Nota Rapid Labeling Kit  

NOTA is an organic molecule capable of chelating heavy metal ions, forming stable complexes with radioactive isotopes such as eridium-68, eridium-44, eridium-52, and gallium-68. These complexes serve as radiotracers for PET scanning, SPECT scanning, radiotherapy, and other nuclear medicine applications. The NOTA Rapid Labelling Kit enables NOTA labelling of proteins. Based on NHS ester chemistry, NHS ester-activated fluorescent dyes react with primary amines on the antibody/protein to be labelled in 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 NOTA molecules. The entire procedure can be completed within two hours.

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.