Protein Staining Procedures
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TIP: All washes and incubations are done with constant, gentle shaking.
- Immerse gel in 50% ethanol/10% acetic acid for at least 1 hr.
- Soak in 5% ethanol/5% acetic acid overnight or for a minimum of 2 hours.
- Wash in diH 2 O for 1 hr.
- Add Gel-Code Blue Stain reagent (Pierce, #24592) for at least 3 hrs.
- Wash in diH 2 O twice, 15 min each.
- Rinse in diH 2 O for 1 hr.
- Gels can be stored at 4°C.
<center> <p> </p> <table> <tbody> <tr> <td> </td> <td> <a name="Silver">Solution A </a></td> <td> </td> <td> </td> <td> <p> <a name="Silver">Solution B </a></p><a name="Silver"> </a></td> <td> </td> <td> </td> <td> </td> </tr> <tr> <td> <p> <a name="Silver"> diH <sub>2</sub> O (ml) </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">10 N NaOH (ml) </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver"> NH <sub>4</sub> OH (ml) </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">Silver Nitrate (g) </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver"> diH <sub>2</sub> O (ml) </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">Final Volume (ml) </a></p><a name="Silver"> </a></td> </tr> <tr> <td> <p> <a name="Silver">21 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">0.2 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">1.3 </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">1.5 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">4 </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">100 </a></p><a name="Silver"> </a></td> </tr> <tr> <td> <p> <a name="Silver">31.5 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">0.3 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">1.95 </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">2.25 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">6 </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">150 </a></p><a name="Silver"> </a></td> </tr> <tr> <td> <p> <a name="Silver">105 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">1 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">6.5 </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">7.5 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">20 </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">500 </a></p><a name="Silver"> </a></td> </tr> <tr> <td> <p> <a name="Silver">210 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">2 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">13 </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">15 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">40 </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">1000 </a></p><a name="Silver"> </a></td> </tr> <tr> <td> <p> <a name="Silver">315 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">3 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">19.5 </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">22.5 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">60 </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">1500 </a></p><a name="Silver"> </a></td> </tr> <tr> <td> <p> <a name="Silver">420 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">4 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">26 </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">30 </a></p><a name="Silver"> </a></td> <td> <p> <a name="Silver">80 </a></p><a name="Silver"> </a></td> <td> </td> <td> <p> <a name="Silver">2000 </a></p><a name="Silver"> </a></td> </tr> </tbody> </table> </center>1. Ammonical silver nitrate staining solution:
Prepare solutions A and B as described below and, after mixing together, make to the final volume. Dissolve each solution WELL by vortexing vigorously.
Add solution B to solution A slowly. A brown precipitate will appear then disappear. If disappearance of the brown precipitate is slow, add drops of NH4OH. Bring to final volume.
TIP: diH 2 O should be high quality. Otherwise, silver nitrate will precipitate.2. Developing solution ( 0.1% formaldehyde, 0.01% citric acid):
100 µl formaldehyde and 0.01 g of citric acid /100 ml diH 2 O
3. Stop solution (2% acetic acid):
2 ml acetic acid plus 98 ml diH 2 O
