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