General LaCava lab protocol, Dec 6 2019
Conjugation of antibody Dynabeads
1. Take 125ul of the antibody; desalt into 0.1M NaPO4, pH7.4, using a Zeba 7k colum (0.5ml) follow the manufacturer’s instruction
2. Check the concentration before and after desalting by Bradford Assay and use desalted antibody to make beads
3. Antibody mix: 10ug of antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads
a. .....ul anti-ORF1 antibody
b. .....ul 3M AmSO4 (final concentration 1M)
c. .....ul 0.1M NaPO4, pH7.4]]
4. Filter the resulting antibody mix using Costar Spin X column (0.45um)
5. Load <500ul on each column (500ul capacity; The column can be use repeatedly for the same antibody mix. Load the column multiple times if the volume is more than 500ul)
6. Spin 12,000g, 1min (16,000g maximum speed)
7. Take 1ul of antibody mix before coupling for gel analysis
8. Keep the rest of the antibody mix @ 4C
9. Add antibody mix to beads pre-equilibrated with 0.1M NaPO4, pH7.4; mix well
10. Leave the tube on a thermomixer at 37C, O.N. (18 - 24hr)
11. Wash the beads 1x 1ml 100mM Glycine-HCl, pH 2.5
12. Wash the beads 1x 1ml 10mM Tris, pH 8.8
13. Wash the beads 1x 1ml fresh 100mM Triethylamine
14. Wash the beads 4x 1ml 1x PBS (5’ each)
15. Wash the beads 1x 1ml 1x PBS + 0.5% Triton X-100, 5’
16. Wash the beads 1x 1ml 1x PBS + 0.5% Triton X-100, 15’
17. Resuspend beads in 50% Glycerol/1xPBS/0.5mg/ml BSA for long-term storage at -20C (1 ml buffer per 150 mg of beads)