Acid-Urea Gel Electrophoresis
Categorized: Protein Purification and Gel Electrophoresis
This technique comes from M. Selsted and has been modified slightly. The technique is used for separating small cationic peptides. Generally, a 15% gel is used although 12% gels are not uncommon.
Amount for 1 large gel or two small gels
|15%||X 2||12%||7.5% stacking gel|
|Urea||3.2 g||6.4 g||3.2 g||1.2 g|
|30:0.8 Acrylamide||5.33 ml||10.66 ml||4.25 ml||1 ml|
|Water||1.33 ml||2.66 ml||2.40 ml||1.5 ml|
|Solution B||1.33 ml||2.66 ml||1.33 ml||0.5 ml|
|10% APS||0.2 ml||0.4 ml||0.2 ml||150 ul|
|TEMED||30 µl||60 µl||30 µl||30 µl|
Solution B: 43% (v/v) acetic acid
- Set up gel plates as you would protein gels
- Mix all ingredients except TEMED. Make sure all urea is dissolved.
- Add TEMED and pour the gels. For mini-gels, a stacking gel is not necessary so you can put the comb directly into the top of the running gel. For large gels, a stacking gel is recommended, so overlay the running gel with iso butanol (as done with SDS PAGE gels) and pour the stacking gel later.
- Since the solubilization of the urea in the gel mix is an endothermic reaction, it is often useful to place the gel in the warm room to enhance polymerization. If this is not done, often the wells do not form properly.
- Once the gel is set, you can pre-electrophorese it. This is necessary to remove acetate ions from the gel. The running buffer is 5% acetic acid.
REMEMBER: RUN THESE GELS IN REVERSE POLARITY, IE. RUN THEM RED TO BLACK !!!!!
For large gels, pre-electrophorese at 150V (constant voltage) overnight, and for small gels, 150V for 1-2 hours is sufficient. During this time, the current will drop to a very low level. When the current no longer drops, pre-electrophoresis is complete, and the buffer can be discarded and replaced with new 5% acetic acid. Alternatively, at this point the gels can now be wrapped in Saran Wrap ( to prevent drying out) and placed in the cold room for up to 3 weeks (give or take). To cut down on the work, it is often prudent to make 4 mini-gels, pre-electrophorese them and store them. This way you have them ready when you want.
NOTE: Make sure that you are not hogging all the spacers and gel plates.
To prepare the sample solution:
Make 5 mls of 10 M urea (dissolve completely) and treat with Bio-Rad AG501-X6 mixed bed resin (ie. add some beads, mix gently for 15 min, let the beads settle and remove the urea). For every 900 µl of treated 10 M urea, add 50 µl of water and 50 µl acetic acid to end up with a 9M urea / 5% acetic acid solution. Add a pinch of methyl green to act as a tracking dye. Freeze this solution in small aliquots to prevent cyanate production.
The samples to be loaded on the gel should be in 5% acetic acid. Mix the sample 2:1 with sample solution and load into the wells. Run large gels at 250V and mini-gels at 120-150V until the methyl green has run off the bottom of the gel. Stain as usual.
NOTE: For blots use reverse polarity