WinterVomitingLab:Protocols/HBGA binding with magnetic beads

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Overview

Study the capture of human norovirus to synthetic HBGAs.

Materials

• Streptavidin-coated magnetic beads (MyOne) or 50 µl (M270)(Dynal)
• PBS-T (PBS containing 0.05% Tween 20)
• biotinylated HBGA (Glycotech)
• blocking buffer of choice, such as;
  • SuperBlock (Pierce)
  • 5% Blotto
  • 5% Fetal Bovine Serum
  • 5% BSA
• stool suspension containing human norovirus

Procedure

1. Vortex streptavidin-coated magnetic beads (Dynal) until re-suspended.
2. To 1.5 ml tubes, add 25 µl (MyOne) or 50 µl (M270) of beads for each sample to be tested.
3. Add PBS up to 1 ml and mix using an end-over-end or Hula (Dynal) mixer for 1 min.
4. Pellet beads against the magnet (Dynal), waiting for the solution to become clear (~ 1 min) before drawing off the supernatant.
5. Repeat wash procedure 2 additional times, suspending the beads in blocking buffer (we use SuperBlock or 5% Blotto) the final time.
6. Add 50 µg of synthetic biotinylated HBGA (Glycotech) to each tube. Be sure to include uncoated control beads for determining non-specific binding of virus to uncoated beads.
7. Incubate for 1.5 hr at RT.
8. Wash 3X with PBS-T.
9. Suspend beads in 980 µl of blocking buffer containing 0.25% Tween.
10. Incubate for 1 hr at RT and then overnight at 4°C on an end-over-end rotator. Note: the overnight blocking step may be replaced by incubating for 2-4 hr at RT.
11. Wash 3X with PBS-T.
12. Suspend beads in 980 µl of blocking buffer (without Tween).
13. Add 1 µl (or appropriate volume) of virus suspension (if adding dilution series, it should be done prior to adding virus to beads).
14. Incubate for 4 h at RT using an end-over-end rotator.
15. Wash 5x with PBS-T. Note: Be sure to discard spent wash solution in biohazard waste container.
16. Wash 3X with PBS. See note in step above.
17. Re-suspend beads in 50 µl sterile PBS.
18. Proceed to RNA extraction step of choice.

Notes

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References

Relevant papers and books

1. Mendez II, Hermann LL, Hazelton PR, and Coombs KM. A comparative analysis of freon substitutes in the purification of reovirus and calicivirus. J Virol Methods. 2000 Oct;90(1):59-67. DOI:10.1016/s0166-0934(00)00217-2 |

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