b'The acrylamide matrix Electrophoresis conditionsLinear vs. gradient gels The separation of proteins is dependent on the Gels that have a single acrylamide percentage are referredelectrophoresis conditions used, some of which are to as linear gels, and those with a range are referred to asdescribed below. gradient gels. The advantage of using a gradient gel is that it allows the separation of a broader range of proteins thanDenaturing conditions (SDS-PAGE)does a linear gel. Electrophoresis is performed under denaturing conditions using an anionic detergent such as sodium dodecyl sulfate Continuous vs. discontinuous gels(SDS). SDS denatures and unfolds the protein by wrapping Researchers occasionally refer to gels as continuous oraround the hydrophobic portions. SDS binds at a ratio of discontinuous. A continuous gel is a gel that has been~1.4 g SDS per gram of protein. The resultant SDSprotein formed from a single acrylamide solution in the entirecomplexes are highly negatively charged and are resolved gel cassette. A discontinuous gel is formed from twoin the gel based on their size.acrylamide solutions: a small, low-percentage stacking gel where the protein wells reside, and a larger portion of gelNondenaturing conditions (native PAGE)that separates the proteins. In the traditional Tris-glycineElectrophoresis is performed under nondenaturing (native) protein gel system, the proteins are stacked in the stackingconditions using buffer systems that maintain the native gel between the highly mobile leading chloride ions (inprotein conformation, subunit interaction, and biological the gel buffer) and the slower, trailing glycine ions (in theactivity. During native electrophoresis, proteins are running buffer). The reason for using the stacking gel isseparated based on their charge-to-mass ratios.to improve the resolution of the bands in the gel. These stacked protein bands undergo sieving once they reach theReducing conditionsseparating gel. Electrophoresis is performed under reducing conditions using reducing agents such as dithiothreitol (DTT), Mini vs. midi protein gels -mercaptoethanol (-ME), or tris(2-carboxyethyl)Commercial gels are available in two size formats: mini gelsphosphine (TCEP). The reducing agents completely unfold and midi gels. Both gels have similar run lengths, but midithe denatured proteins into their subunits by cleaving the gels are wider than mini gels, allowing midi gels to havedisulfide bonds between cysteine residues. more wells or larger wells. The additional wells in the midi gels permit more samples or large sample volumes to be loaded onto one gel.Buffer systemsElectrophoresis is performed using continuous or discontinuous buffer systems. A continuous buffer system utilizes only one buffer in the gel and running buffer. A discontinuous buffer system utilizes a different gel buffer and running buffer [1]. This system may also use two gel layers of different pore sizes and different buffer composition (the stacking and separating gel). Electrophoresis using a discontinuous buffer system results in concentration of the sample in the stacking gel and higher resolution as a result.5'