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Team:Calgary/Notebook/Protocols/NativePAGEGel
From 2013.igem.org
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<p class="noIndent"><b>5% Native PAGE Gel (Separating)</b></p> | <p class="noIndent"><b>5% Native PAGE Gel (Separating)</b></p> | ||
<ul> | <ul> | ||
| - | <li> | + | <li>833 mL Acryl/Bis (40%)</li> |
| - | <li> | + | <li>4112 mL 0.375M Tris (pH 8.8)</li> |
<li>50 µL 10% APS </li> | <li>50 µL 10% APS </li> | ||
<li>5 µL TEMED </li> | <li>5 µL TEMED </li> | ||
| Line 22: | Line 22: | ||
<p class="noIndent"><b>4% Native PAGE Gel (Stacking)</b></p> | <p class="noIndent"><b>4% Native PAGE Gel (Stacking)</b></p> | ||
<ul> | <ul> | ||
| - | <li> | + | <li>400 mL Acryl/Bis (40%)</li> |
| - | <li> | + | <li>2567 mL 0.375M Tris (pH 8.8)</li> |
<li>30 µL 10% APS </li> | <li>30 µL 10% APS </li> | ||
<li>3 µL TEMED </li> | <li>3 µL TEMED </li> | ||
| Line 39: | Line 39: | ||
<li>25% Glycerol</li> | <li>25% Glycerol</li> | ||
<li>1% Bromophenol Blue </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> | </ul> | ||
| Line 55: | Line 62: | ||
<li>Load samples, <b>DO NOT HEAT SAMPLES</b> </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>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, | + | <li>When the gel is done running, remove gel and stain with either the protein stain or iron stain</li> |
| - | + | ||
| - | + | ||
</ol> | </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 Gel
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
- Put two gel glass pieces together in the correct orientation (should be small space between them) and clip into the gel apparatus
- Add about 5 mL of separating gel in between the two glass pieces (ensure it does not leak)
- Add a thin layer of isopropanol on top of the separating gel to form a smooth line
- Wait for the gel to solidify (~ 20 minutes)
- Rinse out the isopropanol with water
- Add about 2 mL of the stacking gel on top of the separating gel and add the gel comb
- Wait for the gel to solidify (~ 20 minutes)
- 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
- 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
- Load samples, DO NOT HEAT SAMPLES
- 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
- When the gel is done running, remove gel and stain with either the protein stain or iron stain
