Native Gel TBE

Native PAGE separations are run in non-denaturing conditions. Detergents are used only to the extent that they are necessary to lyse lipid membranes in the cell. Complexes remain—for the most part—associated and folded as they would be in the cell. One downside, however, is that complexes may not separate cleanly or predictably, since they cannot move through the polyacrylamide gel as quickly as individual, denatured proteins.

Contents

1. Recipe (6-8 gels)
2. Procedure
3. Running and Imaging

1. Recipe (6-8 gels)

Resolving Layer	5%	6%	8 %	10%	12%	15%
H2O (ml)	38.25	37	34.5	32	30	25.75
5X TBE (ml)	5	5	5	5	5	5
40% Acryl (ml)	6.25	7.5	10.0	12.5	15.0	18.75
10% APS (ml)	0.5	0.5	0.5	0.5	0.5	0.5
TEMED (ul)	20	20	20	20	20	20

2. Procedure

1. Prepare the glass plates, combs and casting station before you combine any chemicals. Read the manual to learn how to use the casting system.
2. Adding a 1000 mL pipette tip between the casting station clip will help keep the gel from leaking (it can still happen though).
3. Combine the H2O, 5x TBE, and Acrylamide in a 50 mL disposable falcon tube and invert several times or vortex to mix thoroughly.
4. Add APS and TEMED, invert several times and use a Pipette-Aid to pour all the way to the top. Polymerization initiates as soon as you add APS & TEMED so you only have a few minutes before the gel solidifies.
5. Insert the comb and let the gel polymerize.
6. Wait ~15 minutes for the gel to solidify.
7. Check for leakage by lifting the well comb and determine whether it is still viable.
8. Wrap the gel plates with a damp paper towel and place the gel stack into a plastic bag or wrapped by cling film.
9. Label the bag or cling film with initials, date, and gel type.

3. Running and Imaging

1. Run TBE gel in 0.5X TBE buffer http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8458.
2. Pre-run the gel for 10 minutes at 80V. This ensures acrylamide won’t react with protein samples (for our lab). It also heats the gel up so your DNA stays denatured while running the sample.
3. Run the gel at 80V for 45-60 minutes. Be sure to watch the solvent front.
4. Remove the gel from the casing and soak for 15-20 minutes in 0.5% Ethidium bromide. Gel is gooey, please be careful.
5. Remove from Ethidium Bromide and rinse with water 5 times. Let soak in water for 15 minutes.
6. Image the gel as you would a agarose gel.