Sack: Alyknefluor Azide Beads

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