Team:Calgary/Notebook/Protocols/NitrocelluloseAssay

From 2013.igem.org

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   <li>Cut out an appropriately sized piece of nitrocellulose.</li>
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   <li>Cut out an appropriately sized piece of nitrocellulose</li>
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   <li>Add a piece of filter paper under the nitrocellulose (remove for washes).</li>
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   <li>Place matching filter paper under the nitrocellulose</li>
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   <li>Add 20 μL of TALE protein fused with a K-coil (Commerical Prussian blue ferritin and bovine serum albumins can be added and act as controls).</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>Wait for 20 minutes for the blot to dry.</li>
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   <li>Let the nitrocellulose dry for 15 minutes</li>
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   <li>Do two washes over 20 minutes with TBS-Tween.</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>Block for 20 minutes with 5% skim milk solution.</li>
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   <li>Record results via photographs every two minutes</li>
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  <li>Do two washes over 20 minutes with TBS-Tween.</li>
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  <li>Add a new piece of filter paper under the nitrocellulose (remove for washes).</li>
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  <li>Add the recombinant Prussian blue ferritin to the same blot spots as the intial protein was added to. </li>
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  <li>Wait for 20 minutes for the blot to dry.</li>
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  <li>Do two washes over 20 minutes with TBS-Tween.</li>
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  <li>Wait for 20 minutes for the blot to dry.</li>
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   <li>Add 20 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).</li>
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   <li>Take pictures of the blot over set time intervals.</li>
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Latest revision as of 00:28, 29 October 2013

Nitrocellulose Assay

Reagents and Materials

  • 10 mg/mL ABTS or 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
  • 0.022 mg/mL Horse Spleen Ferritin

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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