Ultrathin‐layer sodium dodecyl sulfate disc electrophoresis of proteins in the range from 10 to 220 kDa in homogeneous, low‐concentrated polyacrylamide gels

Abstract

This study describes an ultrathin-layer sodium dodecyl sulfate (SDS) disc electrophoresis in polyacrylamide gels of a thickness of only 150 microm. By use of 2-amino-2-methyl-1,3-propanediol/glycine instead of traditional Tris/HCl buffer in the resolving phase of the gel, proteins with a wide range of molecular sizes (10 kDa to over 220 kDa) are separated in unusually low-concentrated gels (4%T, 3.3%C). 2-Amino-2-methyl-1,3-propanediol in the resolving part of the gel contributes to stabilization of the pH value at 8.8, while glycine improves destacking as well as separation of small proteins from the bulk of stacked SDS. This method combines both the advantages of conventional slab-gel electrophoresis and capillary gel electrophoresis. It is easy to apply and well suited for all further miniaturization attempts.

SDS disc electrophoresis of proteins in homogeneous, low‐concentrated polyacrylamide gels

“…In the case of the glycine-based system, buffers of higher pK (2-amino-2-methyl-1,3-propanediol, AMPD) have to be used [12]. Unfortunately, AMPD in combination with glycine gives poor acrylamide polymerization.…”

“…Unfortunately, AMPD in combination with glycine gives poor acrylamide polymerization. For this reason the gels have to be polymerized in another buffer systems, washed, dried and rehydrated [12], a time-consuming endeavor.…”

“…Taken together, we propose here taurine and Tris as buffering components in low-concentrated, homogeneous polyacrylamide gels for the resolution of low-and highmolecular proteins because: (i) taurine in combination with Tris gives excellent acrylamide polymerization; this allows simplified handling and easy use of the gels as compared to AMPD/glycine system [12], (ii) the system has a wide separation interval and affords with a single-gel concentration of 4.5%, T, 2.6% C a linear resolution of proteins from 250 to 10 kDa, (iii) the taurine-based buffer system is compatible with all types of staining, including ammoniacal silver staining [13], (iv) the system works at relatively low pH (pH 8.6 in the resolving zone and standard pH of 6.8 in the stacking zone), and (v) the buffer system introduced here can be used in the horizontal method as described in this paper or in the vertical approach; in this case the resolving and stacking gel should be cast independently. Since low-concentrated polyacrylamide gels are fragile, we recommend to use gels covalently attached to glass or to plastic support.…”