Continuous Native Gel Electrophoresis
Ref: An electrophoretic procedure for detecting proteins that bind to actin monomers. (1989) Analytical Biochemistry 178, 32-37.
10X Running buffer
250mM Tris 30.3g/litre
1.94 M Glycine 145.6g/litre
2mM EGTA 10mls 0.2M/liter
pH generally 8.6-9.0, but can be varied. At this pH proteins are negatively charge and move towards the anode.
Gel mix for 10 gels:
10x running buffer 6ml
30% acrylamide 20ml-10%
0.2M EGTA 60ul-0.2mM gives cleaner results
10% APS 300ul
TEMED 40ul
Water to 60ml
Prepare the gel mix in a side arm flask: add the running buffer, the 30% acrylamide, water and the EGTA, mix well. Connect the side arm flask to a vacuum line, cover the flask with a rubber stopper and while swirling "degas" the gel solution for 5 min.
Add the polymerization catalysts to the gel solution: 40 ml TEMED and 300 ml ammonium persulfate (APS). After adding each of the catalysts, quickly swirl the flask to mix. Immediately after adding the catalysts, fill the glass plate sandwich with the gel solution using a pasteur pipet. Insert the comb until its sides touch the tops of the spacers. Rinse out the flask so that the remaining gel solution does no polymerize in it. Allow the gel to polymerize for about 45 min. Cover the top of the gel with plastic wrap (with the comb still in place) and store it in the cold box until the next use.
Samples:
prepare in 10% glycerol and load about 4ug per lane. Bromophenol Blue can be used as tracking dye, but do not add it to the protein samples, since we observed that it migrates as complex with some protein.
Notes:
As a rule use 10% gels as they can get very fragile, can go down to 7%.
Pre-run the gels at a constant 300V for 15min, then run for 1hr 15min at 300V.
Better results are obtained if the native gels are left to 'age' for several days at 40C
before use.
