An improved horizontal slab gel electrophoresis apparatus for DNA separation

An improved horizontal slab gel electrophoresis apparatus was developed for the separation of DNA restriction fragments. The apparatus was designed for both analytical and preparative runs. The use of agarose or polyacrylamide wicks rather than paper wicks simplifies the use of and increases the capabilities of horizontal slab gel electrophoresis.     

Polyacrylamide gel electrophoresis on micro slabs

An electrophoretic technique using micro polyacrylamide flat gels is described and its usefulness demonstrated. The gels are vertically cast and electrophoresed in slab form (75 × 18 × 0.75 mm) in closed thin glass cells (cuvets) made from detachable microscope slides. The main features of the method are: requirement of small sample quantities (0.1–20 μg contained in <1–5 μl solution), simultaneous analysis of several samples in a single gel, relatively brief running periods, easy removal of the gel for rapid staining due to the two-piece gel mold, little pattern diffusion, convenient optical evaluation, drying, autoradiography and other contact print methods, and easy application of immunodiffusion techniques. Continuous gradient gels can be prepared. The advantage and complications of the technique are discussed and certain applications in biochemistry, clinical chemistry, and medicine are pointed out.     

Polyacrylamide gel electrophoresis of myelin proteins: a caution.

Some variables involved in the preparation of rat brain myelin proteins for polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate were studied. Under mild conditions of solubilization the resultant gel patterns were relatively insensitive to the β-mercaptoethanol (ME) concentration in the protein solvent used for solubilization of myelin proteins. However, if the samples were boiled in the presence of ME (a standard procedure for disruption of metastable aggregates of membrane proteins), a major myelin protein, proteolipid protein, as well as some minor proteins were preferentially excluded from the gel. This effect was proportional to the ME concentration.     

Polyacrylamide gel electrophoresis: recovery of non-stained and stained proteins from gel slices

Following electrophoresis or isoelectric focusing in gels of polyacrylamide the protein band of interest is cut out and placed above a sucrose gradient column, containing carrier ampholytes (Pharmalyte). By electrophoresis, isoelectric focusing or displacement electrophoresis the proteins migrate out of the gel slice and into the isoelectric focusing column for concentration and further purification. From this column, the proteins can be withdrawn and their isoelectric points determined. Even after staining with Coomassie Brilliant Blue at least some proteins can be recovered by this technique and used for further analyses, for instance amino acid determinations. The focusing in a pH gradient by carrier ampholytes can be replaced by an electrophoresis in a conductivity gradient column. However, in comparison with isoelectric focusing, this concentration technique has the drawback of not permitting further purification of the eluted protein.     

Two-dimensional polyacrylamide gel electrophoresis: an improved method for ribosomal proteins

A two-dimensional electrophoresis system for analysis of ribosomal proteins with several advantages over previous systems is described. The general features of this system are: (1) first-dimension separation on the basis of mobility at pH 5.0 in 8 m urea and 4% polyacrylamide; (2) second-dimension separation on the basis of molecular weight using dodecyl sulfate detergent; (3) rapid electrophoretic shift between first- and second-dimension separation conditions; (4) high resolution separation can be obtained on 10-cm² slabs with proteins from approximately 100 μg of ribosomal subunits; (5) capacity for handling up to 10 samples at a time, with electrophoresis complete within about 10 hr; and (6) the apparatus is relatively simple and inexpensive to construct and use.     

Modification of commerical and custom-built slab gel electrophoresis units for two-dimensional polyacrylamide gel electrophoresis

Many commercial and custom-built slab gel electrophoresis units can be modified to function as two-dimensional polyacrylamide gel electrophoresis units with the insertion of Plexiglas adapters. These adapters can be made for about $50 a pair and can be used for either temporary or permanent modification of the slab gel units. The physical dimensions of the adapters can be varied to permit great flexibility in the diameter of cylinder gels and the thickness of slab gels that can be run together. For example, proteins from 6-mm cylinder gels can be easily separated on 1-mm slab gels, which can then be dried for autoradiography.     

A method for horizontal polyacrylamide slab gel electrophoresis

We present a simplified method of preparation of polyacrylamide gels which is totally analogous to the procedure now widely used to pour and run horizontal agarose gels. The acrylamide is poured into an open air gel mold consisting of a glass plate with a masking tape border and a comb. It is subsequently run in a submarine horizontal electrophoresis apparatus. The electrophoretic mobility and resolution of DNA fragments obtained in such gels are identical to results obtained with gels poured and run in the vertical configuration. Numerous advantages of horizontal polyacrylamide gel electrophoresis are discussed.     

Highly simplified analytical or preparative slab gel electrophoresis

A simple, rapid, and efficient procedure has been developed for performing analytical or preparative slab gel electrophoresis using only common laboratory materials which can be assembled without special tools or equipment. From one to four gel slabs of variable size can be made between glass plates embedded inside a watertight, supple plastic bag which is then used as the upper electrode buffer chamber. This technique has been applied, with different electrophoretic systems, for both the analysis and isolation of serum proteins and rat liver histones.     

Polyacrylamide gel electrophoresis of visna virus polypeptides isolated by agarose gel chromatography.

The proteins of visna are separated into nine major peaks by agarose gel chromatography in 6 M guanidine hydrochloride (GuHCl). The polypeptides in eack peak were isolated by acid precipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns of SDS-PAGE show that the excluded material from the GuHCl column contains an aggregate of 10 non-glycosylated polypeptides. It is shown that this aggregate represents virus substructures that are not completely solubilized by GuHCl. Two glycoproteins, gp175 and gp115, were isolated from the column eluate. The major glycoprotein gp115 was coeluted with P90, P68, and P61 in GuHCl 4. Each of the four major peaks (GuHCl 5 to 8) contains more than one nonglycosylated polypeptide. However, a small polypeptide, P12, can be isolated in a homogeneous form in the last peak, GuHCl 9. Analysis of the virus proteins (100 microgram) by SDS-PAGE shows that 20 radioactive bands can be recognized. During fractionation of the protein on agarose gel columns followed by analysis with SDS-PAGE, a number of minor polypeptides that were not detected before became clearly recognizable. Thus, the combined use of column chromatography and SDS-PAGE shows that visna virus is composed of 25 proteins.