b'Choosing the right protein gel Bis-Tris chemistry vs. Tris-glycine chemistry Unlike traditional Tris-glycine gels, Invitrogen NuPAGE and Bolt gels are Bis-Tris HCIbuffered (pH 6.4) and have an operating pH of about The most widely used gel system for separating a broad range of proteins7.0. The neutral operating pH of the by SDS-PAGE is Tris-glycine gels (Laemmli system), comprising a stackingBis-Tris system provides the following gel component that helps focus the proteins into sharp bands at theadvantages over the Laemmli system:beginning of the electrophoretic run and the resolving gel component that separates the proteins based on size. This classic system uses a Longer shelf life of 812 months, discontinuous buffer system where the pH and ionic strength of the bufferdue to improved gel stabilityused for running the gel (Tris, pH 8.3) is different from the buffers used in the Improved protein stability during stacking gel (Tris, pH 6.8) and resolving gel (Tris, pH 8.8). The highly alkalineelectrophoresis at neutral pH, operating pH of the Laemmli system may cause band distortion, loss ofenabling sharper band resolution resolution, or artifact bands (Figure 4). and accurate results [2]The major causes of poor band resolution with the Laemmli system are:Complete reduction of disulfides under mild heating conditionsHydrolysis of polyacrylamide at the high pH of the resolving gel, resulting(70C for 10 minutes) and absence in a short shelf life of 8 weeks of cleavage of Asp-Pro bonds Chemical alterations such as deamination and alkylation of proteins Reduced state of the proteins due to the high pH of the resolving gel maintained during electrophoresisReoxidation of reduced disulfides from cysteine-containing proteins,and blotting of the proteins as the redox state of the gel is not constant when using Invitrogen NuPAGE Antioxidant or InvitrogenCleavage of Asp-Pro bonds of proteins when heated at 100C in LaemmliBolt Antioxidantsample buffer, pH 5.2Denaturing gel systemsA 1 2 13 24 35 46 57 68 79 810 9 10 B 1 2 1 3 2 43 5 4 6 5 76 87 98109 10Invitrogen NuPAGE Bis-Tris and Bolt Bis-Tris are well suited for separating a broad range of protein sizes. To separate high-abundance proteins, select our robust Invitrogen Novex Tris-Glycine gel chemistry. Tris-acetate gel chemistry, offered in Invitrogen NuPAGE Tris-Acetate gels, is Figure 4. Protein separation using (A) an Invitrogen Bolt Bis-Tris Plus gel andrecommended for the separation of (B) Bio-Rads Tris-glycine gel. high molecular weight proteins up to 500 kDa. Tricine gel chemistry is designed for the separation of low molecular weight proteins and peptides. Invitrogen Novex tricine gels provide increased resolution of proteins with molecular weights as low as 2.5 kDa.8'