J Biol Chem 250(10): 4007-4021

High Resolution Two-Dimensional Electrophoresis of Proteins<sup><a href="#FN2" rid="FN2" class=" fn">*</a></sup>

Patrick H. O'Farrell, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80302;

^{Present address, Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143.}

Summary

A technique has been developed for the separation of proteins by two-dimensional polyacrylamide gel electrophoresis. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources. Proteins are separated according to isoelectric point by isoelectric focusing in the first dimension, and according to molecular weight by sodium dodecyl sulfate electrophoresis in the second dimension. Since these two parameters are unrelated, it is possible to obtain an almost uniform distribution of protein spots across a two-dimensional gel. This technique has resolved 1100 different components from Escherichia coli and should be capable of resolving a maximum of 5000 proteins. A protein containing as little as one disintegration per min of either ^{C or}^{S can be detected by autoradiography. A protein which constitutes 10}^{to 10}^{% of the total protein can be detected and quantified by autoradiography. The reproducibility of the separation is sufficient to permit each spot on one separation to be matched with a spot on a different separation. This technique provides a method for estimation (at the described sensitivities) of the number of proteins made by any biological system. This system can resolve proteins differing in a single charge and consequently can be used in the analysis of}in vivo modifications resulting in a change in charge. Proteins whose charge is changed by missense mutations can be identified. A detailed description of the methods as well as the characteristics of this system are presented.

Summary

Polyacrylamide gel electrophoresis has been extremely useful as an analytical tool for the separation and quantification of protein species from complex mixtures. In bacteriophage, where a major proportion of the viral proteins can be resolved, the combination of genetics and analysis by electrophoresis has yielded significant information concerning gene regulation and phage morphogenesis (for example, Refs. 1–5). In systems more complex than bacteriophage the response to pleiotropic effectors, developmental transitions or mutation cannot be adequately analyzed by means of any one-dimensional technique for protein separation unless the analysis of a very restricted subset of the total proteins is acceptable. In order to provide a suitable technique for a more extensive analysis of complex systems, I have developed this technique for the separation of total protein.

In terms of the number of components resolved, previous techniques for two-dimensional electrophoretic protein separation (for example, Refs. 6–11) were not significantly better than one-dimensional separation. Only the procedure of Kaltschmidt and Wittman (12) has been widely used. Although this technique is of limited resolution and applicability, it has been used as the basis for many investigations of ribosomal assembly and structure (for example, Refs. 13–16).

To optimize separation, each dimension must separate proteins according to independent parameters. Otherwise proteins will be distributed across a diagonal rather than across the entire surface of the gel. Isoelectric focusing and a discontinuous SDS¹ gel system (1) were chosen because of the high resolution of each system and because they separate proteins according to different properties. Since the procedure is intended for analysis of total proteins, denaturation agents which solubilize most proteins are present during electrophoresis in both dimensions. This system permits simultaneous determination of molecular weights and approximate isoelectric points of proteins. More than 1000 proteins can be resolved and a protein species representing as little as 10^{to 10}^{of 1% of the total protein can be detected and quantified. Since the position of a spot changes detectably if a single charge is altered, some missense mutations can be detected.}

Footnotes

^{This work was supported by National Science Foundation Grant GB-37949.}

^{The abbreviations used are: SDS, sodium dodecyl sulfate; TEMED,}N,N,N′,N′-tetramethylethylenediamine; IF, isoelectric focusing.

^{All autoradiograms of two-dimensional gels were photographed with a metric ruler along two edges of the autoradiogram. These rulers establish a coordinate system which is used to give spot positions. The verticle scale is given in units from top to bottom. The horizontal scale is given in units from left to right. The coordinates are given as horizontal × vertical.}

^{P. H. O'Farrell, in preparation.}

Footnotes

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