Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

Abstract

An inexpensive Plexiglas apparatus which allows a simple and rapid preparation of horizontal polyacrylamide gels of different dimensions for different purposes, is described. Preparation of such gels is as easy and rapid as agarose gel preparation, and polymerized polyacrylamide gels are used to fractionate proteins or small DNA fragments using a common horizontal electrophoretic tank. This apparatus was used to electrophoretically fractionate proteins or DNA for immuno-blot analyses, particularly in the study of the allergenic response to Parietaria judaica pollen in senescence, for Southern-blot hybridizations and in the study of DNA polymorphisms.

Figure 1

Schematic drawing of the apparatus A). Schematic section of the teeth and border frame B). The acrylamide solution was poured through one of the slots to fill the volume between the milled Plexiglas surface and the silanized glass plate.

Figure 2

Low and high molecular mass marker proteins (Biorad, USA) were electrophoretically fractionated on a 10% (left) and 7% (right) H-SDS-PA gel, in 1X TBE, 0.1% SDS buffer until the dye ran off the gel. Gels were stained with coomassie brilliant blue as described in Materials and Methods.

Figure 3

A) 1, 2 and 4 μg of 16HBE cell extracts (lanes 1, 2, and 3, respectively) were fractionated on a 10% H-SDS-PA gel and probed with mouse anti HSP27 monoclonal antibody as described in Materials and Methods. B) 0.5, 2.5 and 12.5 μg of Parj2 E. Coli lysate (lanes 4, 5, and 6, respectively) were fractionated on a 10% H-SDS-PA gel and probed with pooled allergic human sera, as described in Materials and Methods.

Figure 4

Different DNA molecular mass markers, 100 bp ladder (BioLabs, U.S.A.) (lane 1), Marker V (Roche, USA) (lane 2), 50 bp ladder (Amersham, USA) (lane 3), were electrophoretically fractionated on 5% or 10% polyacrylamide gel with or without SDS, until the dye ran off the gel. Gels were stained with ethidium bromide as described in Materials and Methods.

Figure 5

Plates of different breadth (A), and different positioning of the glass plate (B) allow to obtain gels of different number of wells and of different length, respectively.