b'Several forms of PAGE exist, each providing different types ofA stacking gel is not necessary when using a gradient gel, as the information about the proteins under investigation. Nondenaturinggradient itself performs this function.PAGE, also called native PAGE, separates proteins based on their charge and size, preserving their conformation andLinear vs. gradient gelsactivity. Denaturing and reducing SDS-PAGE, the most widelyGels that have a single acrylamide percentage are referred to used electrophoresis technique, separates proteins by mass.as linear gels, and those with a range are referred to as gradient Two-dimensional (2D) PAGE separates proteins by isoelectricgels. The advantage of using a gradient gel is that it allows the point in the first dimension and by mass in the second dimension. separation of a broader range of proteins than does a linear gel.SDS-PAGE separates proteins primarily by mass. The ionic detergent sodium dodecyl sulfate (SDS) binds to the proteins inMini vs. midi protein gelsthe mixture shielding their respective charge and making themCommercial gels are available in two size formats: mini gels and all uniformly negatively charged. Thus, when a current is applied,midi gels. Both gels have similar run lengths, but midi gels are all SDS-bound proteins in a sample will migrate through the gelwider than mini gels, allowing midi gels to have more wells or toward the positively charged electrode. Proteins with less masslarger wells. The additional wells in the midi gels permit more travel more quickly through the gel than those with greater masssamples or large sample volumes to be loaded onto one gel.because of the sieving effect of the gel matrix.Once separated by electrophoresis, proteins can be detected in aHomemade vs. precast gelsgel with various stains, transferred onto a membrane for detectionTraditionally, researchers poured their own gels using standard by western blotting, or excised and extracted for analysis byrecipes that are widely available in protein method books. Most mass spectrometry. Protein gel electrophoresis is, therefore, alaboratories now depend on the convenience and consistency common step in many kinds of proteomic analyses. afforded by commercially available ready-to-use precast gels. Precast gels are available in a variety of percentages; these Gel matrix include difficult-to-pour gradient gels that provide excellent Acrylamide is the base component used in electrophoretic gels toresolution and separate proteins over the widest possible range separate proteins by size; when mixed with bisacrylamide in theof molecular weights. Technological innovations in buffer and gel presence of ammonium persulfate, it forms a crosslinked polymerpolymerization methods enable manufacturers to produce gels network. Ammonium persulfate acts as a polymerizing agentwith greater uniformity and longer shelf life than with traditional by producing free radicals, while TEMED (N,N,N,N-tetramethylequipment and methods. In addition, precast polyacrylamide gels ethylenediamine) is added to catalyze and speed up the reaction.obviate the need to work with the acrylamide monomera known The size of the pores created in the gel is inversely related to theneurotoxin and suspected carcinogen.amount of acrylamide and the bisacrylamide to acrylamide ratio.Protein standardsElectrophoresis gels are formulated in buffers that conduct an electrical current through the matrix. The solution is poured intoTo assess the relative molecular weight (MW) of a protein on a the thin space between two glass or plastic plates of an assemblygel, protein MW markers are run in the outer lanes of the gel. A called a cassette. Once the gel polymerizes, the cassette isstandard curve can be constructed using the distances migrated mounted into an apparatus so that opposite edges (top andby each marker protein. The distance migrated by the unknown bottom) are placed in contact with buffer chambers containingprotein is then plotted, and the molecular weight is interpolated a cathode and anode, respectively. When proteins are addedfrom the standard curve.in wells at the top edge and current is applied, the proteins areSeveral kinds of ready-to-use protein MW markers are drawn by the current through the matrix slab and separated by itsavailable that are labeled or prestained for different modes sieving properties. of detection. These are pre-reduced and, therefore, primarily To obtain optimal resolution of proteins, a stacking gel is castsuited for SDS-PAGE rather than native PAGE. MW markers over the top of the resolving gel. The stacking gel has a lowerare detectable via their specialized labels or by ordinary protein concentration of acrylamide, lower pH, and a different ionicstaining methods.content. This allows the proteins in a loaded sample to be concentrated into a tight band during the first few minutes of electrophoresis before entering the resolving portion of a gel. 42 Protein gel electrophoresis thermofisher.com/proteinbiology'