Proteintech's Biotin Labeling Kit provides a complete set of reagents required for biotin labeling. The kit is designed for labeling antibodies containing amino groups (NH₂-). The biotin included in the kit is pre-activated and ready for direct use, and can be used to label 0.1–5 mg of antibody. The kit also includes buffer, ultrafiltration tubes, and protective solution. Antibodies do not require dialysis, the procedure is simple, and the entire workflow can be completed in only 90 minutes.

Basic Information:

Structural Formula:

Molecular Weight: 341.4

Product Features:

1. Comprehensive: This kit provides all components required for biotin labeling.

2. Fast: The entire process takes only 90 minutes.

3. Convenient: No dialysis required for antibodies.

4. Flexible: Wide labeling range, 0.1–5 mg of antibody.

5. Stable: The product has passed an accelerated stability test at 37 °C for 4 days with qualified results.

Recommended Applications: This kit is used for biotin labeling of antibodies or proteins. The labeled antibodies, upon validation, are suitable for the vast majority of ELISA applications and some IHC applications.

Component	Size	Size
	2 Labeling Reactions	5 Labeling Reactions
Maximum Antibody Amount	2 mg	5 mg
NHS-bio	0.1 mg/vial × 2	0.1 mg/vial × 5
Labeling Buffer	15 mL × 1	30 mL × 1
DMF	200 uL × 1 vial	200 uL × 1 vial
Ultrafiltration Tube	0.5 mL × 4	0.5 mL × 10
Antibody Protective Solution	1 mL	2 mL

Note: Unopened kit can be stored at 2–8 °C for 1 year. Dissolved biotin can be stored at 2–8 °C for 1 week.

Materials Required But Not Provided:

1. Pipettes and disposable tips.

2. 37 °C constant-temperature incubator.

3. Centrifuge.

Labeling Principle:

Instructions:

Usage

Pre-Experiment Preparation:

1. Equilibrate the kit at room temperature for 15–30 minutes.

2. Calculate the required amount of dissolved biotin.

Experimental Procedure:

1. Dissolve each 0.1 mg of biotin lyophilized powder in 10 uL of DMF. The dissolved biotin concentration is 10 mg/mL.

2. Add the antibody to be labeled into an ultrafiltration tube, and add an appropriate volume of Labeling Buffer to a total volume of 0.5 mL. Centrifuge at 12,000 × g for 10 minutes. If glycerol is present, repeat this step 2–3 times. In the final wash, resuspend the antibody with Labeling Buffer to a final concentration of 2 mg/mL. (Note: The total volume added must not exceed the maximum volume of the ultrafiltration tube, 0.5 mL.)

3. Add an appropriate amount of dissolved biotin (recommended antibody:biotin mass ratio is 10:1). For example, for 0.5 mL of 2 mg/mL antibody (MW 150,000), add 10 uL of 10 mg/mL biotin, mix gently by pipetting, and incubate in a 37 °C incubator protected from light for 30 minutes.

4. After incubation, centrifuge at 12,000 × g for 10 minutes.

5. Washing: Add an appropriate amount of Labeling Buffer to the above ultrafiltration tube to a final volume of 0.5 mL, mix gently by pipetting, centrifuge at 12,000 × g for 10 minutes, and repeat this operation once.

6. Recycling: After centrifugation, add 0.2 mL of Labeling Buffer to the above ultrafiltration tube, pipette gently, invert the filter into another clean centrifuge tube, and centrifuge at 6,000 × g for 10 minutes.

7. Volume adjustment: Collect the solution in the centrifuge tube, which is the biotin-labeled antibody. If needed, add Labeling Buffer to adjust the concentration, then add an equal volume of protective solution, and store at −20 °C.

Cautions

1. This kit can also label other amino-containing antigens, HRP, and peptides. The specific labeling ratio depends on the number of amino groups in the substance to be labeled. If labeling proteins, it is recommended to adjust the protein concentration to 2 mg/mL, at which point the protein: biotin molar ratio is 1:20.

Calculation method for the amount of biotin to be added:

2. For a 10 mmoL biotin solution, the calculation method for the volume of biotin to be added to the reaction:

3. DMF should be stored sealed and dry.

4. The ultrafiltration tubes provided in this kit have a molecular weight cutoff of 10 KD. Labeled antigens or peptides should have a molecular weight of 20 KD or above.

5. Please calculate the required amount of biotin according to the amount of antibody to be labeled before labeling.