Biotinylated Rapid Labeling Kit
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The Biotinylation Rapid Labelling Kit enables biotinylation of proteins. Based on NHS ester chemistry, the NHS ester-activated fluorescent dye reacts with primary amines in the antibody/protein to be labelled within a pH 7-9 solution, forming stable amide bonds to achieve conjugation with the antibody/protein. Typically, a single IgG molecule can bind 2-8 molecules of biotin. The entire procedure can be completed within two hours.
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Storage :
4°C 1 year
- Manual (1555KB)
- COA
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To place orders, for customer services and technical support, please contact: MedChemExpress USA
Tel: 609-228-6898 E-mail: [email protected] [email protected]
Description & Advantages
Biotinylation labelling services involve the covalent attachment of biotin to a specific functional group on the target molecule under investigation. Leveraging biotin's ability to undergo specific recognition with streptavidin, this technique enables qualitative, localisation, and quantitative analysis of labelled targets. It offers advantages including absence of radioactive contamination, straightforward operation, and ease of observation. Its applications have expanded into pharmacology, physiology, environmental science, and information science. It also finds extensive application in protein functional studies and drug screening.
The Biotinylation Rapid Labelling Kit enables biotinylation of proteins. Based on NHS ester chemistry, NHS ester-activated fluorescent dyes react with primary amines in pH 7-9 solutions to form stable amide bonds, thereby achieving conjugation with antibodies or proteins. Typically, a single IgG molecule can bind 2-8 biotin molecules. The entire procedure can be completed within two hours.
Protocol
3.1 Sample Preparation
Prepare labelled antibodies/proteins
1) Buffer pH requirement: pH 6.5–8.5.
2) Buffer component requirements: Ideal buffers: PBS; HEPES; potassium salts; sodium salts.
3) Restricted components: Tris ≤ 50 mM / 0.6%; BSA ≤ 0.1%; glycerol ≤ 10%.
4) Prohibited components: arginine; glutathione; DTT.
Note: If buffer specifications cannot be met, sample must first be dialysed in PBS. Microdialysis cups may be used for small samples.
5) Protein concentration must exceed 1 mg/mL, with 2 mg/mL being optimal. If concentration is too low, concentrate using an ultrafiltration tube.
3.2 Preparation of Dye Stock Solution
Dissolve lyophilised powder in biotin solvent. Add dye solvent to the dye and prepare for use.
1) Kit specification (50–200 μg): Add 25 μl;
2) Kit specification (200 μg–1 mg): Add 110 μl.
3.3 Labelling
Antibody (protein) quantity: Add the dissolved biotin to the antibody/protein to be labelled* at a volume ratio of 1:10 (e.g., add 5 μl dye per 50 μg antibody/protein). Mix thoroughly, place in a light-protected bag, and incubate on a shaker or rotator for 30 minutes to 1 hour.
*Minor flocculation may occur during addition; gently pipette several times or shake to disperse, which will resolve.
*A 30-minute rotation achieves 90% reaction efficiency; 3 hours yields >98%. To enhance efficiency, extend reaction time or incubate overnight at 4°C.
3.4 Purification
3.4.1 Purification Column Preparation
Remove the sealing foil. Centrifuge at 1000 g for 2 minutes. Discard the supernatant from the outer tube. Transfer the inner tube of the purification column to a 1.5 ml collection tube and set aside.
3.4.2 Collection
After brief centrifugation, transfer the entire contents to the inner tube of the purification column. Allow to stand for 1 minute, then centrifuge at 1000 g for 2 minutes. Collect the labelled product and discard the inner tube of the purification column.
Storage
4°C 1 year
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