Agarose Gel Electrophoresis of Proteoglycans
Dominik Haudenschild August 4, 1994 at CBRC
Radiolabel cell culture or organ culture in a medium containing 50 uCi/ml S35-H2SO4 overnight. Extract tissue or cells with 4M GdnHCl, 50mM NaOAc (pH 5.8) plus protease inhibitors at 4oC. Dialyze medium or tissue extracts in 0.5M NaOAc, 0.05 M Na2SO4 (pH 7.0) overnight at 4oC. These samples are ready for electrophoresis.
Materials
Running Buffer. 0.1 M Tris Cl pH 6.8; 2.5 mM Na
2SO4; 0.25% SDS.6x sample buffer. 60 % (
w/v) dextrose; 0.05% Bromophenol Blue; in running buffer.1. Prepare 1% agarose gel using Agarose Type II from Sigma in running buffer (0.1 M Tris Cl pH 6.8, 2.5mM Na
2SO4, 0.25% SDS). Pour the gel into a typical mini-gel apparatus for DNA eletrophoresis.2. Pre-run gel at 75 mA for 2hr at +4
oC (cold room).The running will start foaming. When loading samples, you can remove the some of the foam and replace with fresh (cold) running buffer.
3. Into 20µL of
35S labeled sample add 0.2µL of10% SDS (0.1% SDS).4. Incubate at 37
oC for 1 hr.5. Add 4µL of sample buffer to each sample.
6. Load samples onto gel and continue electrophoresis for 3 hrs at 4
oC.7. Fix gel with 50% methanol + 10% acetic acid for at least1 hr.
8. Discard fixative and wash gel in E
3Hance for 1 hr.Enhancer is very toxic and has a very noxious odor. Discard Enhancer safely. Ensure that the washing container will not react with the Enhancer and that odor will not leak.
9. Wash gel 15 min in water. The color of the gel will change to a white chalky color.
10. Cover gel with plastic wrap and dry gel for 1
1/2 hr with heat at 80oC.11. Expose the gel over night (or longer as necessary).
Adapted from Qian Chen by David Johnson 8/3/94