Team:Calgary/Notebook/Protocols/CoilBindingNitrocelluloseAssay

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<h1>Coil Binding Nitrocellulose Assay</h1>
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  <li>10 mg/mL TMB</li>
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  <li>0.2 M pH 3.6 Acetate buffer</li>
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  <li>30% Hydrogen Peroxide</li>
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  <li>Nitrocellulose</li>
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  <li>Filter Paper</li>
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<li>0.022 mg/mL Prussian Blue Ferritin</li>
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<li>1 mg/mL Bovine Serum Albumin</li>
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<li>0.5 mg/mL Recombinant Prussian Blue Ferritin</li>
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<li>5% Skim Milk solution</li>
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<li>1x 0.1% TBS-Tween</li>
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</ul>
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<h2>Protocol</h2>
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  <li>Cut out an appropriately sized piece of nitrocellulose</li>
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  <li>Place matching filter paper under the nitrocellulose</li>
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  <li>Add 5 uL of sample to (Prussian blue ferritin, ferritin, negative BSA control) to each wanted spot</li>
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  <li>Let the nitrocellulose dry for 15 minutes</li>
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  <li>Add 7.5 uL 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)</li>
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  <li>Record results via photographs every two minutes</li>
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</ol>
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Revision as of 00:20, 28 September 2013

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 uL 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 uL 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

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