Gel electrophoresis

About: Gel electrophoresis is a research topic. Over the lifetime, 26026 publications have been published within this topic receiving 1113565 citations.

Evaluation of Non-Formalin Tissue Fixation for Molecular Profiling Studies

Abstract: Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).

Rapid separation and purification of oligonucleotides by high-performance capillary gel electrophoresis

Abstract: Picomole amounts of oligodeoxynucleotides [polydeoxyadenylic acids, (dA)40-60] were baseline resolved and analyzed in less than 8 min by high-performance capillary electrophoresis with polyacrylamide gels. In addition, fast analysis of a crude 70-mer oligodeoxynucleotide and a slab gel-purified 99-mer oligodeoxynucleotide was accomplished, demonstrating the ability of high-performance capillary electrophoresis to characterize rapidly synthesized oligonucleotides. Besides analytical separations, 800 ng of a primer (20-mer) was isolated in less than 20 min. The purified species was collected in water and subsequently used as a probe in a standard dot-blot analysis. The use of high-performance capillary electrophoresis for the analysis and purification of a variety of biopolymers is simple, rapid, and has the potential for automation.

Acrylamide gel electrophoresis.

Abstract: This symposium on gel electrophoresis includes in its program papers on starch gel electrophoresis, agar gel electrophoresis, acrylamide gel electrophoresis, and the specialized technique of disc electrophoresis. It is appropriate to consider what characteristic distinguishes gel electrophoresis from other techniques and makes it especially useful in the analysis of protein and other complex mixtures. In my opinion it is the “sieve” or “molecular filtration” effect, first described by 0. Smithies,’ that is the essential characteristic of gel electrophoresis. The combination of electrophoretic effects and molecular filtration effects produces new and startling resolutions that cannot be obtained by other electrophoretic procedures. This combination‘of effects is not present in agar gel electrophoresis. Therefore agar techniques should logically be classified with paper electrophoresis techniques, which they most closely resemble with respect to the type of pattern produced. The specialized technique of “disc electrophoresis” intrinsically applies a discontinuous buffer system to sharpen the electrophoretic zones of the individual components of a mixture but does not in itself make use of the special feature of gel electrophoresis, which is the molecular filtration effect. Immunoelectrophoresis is also a specialized technique, namely a technique of detecting and identifying the zones separated by electrophoresis. It could equally well be applied to a paper electrophoresis type of separation, as it is in agar gel, or to a molecular filtration type of separation as is produced by starch gel or acrylamide gel. These considerations provide a rational basis for focusing our attention on the special problems that have arisen in gel electrophoresis (defined as electrophoresis technique combining both electrophoretic and molecular filtration effects) and the special results available from it. It is, however, beyond the scope of this paper to consider the theoretical problems of electrophoresis as modified by the filtration effect. Instead, this paper is limited to the applications of gel electrophoresis and to the problems we have encountered with it since our first publication in 1959.2 It is evident from a quick perusal of the program that we are devoting most of our attention to the disc electrophoresis technique of Ornstein. Since, apparently, I am the only speaker to describe the vertical slab technique I must begin with a description of our procedure and apparatus. Our procedure starts with a vertical gel slab prepared in a specially designed cell (FIGURE combining gel mold, buffer reservoirs and electrodes, and cooling plates all in one unit. We prefer a flat slab in contrast to a cylindrical gel for several reasons. First, the flat slab provides maximum surface area for cooling the gel. Second, the resulting patterns are easier to quantitate in standard recording densitometers. Third, a large number of samples can be processed in a single gel, making the technique easier to carry out in the laboratory and facilitating the direct intercomparison of specimens processed under identical conditions all the way through to the final densitometer recording. Fourth, and most important, the flat slab permits the application of two-dimensional techniques, which is impossible in the vertical tube apparatus. We have never observed any difficulties with stress relief or separation from