Biotin (cleavable) Rapid Coupling Kit

Biotin (Biotin, cleavable) Rapid Conjugation Kit provides a simple and rapid conjugation protocol for conjugating biotin derivatives (Biotin-S-S) to antibodies, proteins, peptides, or other ligands containing free amino groups. The reagent reacts effectively with primary amino groups (-NH2) in a buffer at pH 7-9 to form stable amide bonds. Proteins, including antibodies, typically have 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 coupling agent contains a disulfide group in the linear chain. Reducing agents such as DTT, BME, and TCEP can cleave the disulfide bond in the linker, thereby removing the biotin label and providing further functionality to the reagent. For example: it can be used for purifying antibodies or proteins, cleaving the binding with streptavidin probes, or serving as a cleavable ADC linker for the synthesis of antibody-drug conjugates (ADCs).

Applications
(1) Rapid labeling of proteins (antibodies), etc.

Advantages
(1) High sensitivity and high labeling efficiency;
(2) Providing desalting columns and usage protocols;
(3) Providing methods for detecting labeling efficiency;
(4) This kit provides a complete set of reagents required for rapid labeling, and the reagents are easy to store.

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

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

2mg Usage Instructions
1. 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), pipette repeatedly to mix well, avoiding the generation of bubbles;
2. Add Reagent C (50µL) to the Reagent B tube, dissolve and mix well to prepare a 5mg/mL coupling agent solution (prepared and used immediately);
3. Continue to add the freshly prepared coupling agent to the mixture from Step 1 (add 10µL of coupling agent B solution per 0.5mg of protein/antibody; if the amount of labeled protein/antibody increases, the amount of coupling agent added can be increased proportionally), pipette to mix well, avoiding the generation of bubbles;
4. React at room temperature for 2 hours, or slowly mix on a mixer;
5. Dilute the Reagent D concentrate 20-fold with distilled water or purified water to prepare column buffer and dialysis solution.
6. Desalting and purification: ① Equilibrate the desalting column with the diluted solution of Reagent D, with an equilibration volume of 30-40mL. ② Supplement the volume of the coupled protein/antibody to 2.5mL with column buffer, add it to the desalting column, let it flow dry, and do not collect the filtrate. ③ Add another 3.5mL of column buffer to the desalting column, and collect 3.5mL of filtrate at the same time.
7. Dialysis (optional): PBS dialysis solution (10mM, pH7.2-7.4) prepared by diluting Reagent D or other dialysis solutions can be used. Dialyze the reaction mixture at 2-8°C for 24-36 hours, and replace the buffer 3-4 times. During dialysis, the buffer should be gently stirred.
8. Cleaving the disulfide bond in the spacer arm: ① Preparation of reducing agent: Add Reagent F (1mL) to the Reagent E tube, dissolve and mix well to prepare a 50mM 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 50mM DTT or 2-mercaptoethanol at room temperature for 2 hours and 30 minutes.

10mg Usage Instructions
1. 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 repeatedly pipette to mix well to avoid generating bubbles;
2. Add Reagent C (220µL) to the Reagent B tube, dissolve and mix well to prepare a 5mg/mL coupling agent solution (prepared and used immediately);
3. Continue to add the freshly prepared coupling agent to the mixture from step 1 (add 10µL of coupling agent B solution per 0.5mg of protein/antibody; if the amount of labeled protein/antibody increases, the amount of coupling agent added can be increased proportionally), pipette to mix well to avoid generating bubbles;
4. React at room temperature for 2 hours, or gently mix on a mixer;
5. Dilute the Reagent D concentrate 20 times with distilled water or purified water to prepare column buffer and dialysis buffer.
6. Desalting and purification: ① Equilibrate the desalting column with the diluted solution of Reagent D, with an equilibration volume of 30-40mL. ② Supplement the volume of the coupled protein/antibody to 2.5mL with column buffer, add it to the desalting column, let it flow dry, and do not collect the filtrate. ③ Add another 3.5mL of column buffer to the desalting column, and collect 3.5mL of filtrate at the same time.
7. Dialysis (optional): PBS dialysis buffer (10mM, pH7.2-7.4) prepared by diluting Reagent D or other dialysis buffers can be used. Dialyze the reaction mixture at 2-8°C for 24-36 hours, and replace the buffer 3-4 times. During dialysis, the buffer should be gently stirred.
8. Cleavage of disulfide bonds in the spacer arm: ① Preparation of reducing agent: Add Reagent F (2mL) to the Reagent E tube, dissolve and mix well to prepare a 50mM TCEP.HCL reducing agent solution, pH 5.5. ② Incubate the sample at room temperature for 30 minutes. ③ The sample can also be incubated in 50mM DTT or 2-mercaptoethanol at room temperature for 2 hours and 30 minutes.

Instructions for Using the Gravity Desalting Column
1. Preparation before using the gravity desalting column
1). Remove the top cover of the desalting column and pour out the column storage solution;
2). Cut off or slice off the sealed end at the bottom of the column.
2. Desalting and purification
1). Equilibrate the desalting column with a 20-fold diluted solution of Reagent D or other buffer solutions, with an equilibration volume of 35-40mL, and discard the eluate;
2). Supplement the volume of the conjugated protein/antibody to 2.5mL with column buffer, add it to the desalting column, let it flow dry, and do not collect the filtrate;
3). Add another 3.5mL of column buffer to the desalting column, and collect 3-3.5mL of filtrate at the same time (EP tubes can be used, with 1mL collected in each tube);
3. Operating conditions, regeneration and storage of the desalting column
1). Operating 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). It is recommended to use it once (it can also be reused after regeneration);
3). Regeneration and storage of the desalting column: After passing through the column, wash it thoroughly, and store the column with 20% ethanol, 0.05% NaN3 or 0.05mol/L NaOH.

1. Requirements for buffer and concentration of antibodies/proteins to be conjugated: Antibodies/proteins should be stored in 10mM phosphate buffer (1xPBS, pH 7.2-7.4) with a recommended concentration range of 2-4mg/mL. The buffer must not contain amino components to avoid competitive reactions with the coupling agent, which would affect the conjugation effect. Specifically, this includes primary amines (such as Tris, glycine, ammonium salts, EDTA) and protein stabilizers (such as BSA, gelatin), etc. If the sample contains substances that may interfere with labeling, it is recommended to replace the buffer with PBS and concentrate to the recommended concentration.
2. For most IgG, in a cuvette with a 1cm optical path, the absorbance of a 1mg/mL solution at 280nm is approximately 1.3-1.4. The coupling agent has no significant absorption at 280nm. Therefore, the concentration (mg/mL) of biotin-labeled antibodies can be calculated by dividing the absorbance value of the dialyzed sample at 280nm by 1.4.
3. It is recommended to store the conjugated products under refrigeration. If the final concentration of the purified antibody/protein conjugate is less than 1mg/mL, add bovine serum albumin (BSA) or other protein stabilizers to a concentration of 1-10mg/mL, which can stabilize for several months at 2-8°C. For long-term storage, the labeled solution can be aliquoted according to usage needs and stored frozen at -20°C in the dark, avoiding repeated freezing and thawing.
4. If the coupling agent solution prepared by adding the cosolvent in this kit is not fully used, please seal it and store at -20°C. It can be used normally within one week. If it exceeds one week but is within one month, the usage amount should be appropriately increased; do not use it if it exceeds one month.
5. For small peptide molecules with a molecular weight less than 5KD, the amount of coupling agent solution added can be appropriately increased to improve the coupling efficiency.
6. Instructions for using the desalting column (>5000 Mr) in this kit: Column volume is 8.3mL, eluent is 3.5mL. For gravity desalting: sample loading volume is 1.0-2.5mL, desalting efficiency is >98%; for centrifugal desalting: sample loading volume is 1.75-2.5mL, desalting efficiency is >90%.
7. If reagent D has salt crystals when stored at 2°C -8°C, it is a normal phenomenon that the crystals disappear after returning to room temperature and mixing.