturer's instruction.

1. Protein concentration can be determined from the extinction coefficient by measuring absorbance at 280 nm.

Section 1: Coupling RLR Biotinylated Protein to a Resin

1. Select a streptavidin-Resin suitable for your application.

2. Wash and equilibrate the resin by adding 1xPBS or a suitable wash buffer.

3. Add appropriate amounts of RLR Biotinylated protein and incubate for 30 minutes.

4. Wash the resin to remove unlabeled protein and equilibrate with PBS.

Section 2: Pull-down the Target Protein

1. Add the sample containing the target protein to the resin from the above section.

2. Incubate for 60 minutes.

3. The target protein will be pulled down by RLR Biotinylated protein resin from Section 1.

1. Centrifuge the resin to remove the supernatant and wash the resin by adding 1xPBS buffer (pH=7.2~7.4) or a suitable wash buffer.

2. Repeat washing as needed.

3. Add elution buffer (4 mM d-biotin in 20 mM Tris-HCl Buffer (pH=7.5) with 50 mM NaCl) and incubate at 37°C for 10 minutes or longer. Repeat three times or as needed.

4. Pool all the elution, and the target protein and RLR biotinylated protein complex will be ready for further analysis.