Sack: Alyknefluor Azide Beads

From OpenWetWare

Jump to: navigation, search

Copper-click Alkyne / Azide Condensation Reaction with peptide or tentagel beads

Notes: keep fluors in dark, dry DMSO dry.

Materials for 1 reaction:

  • 2uL 2 mM label, containing azide or alkyne, in DMF or DMSO
  • 50uL peptide or bead sample containing complementary azide or alkyne.

Up to 40 uM concentration (for 2:1 label:macromolecule) in 50 mM Tris pH 8

  • Click-iT reaction buffer components stored in click-it box in -20C freezer:
    • 100 uL Component A:
    • 10 uL Component B: CuSO4, treat as hazardous waste.
    • 10 uL additive 1: Component C + water. 100uL aliquots stored, use open one.
      • Additive 1 contains a reducing agent that can oxidize. Be aware of color change, additive turns from colorless to brown.
    • 20 uL additive 2: component D + water.

Protocol

  1. Put 98 uL component A in a 1.5 ml tube
  2. Add 2uL label, for a final concentration of 40uM. Total amount: 4 nmole label.
  3. Add 50uL of peptide or bead in 50 mM Tris pH 8, cap tube, vortex ~ 5 seconds
  4. Add 10uL of CuSO4 (component B), then vortex ~ 5 seconds.
  5. Add 10uL of additive 1, vortex ~ 5 secs.
    • Wait for 2-3 mins. but no longer than 5 mins, before the next step.
  6. Add 20uL of additive 2, vortex ~ 5 secs.
    • Solution color change will occur, generating a bright orange color.
  7. Using the rotisserie, rotate end over end for 20 mins using a rotator.
    • If light sensitive, protect sample from light with foil paper wrapped around tube.
  8. Remove products from reaction ASAP, filter and wash beads, freeze peptide at -80 or inject on HPLC


Personal tools