Table 1 Technical protocol 125 I labelling of BoNT-A according to IODOGEN-coated method
Step | |
|---|---|
1. | X μL 125I is added to wobbling reaction vial |
2. | 250 μL phosphate buffer (0.5 M, pH 7.4) is added |
3. | 50 μL BoNT-A/ phosphate buffer (=50 μg BoNT-A) is addeda |
4. | 115-X μL phosphate buffer (0.1 M, pH 6.8) is added to adjust the reaction volume to 215 μL |
5. | 17.5 μL freshly prepared IODOGEN/acetonitrile solution (2.5 μg/35 μL) is added into the reaction mixture (start of reaction, t = 0) |
6. | At t = 45 s, another 17.5 μL IODOGEN/acetonitrile solution (2.5 μg/35 μL) is added |
7. | At t = 90 s, 100 μL ascorbic acid solution (25 mg/mL, pH 5.0) is added to stop the reaction and to reduce any formed S-Cl bonds |
8. | At t = 4.30 min, 10 μL BSA (50 mg/mL) is addedb |
9. | At t = 5.30 min, samples are taken for ITLC for determination of the labelling efficiency |
10. | At t = 10 min, 360 μL reaction mixture is transferred to a syringe connected to a filterc |
11. | The reaction vial is rinsed by 640 μL phosphate buffer (0.1M, pH 6.8) and also transferred to the syringe connected to the filterc |
12. | The combined solution in the syringe is filtered and purified on a PD10 column with ascorbic acid solution (5 mg/mL, pH 5.0) as eluent, collected fractions were 0.5 mL |
13. | Fractions with highest amount of 125I-BoNT-A and the highest radiochemical purity are pooled |
14. | Samples of the pooled 125I-BoNT-A (fractions 6, 7, 8) are taken for ITLC |
15. | Final product is diluted by BSA (1 mg/mL) till a concentration of 1 μg/100 μL and stored at 20°C |