BASICNATIVE GEL Protocol
来自 : 蚂蚁淘
Stock Solutions
1) 1.5M TrisHCl pH 8.9 - Keep RT. 2) 30% Acrylamide 0.8% Methylene bis Acrylamide. Keep 4°C. 3) 0.5M TrisHCl pH 6.8 - Keep RT. 4) 10% Ammonium Persulfate (APS). Keep 4°C less than 1 month. Running Buffer x1
| Trizma | 0.05M | 1.21gr |
| Glycine | 0.38M | 5.32gr |
| H2O up to | | 200ml |
| Adjust pH to | | 8.9 |
Keep at RT
Dissolving Buffer x5
| Glycerol | 5ml |
| H2O | 2.7ml |
| 0.5M TrisHCl pH 6.8 | 2.13ml |
| Bromo Phenol Blue | traces |
Keep in aliquots of 1ml at -20C
Separating Gel
| % Acrylamide | 10% | 10% | 12% | 12% | 15% |
| Number of Minigels | 5 | 8 | 5 | 8 | 5 |
| 1.5M TrisHCl pH 8.9 | 7.0 ml | 10.5 ml | 7.0 ml | 10.5 ml | 7.0 ml |
| 30% Acrylamide 0.8% Methylene bis Acrylamide | 9.3 ml | 13.9 ml | 11.3 ml | 16.9 ml | 13.9 ml |
| H2O | 12.3 ml | 18.4 ml | 9.3 ml | 13.9 ml | 6.3 ml |
| 10% APS | 100 ul | 150 ul | 100 ul | 150 ul | 100 ul |
| TEMED | 23 ul | 35 ul | 23 ul | 35 ul | 23 ul |
Add TEMED and APS at the end. Gently swirl the flask to mix, being careful not to generate bubbles. Pipette the solution to a level of 4cm of the top. Add 0.3ml of n-buthanol. A very sharp liquid interface will be visible within 10-20min. Let polymerize the gel for another hour at least. Rinse the surface of the gel with H2O before pouring the stacking gel.
Stacking Gel
| Number of Minigels | 2 | 5 | 8 |
| 0.5M TrisHCl pH 6.8 | 2.5 ml | 4.0 ml | 5.2 ml |
| 30% Acrylamide 0.8% Methylene bis Acrylamide | 1.0 ml | 1.5 ml | 2.0 ml |
| H2O | 6.4 ml | 9.6 ml | 12.8 ml |
| 10% APS | 100 ul | 150 ul | 200 ul |
| TEMED | 10 ul | 15 ul | 20 ul |
Fill each sandwich with stacking gel solution and insert a comb into each place taking care not to trap any bubbles bellow the teeth. The gel should fully polymerized after 1hour. Cover gel with a wrap nylon. Keep gel at 4C no more than 2 days; use only fresh gels. Running conditions: 30mA / 250V max.
Sample Preparation
Prior to adding the sample buffer, keep samples at 0°C. Add the sample buffer (RT) to the sample (still on ice), and load immediately. For a gel thickness of 0.75mm and 15 wells applied 0.5 to 5ug for Coomasie Blue stain and 10 to 100-fold less protein for silver stain. If you do not know the electrophoretic pattern of your protein, load same sample in parallel wells at different times during the run. Use only purified or paritally purified material.
Staining Solution
| Methanol CP | 500ml | 50% |
| Acetic Acid CP | 100ml | 10% |
| H2O | 400ml | |
| Coomasie Brilliant Blue R | 2.5gr | 0.25% |
Keep flask on dark at RT
Destaining Solution
| Methanol CP | 150ml | 15% |
| Acetic Acid CP | 100ml | 10% |
| H2O | 750ml | |
Keep flask on dark at RT
Stain overnight at RT or put gel with staining solution few seconds in microwave on the high position, and then shake for another 15min at RT. Wash with water 2-3 times and distain by several changes of destaining solution in the presence of a sponge.
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