WinterVomitingLab:Protocols/HBGA binding with magnetic beads

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Contents

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

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books

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  1. Error fetching PMID 11011081: [Paper1]

Contact

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