1x Running Buffer: 25 mM Tris-HCl 200 mM Glycine 0.1% (w/v) SDS (Approximately vol. of less than 1 liter is needed depending on the type of your electrophoresis system.) SDS PAGE Protocol: 1. Make the separating gel: Set the casting frames (clamp two glass plates in the casting frames) on the casting stands. Prepare the gel solution (as described above) in a separate small beaker. Swirl the solution gently but thoroughly.

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SDS-PAGE, with full name of sodium dodecyl sulfate polyacrylamide gel electrophores, is the most widely used technique to separate proteins from complicated samples of mixture, plays key roles in molecular biology and wide range of subfield of biological research. Being present a electricity, proteins migerate towards the negative anode inside the poly-acrylamide gel under denaturing conditions. In SDS-PAGE, the detergent SDS and a heating step determine that the electrophoretic mobility of a single kind of protein is only affected by its molecular weight in the porous acrylamide gel.

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4. Load prepared samples into wells and make sure not to overflow. Don't forget loading protein marker into the first lane. Then cover the top and connect the anodes.

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Make sure your target protein dissolved in the liquid phase, and no inappropriate ingredients present (e.g. guanidine hydrochloride can interact with SDS and cause precipitation) Generally, to treat your unprepared sample, you can use sonicator, lysis buffer or both to sufficiently make your target protein released, and centrifuge to make supernatant and pellet separated.

6. As for the total running time, stop SDS-PAGE running when the downmost sign of the protein marker (if no visible sign, inquire the manufacturer) almost reaches the foot line of the glass plate. Generally, about 1 hour for a 120V voltage and a 12% separating gel. For a separating gel posessing higher percentage of acylamide, the time will be longer.

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2. Make sure a complete gelation of the stacking gel and take out the comb. Take the glass plates out of the casting frame and set them in the cell buffer dam. Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow untill the buffer surface reaches the required level in the outer chamber.

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The SDS PAGE gel in a single electrophoresis run can be divided into stacking gel and separating gel. Stacking gel (acrylamide 5%) is poured on top of the separating gel (after solidification) and a gel comb is inserted in the stacking gel. The acrylamide percentage in SDS PAGE gel depends on the size of the target protein in the sample. (details shown below)

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An intact SDS PAGE electrophoresis system should include: a tank, lid with power cables, electrode assembly, cell buffer dam, casting stands, casting frames, combs(usually 10-well or 15-well), and glass plates (thickness 0.75mm or 1.0mm or 1.5mm). (Bio-rad brand one is recommended)

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