Biotin Rapid Coupling Kit

The Biotin Rapid Conjugation Kit provides a simple and fast conjugation protocol for coupling biotin to antibodies, proteins, peptides, or other ligands containing free amino groups. The reagents effectively react with primary amines (-NH₂) in buffers at pH 7-9, forming stable amide bonds. Proteins, including antibodies, typically possess several primary amines on the side chains of lysine (K) residues and at the N-terminus of each polypeptide, which can serve as targets for reagent labeling.

Applications
(1) Rapid labeling of proteins (antibodies), etc.
 
Advantages
(1) High sensitivity and labeling efficiency;
(2) Includes a desalting column and usage protocol;
(3) Provides a method for detecting labeling efficiency;
(4) This kit offers a complete set of reagents for rapid labeling, and the reagents are easy to store.

2mg can be used to label 0.5-2mg antibodies (monoclonal, polyclonal) or other proteins:

10mg can be used to label 0.5-10mg antibodies (monoclonal, polyclonal) or other proteins:

2mg Usage Instructions
1. This reagent is sensitive to moisture. Before opening, ensure that each vial of the components is fully equilibrated to room temperature to prevent condensation inside the container.
2. Add Reagent A to the antibody or protein to be labeled (the volume ratio of the protein to Reagent A is 9:1), and pipette up and down repeatedly to mix thoroughly, avoiding the formation of bubbles.
3. Add Reagent C (40 µL) to the Reagent B tube, dissolve and mix well to prepare a 5 mg/mL coupling agent solution (prepare fresh and use immediately).
4. To the mixture from Step 2, add the freshly prepared coupling agent (add 8 µL of Reagent B solution for every 0.5 mg of protein/antibody; if the amount of protein/antibody being labeled increases, proportionally increase the amount of coupling agent added). Mix by pipetting up and down, avoiding the formation of bubbles.
5. React at room temperature for 2 hours, or gently mix on a shaker;
6. Dilute the concentrated Reagent D 20-fold with distilled or purified water to prepare the column buffer and dialysis solution;
7. Desalt using a desalting column (see instructions below); load 2.5 mL of sample, and collect the filtered protein solution in collection tubes—this is the conjugation product.
8. Dialysis (optional): Use PBS dialysis buffer prepared by diluting Reagent D (10 mM, pH 7.2–7.4) or another suitable dialysis buffer, and dialyze the reaction mixture at 2–8°C for 24–36 hours, changing the buffer 3–4 times. Gently stir the buffer during dialysis.

Usage instructions for 10 mg
1. This reagent is sensitive to moisture. Before opening, ensure that each vial of the components is fully equilibrated to room temperature to prevent condensation inside the container.
2. Add Reagent A to the antibody or protein to be labeled (the volume ratio of the protein to be labeled to Reagent A is 9:1), and pipette up and down repeatedly to mix thoroughly, avoiding the formation of bubbles.
3. Add Reagent C (200 µL) to the Reagent B tube, dissolve and mix well to prepare a 5 mg/mL coupling agent solution (prepare fresh before use).
4. Continue by adding the freshly prepared coupling agent to the mixture from Step 2 (add 8 µL of Coupling Agent B solution for every 0.5 mg of protein/antibody; if the amount of protein/antibody being labeled increases, proportionally increase the amount of coupling agent added). Pipette up and down to mix thoroughly, avoiding the formation of bubbles.
5. Allow the reaction to proceed at room temperature for 2 hours, or gently mix on a shaker.
6. Dilute reagent D concentrate 20-fold with distilled or purified water to prepare the column buffer and dialysis solution.
7. Perform desalting using a desalting column (see instructions below); load 2.5 mL of sample, and collect the filtered protein solution in collection tubes—this is the conjugation product.
8. Dialysis (optional): Use PBS dialysis buffer prepared by diluting reagent D (10 mM, pH 7.2–7.4) or another suitable dialysis buffer, and dialyze the reaction mixture at 2–8°C for 24–36 hours, changing the buffer 3–4 times. During dialysis, stir the buffer gently.

Instructions for Use of the Gravity Desalting Column
1. Preparations Before Using the Gravity Desalting Column
1). Remove the top cap of the desalting column and pour out the storage solution;
2). Cut or trim off the sealed end at the bottom of the column.
2. Desalting purification
1). Equilibrate the desalting column with a 20-fold dilution of Reagent D or another buffer, using an equilibration volume of 35–40 mL, and discard the eluate;
2). Adjust the volume of the post-coupling protein/antibody to 2.5 mL with column buffer, add it to the desalting column, allow it to drain completely, and do not collect the filtrate;
3). Add 3.5 mL of column buffer to the desalting column again, while collecting 3–3.5 mL of the filtrate (using EP tubes, collecting 1 mL per tube);
3. Operating conditions and regeneration/storage of the desalting column
1). Working conditions of the desalting column: pH 2–13, 4°C–30°C; stable in commonly used buffer solutions (PBS, Tris-HCl, sodium carbonate–sodium bicarbonate, etc.).
2). Single-use is recommended (may also be reused after regeneration).
3). Regeneration and storage of the desalting column: After passing the sample through the column, wash thoroughly, then store the column in 20% ethanol, 0.05% NaN3, or 0.05 mol/L NaOH.
 

1. Requirements for the antibody/protein buffer to be conjugated and concentration: The antibody/protein should be stored in 10 mM phosphate buffer (1x PBS, pH 7.2-7.4). The recommended concentration range is 2-4 mg/mL. The buffer must not contain amino groups to avoid competitive reactions with the coupling reagent, which may affect conjugation efficiency. Specifically, primary amines (e.g., Tris, glycine, ammonium salts, EDTA) and protein stabilizers (e.g., BSA, gelatin) should be avoided. If the sample contains substances that may interfere with labeling, it is recommended to exchange the buffer with PBS and concentrate to the recommended concentration. 2. For most IgGs, a 1 mg/mL solution in a 1 cm pathlength cuvette has an absorbance of approximately 1.3-1.4 at 280 nm. The coupling reagent does not exhibit significant absorption at 280 nm. Therefore, the concentration (mg/mL) of the biotin-labeled antibody can be calculated by dividing the absorbance of the dialyzed sample at 280 nm by 1.4. 3. It is recommended to store the conjugated product under refrigeration. If the final concentration of the purified antibody/protein conjugate is less than 1 mg/mL, adding bovine serum albumin (BSA) or another protein stabilizer to a concentration of 1-10 mg/mL can stabilize it for several months at 2-8°C. For long-term storage, the labeled solution can be aliquoted as needed and frozen at -20°C in the dark, avoiding repeated freeze-thaw cycles. 4. The coupling reagent solution prepared with the solubilizing agent included in this kit should be sealed and stored at -20°C if unused. It can be used normally within one week. If stored for more than one week but within one month, the amount used should be increased appropriately. Do not use if stored for more than one month. 5. For small peptide molecules with a molecular weight of less than 5 kDa, the amount of coupling reagent solution added can be appropriately increased to improve conjugation efficiency. 6. Instructions for use of the desalting column (>5000 Mr) included in this kit: Column volume 8.3 mL, elution volume 3.5 mL. Gravity desalting: Sample loading volume 1.0-2.5 mL, desalting efficiency >98%. Centrifugal desalting: Sample loading volume 1.75-2.5 mL, desalting efficiency >90%. 7. Reagent D may form salt crystals when stored at 2°C -8°C. This is normal; the crystals will disappear after returning to room temperature and mixing.