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Team:Brown/Notebook Protocols/sdspage
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Latest revision as of 03:01, 22 October 2009
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
The standard method to separate proteins in a complex mixture according to mass (formally, according to their ability to migrate through the gel). The proteins are denatured by SDS and boiling, and must be stained with dyes in order to see, yet the gels can also be blotted to identify specific proteins by antibodies, or lectins. “Bands” of individual proteins can be excised and analyzed by mass spectrometry or, eluted and used to make antibodies.
Components:
- 1) Gel box, electrode buffer, power supply, pre-pored gels.
- 2) Protein molecular weight marker (not 1kb ladder), 2x sample buffer, samples
- The protein MW marker is in sample buffer and ready to load. Simply thaw, and load 10 μl.
- Electrode buffer: per 3 liters – 43.2 grams of Glycine, 9.09 grams of TRIS base, and 15 mls of 20% SDS, pH8.0
- 2x Sample Buffer: 5mM TRIS, pH 6.8; 2% SDS; 5mM EDTA; 20% sucrose; and a pinch of Bromphenol blue
Sample preparation:
- 1) For protein in aqueous solution, add equal volume of 2x sample buffer and treat at 95oC for 5 minutes. Spin for 1 minute at high speed in the microfuge and load supernatant (being careful not to disturb pellet).
- 2) For bacteria: pellet (spin at high speed in the microfuge for 30 seconds) appropriate amount of cells (e.g. 100ul of liquid culture). Resuspend cells in dH2O (e.g. 20 ul) by vortexing vigorously. Add equal volume of 2x sample buffer and incubate at 95oC for 5 minutes with intermittent vigorous vortexing to lyse the cells. Spin for 1 minute at high speed in the microfuge and load supernatant (being careful not to disturb pellet).
- Running gels: Assemble the gels in the gel box. For one gel use a blank to form a reservoir. Remove gel comb (and perhaps label wells for easier loading), add buffer to the two chambers, wash wells with pipette, and load samples (about 10-20 μl, being careful to not bump the gel box). Run 125V for 90 minutes. Turn off power supply before disassembling the gel box.
- Staining gels: Carefully (‘cause it will stain you, your clothes, shoes, teeth, fingers etc) place the gel in Coomassie Blue (or similar choice) for >4 hours on a rotary shaker (not a rocker). Destain to taste.
