General LaCava Lab Protocol
Original Rout Lab protocol here:
https://lab.rockefeller.edu/rout/assets/file/protocols/Conjugation_of_Dynabeads.pdf
Conjugation of antiORF1 Dynabeads
Dynabeads M-270 Epoxy (Invitrogen Dynal, Cat. No. 143.02D)
Check bead expiration date; we have had issues in the past with expired beads.
Before beginning, please allow for enough time to let Dynabeads equilibrate to RT to prevent condensation from forming after removing bottle from 4C.
Beads must be equlibrated into 0.1M NaPO4 pH 7.4. For a 300mg bottle of beads you would resuspend into 16ml of NaPhosphate. Calculate your volume accordingly. Beads should be vortexed for 30 seconds then incubate on nutator or end-over-end mixer for 10 minutes. Then aspirate buffer and wash once more in the same volume. During incubation prepare antibody mix.
This antibody must be desalted (buffer exchanged into 0.1M NaPO4 using Zeba column) before using
1. Take XX ul of the antibody; desalt into 0.1M NaPO4, pH7.4, using a Zeba 7k colum - follow the manufacturer’s instructions
2. Check the concentration before and after desalting by BCA Assay and use desalted antibody to make beads
3. Antibody mix: 15ug of antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads
NOTE: The standard ratio is 10ug of antibody to 20ul of beads, but because we recover unbound antibody after conjugation, and there is some evidence that more antibody improves bead performance with this antibody, we increase the ratio.
| Volume Required | Antibody Mix Component | Notes |
|---|---|---|
| XX | abmart 4H1 antiORF1 | 15ug/20ul beads |
| XX | 3M AmSO4 final cxn 1M | Can use 4M if needed |
| XX | 0.1M NaPO4 pH 7.4 | Can be omitted entirely if necessary |
4. If you observe precipitation in the antibody mix, mix using Costar Spin X column (0.45um). Otherwise proceed without filtering.
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 1-2ul of antibody mix before coupling for gel analysis
8. Add antibody mix to beads pre-equilibrated with 0.1M NaPO4, pH7.4; mix well by vortexing
9. Place beads in the end-over-end mixer or in a thermomixer. Place the end-over-end mixer INSIDE a 37C incubator overnight. If ugina thermomixer it is also best to leave inside a 30C or 37C incubator if there is any possibility of condensation. Condensation can negatively affect the conjugation reaction. The conjugation reaction requires 18 - 24 hours. Bead washing is performed the next day.
Save entire antibody mix flow-through (FT); antibody can be recovered using thiis protocol: insert wiki link here
*Make fresh triethylamine before beginning wash steps. You must make triethylamine fresh every time!
(Make fresh 100 mM Triethylamine by adding
168ul stock from flammables cabinet to 11.156mL of DDH20)
10. Wash the beads 1x 1ml 100mM Glycine-HCl, pH 2.5 - this wash should be on and off as fast as possible
11. Wash the beads 1x 1ml 10mM Tris, pH 8.8
12. Wash the beads 1x 1ml fresh 100mM Triethylamine - this wash should be on and off as fast as possible.
13. Wash the beads 4x 1ml 1x PBS (5’ each) - Five total washes of 5 minutes each.
14. Wash the beads 1x 1ml 1x PBS + 0.5% Triton X-100, 5’
15. Wash the beads 1x 1ml 1x PBS + 0.5% Triton X-100, 15’
16. Resuspend beads in 50% Glycerol/1xPBS/0.5mg/ml BSA for long-term storage at -20C (2 ml buffer per 300 mg of beads)