Team:Calgary/Notebook/Protocols/CoilBindingNitrocelluloseAssay

From 2013.igem.org

Coil Binding Nitrocellulose Assay

  • 10 mg/mL TMB
  • 0.2 M pH 3.6 Acetate buffer
  • 30% Hydrogen Peroxide
  • Nitrocellulose
  • Filter Paper
  • 0.022 mg/mL Prussian Blue Ferritin
  • 1 mg/mL Bovine Serum Albumin
  • 0.5 mg/mL Recombinant Prussian Blue Ferritin
  • 5% Skim Milk solution
  • 1x 0.1% TBS-Tween

Protocol

  1. Cut out an appropriately sized piece of nitrocellulose
  2. Place matching filter paper under the nitrocellulose
  3. Add 5 μL of sample to (Prussian blue ferritin, ferritin, negative BSA control) to each wanted spot
  4. Let the nitrocellulose dry for 15 minutes
  5. Add 7.5 μL of substrate solution (1:1:1 ratio of 30% hydrogen peroxide, 10 mg/mL ABTS or TMB, and 0.2 M pH 3.6 acetate buffer)
  6. Record results via photographs every two minutes