DOTA Rapid Labeling Kit
The DOTA rapid labelling kit enables DOTA labelling of proteins. Based on NHS ester chemistry, NHS ester-activated fluorescent dyes react with primary amines on the antibody/protein to form stable amide bonds at pH 7–9, thereby achieving conjugation with the antibody/protein. Typically, a single IgG molecule can bind 2–8 DOTA molecules. The entire procedure can be completed within 2 hours.
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Storage :
4°C 1 year
- Manual (1546KB)
- COA
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Description & Advantages
DOTA is an organic molecule capable of forming chelate complexes with heavy metal ions, enabling it to bind with radioactive isotopes such as ⁵²Mn, ⁵⁵Co, ⁶⁴Cu, ⁶⁶Ga, ⁸⁶Y, ⁸⁹Zr, and ¹⁵²Tb to form stable chelate complexes. These complexes may be employed in the preparation of radiotracers for use in PET scanning, SPECT scanning, radiotherapy, and other nuclear medicine applications.
The DOTA rapid labelling kit enables DOTA labelling of proteins. Based on NHS ester chemistry, NHS ester-activated fluorescent dyes react with primary amines on the antibody/protein to form stable amide bonds at pH 7–9, thereby achieving conjugation with the antibody/protein. Typically, a single IgG molecule can bind 2–8 DOTA molecules. The entire procedure can be completed within 2 hours.
Protocol
3. Kit Usage Instructions
3.1 Sample Preparation
Prepare antibodies/proteins for labelling
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: Should the buffer fail to meet requirements, sample dialysis in PBS is necessary. Microdialysis cups may be employed for small sample volumes.
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 using the dye dissolving solution. Add the dissolving solution to the dye and prepare for use.
1) Kit specification (50–200 μg): Add 25 μl;
2) Kit specification (200μg–1mg): Add 110μl.
3.3 Labelling
Antibody (protein) quantity: Add the dissolved dye to the antibody/protein to be labelled* at a volume ratio of 1:10 (e.g., add 5μl dye per 50μg 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 and mix thoroughly; flocculation will dissipate.
*Rotation for 30 minutes achieves 90% reaction efficiency; 3 hours yields >98% efficiency. 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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