Team:Calgary/Notebook/Protocols/NativePAGEGel

From 2013.igem.org

(Difference between revisions)
(Created page with "<html> <div id="Banner"><h1>Native PAGE Gel</h1></div> </html> {{Team:Calgary/ContentPage}} <html> <section id="Content"> <h1>Native PAGE Gel</h1> <p>Insert Text Here</p> </s...")
 
(11 intermediate revisions not shown)
Line 6: Line 6:
<section id="Content">
<section id="Content">
-
<h1>Native PAGE Gel</h1>
+
<h1>Native PAGE</h1>
-
<p>Insert Text Here</p>
+
<h2>Reagents and Materials</h2>
 +
<ul>
 +
<li>SDS PAGE Gel apparatus: lid, tank, combos, spacer plates, short plates, casting frame and casting stand</li>
 +
</ul>
 +
<p class="noIndent"><b>5% Native PAGE Gel (Separating)</b></p>
 +
<ul>
 +
<li>833 mL Acryl/Bis (40%)</li>
 +
<li>4112 mL 0.375M Tris (pH 8.8)</li>
 +
<li>50 µL 10% APS </li>
 +
<li>5 µL TEMED </li>
 +
</ul>
 +
 
 +
<p class="noIndent"><b>4% Native PAGE Gel (Stacking)</b></p>
 +
<ul>
 +
<li>400 mL Acryl/Bis (40%)</li>
 +
<li>2567 mL 0.375M Tris (pH 8.8)</li>
 +
<li>30 µL 10% APS </li>
 +
<li>3 µL TEMED </li>
 +
</ul>
 +
 
 +
<p class="noIndent"><b>Native PAGE Running Buffer</b></p>
 +
<ul>
 +
<li>25mM Tris </li>
 +
<li>192mM Glycine</li>
 +
</ul>
 +
 
 +
<p class="noIndent"><b>4x Native PAGE Loading Dye</b></p>
 +
<ul>
 +
<li>62.5mM Tris-HCl (pH 6.8)</li>
 +
<li>25% Glycerol</li>
 +
<li>1% Bromophenol Blue </li>
 +
</ul>
 +
 
 +
<p class="noIndent"><b>Stains</b></p>
 +
<ul>
 +
<li>Coomassie Brilliant Blue</li>
 +
<li>Potassium ferrocyanide</li>
 +
<li>Hydrogen Chloride</li>
 +
</ul>
 +
 
 +
 
 +
<h2>Protocol</h2>
 +
<ol>
 +
<li>Put two gel glass pieces together in the correct orientation  (should be small space between them) and clip into the gel apparatus</li>
 +
<li>Add about 5 mL of separating gel in between the two glass pieces (ensure it does not leak) </li>
 +
<li>Add a thin layer of isopropanol on top of the separating gel to form a smooth line</li>
 +
<li>Wait for the gel to solidify (~ 20 minutes) </li>
 +
<li>Rinse out the isopropanol with water</li>
 +
<li>Add about 2 mL of the stacking gel on top of the separating gel and add the gel comb</li>
 +
<li>Wait for the gel to solidify (~ 20 minutes) </li>
 +
<li>Place the gel into the gel running apparatus and lock it in place. Ensure the side with the larger piece of glass is facing outward. If there is only one gel, add empty glass pieces to the other side</li>
 +
<li>Put the gel into the gel box and fill with 1x Native PAGE Running Buffer. Ensure the middle section between the gels is filled with buffer</li>
 +
<li>Load samples, <b>DO NOT HEAT SAMPLES</b> </li>
 +
<li>Run gel at 100V until samples leave the well and increase the voltage to 150V. Or run the gel at lower voltages (~90V) to get cleaner bands</li>
 +
<li>When the gel is done running, remove gel and stain with either the protein stain or iron stain</li>
 +
</ol>
 +
<br>
 +
 
 +
<p><h3>Protein Stain </h3>
 +
<li>Stain gel in Coomassie Brilliant blue for approximately 1 hour</li>
 +
<li>Destain gel with destaining solution</li>
 +
</p>
 +
<p><h3>Iron Stain </h3>
 +
<li>Prepare 25mL 4% hydrogen chloride and 25mL 2% potassium ferrocyanide</li>
 +
<li>Mix solutions right before use, and stain gel using the resulting solution for approximately 1 hour</li>
 +
<li>This gel does not require a destain</li>
 +
</p>
</section>
</section>
</html>
</html>

Latest revision as of 00:29, 28 September 2013

Native PAGE

Reagents and Materials

  • SDS PAGE Gel apparatus: lid, tank, combos, spacer plates, short plates, casting frame and casting stand

5% Native PAGE Gel (Separating)

  • 833 mL Acryl/Bis (40%)
  • 4112 mL 0.375M Tris (pH 8.8)
  • 50 µL 10% APS
  • 5 µL TEMED

4% Native PAGE Gel (Stacking)

  • 400 mL Acryl/Bis (40%)
  • 2567 mL 0.375M Tris (pH 8.8)
  • 30 µL 10% APS
  • 3 µL TEMED

Native PAGE Running Buffer

  • 25mM Tris
  • 192mM Glycine

4x Native PAGE Loading Dye

  • 62.5mM Tris-HCl (pH 6.8)
  • 25% Glycerol
  • 1% Bromophenol Blue

Stains

  • Coomassie Brilliant Blue
  • Potassium ferrocyanide
  • Hydrogen Chloride

Protocol

  1. Put two gel glass pieces together in the correct orientation (should be small space between them) and clip into the gel apparatus
  2. Add about 5 mL of separating gel in between the two glass pieces (ensure it does not leak)
  3. Add a thin layer of isopropanol on top of the separating gel to form a smooth line
  4. Wait for the gel to solidify (~ 20 minutes)
  5. Rinse out the isopropanol with water
  6. Add about 2 mL of the stacking gel on top of the separating gel and add the gel comb
  7. Wait for the gel to solidify (~ 20 minutes)
  8. Place the gel into the gel running apparatus and lock it in place. Ensure the side with the larger piece of glass is facing outward. If there is only one gel, add empty glass pieces to the other side
  9. Put the gel into the gel box and fill with 1x Native PAGE Running Buffer. Ensure the middle section between the gels is filled with buffer
  10. Load samples, DO NOT HEAT SAMPLES
  11. Run gel at 100V until samples leave the well and increase the voltage to 150V. Or run the gel at lower voltages (~90V) to get cleaner bands
  12. When the gel is done running, remove gel and stain with either the protein stain or iron stain

Protein Stain

  • Stain gel in Coomassie Brilliant blue for approximately 1 hour
  • Destain gel with destaining solution

    • Iron Stain

  • Prepare 25mL 4% hydrogen chloride and 25mL 2% potassium ferrocyanide
  • Mix solutions right before use, and stain gel using the resulting solution for approximately 1 hour
  • This gel does not require a destain

    • Retrieved from "http://2013.igem.org/Team:Calgary/Notebook/Protocols/NativePAGEGel"