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>Place matching filter paper under the 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>Add 5 uL of sample to (Prussian blue ferritin, ferritin, negative BSA control) to each wanted spot</li>
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   <li>Add 20 uL 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>Let the nitrocellulose dry for 15 minutes</li>
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  <li>Wait for 20 minutes for the blot to dry.</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>Do two washes over 20 minutes with TBS-Tween.</li>
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   <li>Record results via photographs every two minutes</li>
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  <li>Block for 20 minutes with 5% skim milk solution.</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>
 +
  <li>Wait for 20 minutes for the blot to dry.</li>
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   <li>Add 20 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>Take pictures of the blot over set time intervals.</li>
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Revision as of 00:22, 28 September 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. Add a piece of filter paper under the nitrocellulose (remove for washes).
  3. Add 20 uL of TALE protein fused with a K-coil (Commerical Prussian blue ferritin and bovine serum albumins can be added and act as controls).
  4. Wait for 20 minutes for the blot to dry.
  5. Do two washes over 20 minutes with TBS-Tween.
  6. Block for 20 minutes with 5% skim milk solution.
  7. Do two washes over 20 minutes with TBS-Tween.
  8. Add a new piece of filter paper under the nitrocellulose (remove for washes).
  9. Add the recombinant Prussian blue ferritin to the same blot spots as the intial protein was added to.
  10. Wait for 20 minutes for the blot to dry.
  11. Do two washes over 20 minutes with TBS-Tween.
  12. Wait for 20 minutes for the blot to dry.
  13. Add 20 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).
  14. Take pictures of the blot over set time intervals.

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