LABELING ANTIBODY WITH BIOTIN
PRINCIPLE
Biotin labeling assay is primarily used for the preparation of biotin-labeled antibody for enzyme immunoassay (EIA). NH2-Reactive Biotin has a succinimidyl ester group, and can easily make a covalent bond with an amino group of the target protein or other macromolecules.
MATERIALS
Reagents
- NH2-Reactive Biotin
- Reaction Buffer
- WS Buffer
Instruments and Consumables
- Filtration Tube
- Incubator
- Microcentrifuge
- Microtube
METHODS
1. Add 100 μl WS buffer and the sample solution containing 100 μg protein to a Filtration tube.
2. Pipette to mix and centrifuge at 8,000g for 10 min.
3. Add 10 μl DMSO to NH2-Reactive Biotin, and dissolve with pipetting.
4. Add 100 μL reaction buffer to the filtration tube, and then add 8 μL NH2-reactive biotin solution to the filtration tube and pipette to mix.
5. Incubate the tube at 37°C for 10 min.
6. Add 100 μL WS buffer to the filtration tube, and centrifuge at 8,000g for 10 min. Discard the filtrate.
7. Add 200 μL WS buffer to the filtration tube, and centrifuge at 8,000g for 10 min. Repeat this step one more time.
8. Add 200 μL WS buffer, and pipette about 10 times to recover the conjugate. Transfer the solution to a microtube, and store at 0-5°C.
9. Collect the labeled solution. Store at 4°C and protect from light.
