Biotin (cleavable) Rapid Coupling Kit

The Biotin (Biotin, Cleavable) Rapid Conjugation Kit provides a simple and fast conjugation protocol for coupling biotin derivatives (Biotin-S-S) 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 to form 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. The conjugation agent contains a disulfide bond in its linear chain, which can be cleaved by reducing agents such as DTT, BME, or TCEP, thereby removing the biotin label and providing additional functionality to the reagent. For example, it can be used for purifying antibodies or proteins, cleaving interactions with streptavidin probes, or serving as a cleavable ADC linker for synthesizing antibody-drug conjugates (ADCs).

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 (mAb, pAb) or other proteins:

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

Usage Instructions for 2mg Kit
1. Add Reagent A to the antibody or protein to be labeled (volume ratio of protein to be labeled to Reagent A is 9:1), pipette up and down repeatedly to mix thoroughly, avoiding bubble formation;
2. Add Reagent C (50 µL) to the Reagent B tube, dissolve and mix well to prepare a 5 mg/mL coupling agent solution (prepare fresh and use immediately);
3. Continue to add the freshly prepared coupling agent to the mixture from Step 1 (add 10 µL of coupling agent solution per 0.5 mg of protein/antibody; if the amount of protein/antibody to be labeled increases, increase the coupling agent dosage proportionally), pipette up and down to mix thoroughly, avoiding bubble formation;
4. React at room temperature for 2 hours, or mix slowly on a mixer;
5. Dilute Reagent D concentrate 20-fold with distilled or purified water to prepare column buffer and dialysis buffer.
6. Desalting purification: ① Equilibrate the desalting column with the diluted Reagent D solution, using an equilibration volume of 30-40 mL. ② Adjust the volume of the conjugated protein/antibody to 2.5 mL with column buffer, load it onto the desalting column, allow it to drain completely, and do not collect the flow-through. ③ Add another 3.5 mL of column buffer to the desalting column and collect 3.5 mL of flow-through.
7. Dialysis (optional): The reaction mixture can be dialyzed against PBS dialysis buffer (10 mM, pH 7.2-7.4) prepared by diluting Reagent D, or other dialysis buffers, at 2-8°C for 24-36 hours, changing the buffer 3-4 times. During dialysis, the buffer should be gently stirred.
8. Cleave the disulfide bond in the spacer arm: ① Reducing agent preparation: Add Reagent F (1 mL) to the Reagent E tube, dissolve and mix well to prepare a 50 mM TCEP·HCl reducing agent solution, pH 5.5. ② Incubate the sample at room temperature for 30 minutes. ③ Alternatively, the sample can be incubated in 50 mM DTT or 2-mercaptoethanol at room temperature for 2 hours and 30 minutes.

Usage Instructions for 10mg Kit
1. Add Reagent A to the antibody or protein to be labeled (volume ratio of protein to be labeled to Reagent A is 9:1), pipette up and down repeatedly to mix thoroughly, avoiding bubble formation;
2. Add Reagent C (220 µL) to the Reagent B tube, dissolve and mix well to prepare a 5 mg/mL coupling agent solution (prepare fresh and use immediately);
3. Continue to add the freshly prepared coupling agent to the mixture from Step 1 (add 10 µL of coupling agent solution per 0.5 mg of protein/antibody; if the amount of protein/antibody to be labeled increases, increase the coupling agent dosage proportionally), pipette up and down to mix thoroughly, avoiding bubble formation;
4. React at room temperature for 2 hours, or mix slowly on a mixer;
5. Dilute Reagent D concentrate 20-fold with distilled or purified water to prepare column buffer and dialysis buffer.
6. Desalting purification: ① Equilibrate the desalting column with the diluted Reagent D solution, using an equilibration volume of 30-40 mL. ② Adjust the volume of the conjugated protein/antibody to 2.5 mL with column buffer, load it onto the desalting column, allow it to drain completely, and do not collect the flow-through. ③ Add another 3.5 mL of column buffer to the desalting column and collect 3.5 mL of flow-through.
7. Dialysis (optional): The reaction mixture can be dialyzed against PBS dialysis buffer (10 mM, pH 7.2-7.4) prepared by diluting Reagent D, or other dialysis buffers, at 2-8°C for 24-36 hours, changing the buffer 3-4 times. During dialysis, the buffer should be gently stirred.
8. Cleave the disulfide bond in the spacer arm: ① Reducing agent preparation: Add Reagent F (2 mL) to the Reagent E tube, dissolve and mix well to prepare a 50 mM TCEP·HCl reducing agent solution, pH 5.5. ② Incubate the sample at room temperature for 30 minutes. ③ Alternatively, the sample can be incubated in 50 mM DTT or 2-mercaptoethanol at room temperature for 2 hours and 30 minutes.① Reducing agent preparation: Add Reagent F (2 mL) to the Reagent E tube, dissolve and mix well to prepare a 50 mM TCEP·HCl reducing agent solution, pH 5.5. ② Incubate the sample at room temperature for 30 minutes. ③ Alternatively, the sample can be incubated in 50 mM DTT or 2-mercaptoethanol at room temperature for 2 hours and 30 minutes.

Instructions for Gravity Desalting Column
1. Preparation before using the gravity desalting column
1). Remove the top cap of the desalting column and pour out the column storage solution;
2). Cut or snip 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 other buffers, using an equilibration volume of 35-40 mL, and discard the eluate;
2). Adjust the volume of the conjugated protein/antibody to 2.5 mL with column buffer, load it onto the desalting column, allow it to drain completely, and do not collect the flow-through;
3). Add another 3.5 mL of column buffer to the desalting column and collect 3-3.5 mL of flow-through (EP tubes can be used, collecting 1 mL per tube);
3. Operating conditions and regeneration/storage of the desalting column
1). Operating conditions for the desalting column: pH 2-13, 4°C -30°C; stable in common buffers (PBS, Tris-HCl, sodium carbonate-sodium bicarbonate, etc.);
2). Recommended for single use (can also be regenerated and reused);
3). Regeneration and storage of the desalting column: After use, wash thoroughly and store the column in 20% ethanol, 0.05% NaN3, or 0.05 mol/L NaOH.

1. Buffer and concentration requirements for antibodies/proteins to be conjugated: Antibodies/proteins 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 amine components to avoid competitive reactions with the coupling reagent, which may affect conjugation efficiency. This includes primary amines (e.g., Tris, glycine, ammonium salts, EDTA) and protein stabilizers (e.g., BSA, gelatin). If the sample contains substances that may interfere with labeling, it is recommended to exchange the buffer with PBS and concentrate it 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 value 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 other protein stabilizers 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 stored 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 not fully used. It can be used normally within one week. If used within one month, the amount should be increased appropriately. Do not use it beyond one month.5. For small peptide molecules with a molecular weight of less than 5 kDa, the amount of coupling reagent solution can be appropriately increased to improve conjugation efficiency.6. Instructions for using the desalting column (>5000 Mr) included in this kit: column volume 8.3 mL, elution volume 3.5 mL. For gravity desalting: sample loading volume 1.0-2.5 mL, desalting efficiency >98%. For centrifugal desalting: sample loading volume 1.75-2.5 mL, desalting efficiency >90%.7. For Reagent D, salt crystallization may occur when stored at 2°C-8°C. This is a normal phenomenon, and the crystals will disappear after returning to room temperature and mixing.