Isotachophoresis in capillary tubes of CSF proteins -- especially gammaglobulins.

Isotachophoresis in polyacrylamide gel tubes (PAG-ITP) and in capillary tubes (Tachophor, LKB) have previously been found by the authors, to be very promising high-separation methods for CSF and serum proteins, especially regarding the diagnosis of MS. PAG-ITP methods for analytical and preparative use have been described by the authors elsewhere, while in this paper proper cationic systems for ITP in capillary tubes for studying gammaglobulins in microliter amounts of CSF and serum are described, i.e. the albumin injection-clog problem is avoided and the preparation time can be forced. By using microdialysis of the CSF samples for desalting, with a technique easy to perform and with high reproducibility, microliter amounts of native CSF can be performed in less than half an hour. The method seems to be even more applicable for clinical and scientific use if the capillary isotachophoretic apparatus is connected to a synchronized equipment (LKB Tachophrac) with a cellulosa acetate strip onto which the separated fractions are ejected for further analysis by immunological tests. The analytical systems used have been especially directed to gammaglobulins in CSF and serum regarding further studies on demyelinating and infectious disorders of the nervous system.

[Protein makeup of rabbit myofibrils determined by a disc eletrophoretic method in the presence of sodium dodecyl sulfate]. / Belkovyi sostav miofibrill krolika, opredelennyi metodom disk-élektroforeza v prisutstvii dodetsilsul'fata natriia

The protein subunit composition of isolated myofibrils of rabbit skeletal muscle is studied by polyacrylamide gel disc-electrophoresis in the presense of sodium dodecyl sulfate (SDS). The method of disc-SDS-electrophoresis is described in detail. The electrophoretic patterns of SDS-solubilized myofibrils obtained by disc-SDS-electrophoresis and by SDS-electrophoresis in continuous buffer system according to Weber and Osborn are compared. The former results in a markedly improved resolution and allows to discover some additional protein components, the origin of these additional components being discussed. A standard curve is given for determination of polypeptide chain molecular weights by disc-SDS-electrophoresis.

An accurate slicer of disc acrylamide gels devised by simple modification of a density gradient fractionator.

A commercially available density gradient fractionator has been modified in a simple and inexpensive way to yield a device for accurately slicing disc acrylamide gels. Attractive features of this device include convenience of operation and the capacity to easily vary thickness of the gel slice.

Further development of an electroosmotic medium pump system for preparative disk gel electrophoresis.

A simple and practical 6.8-cm-diameter (36.30-cm(2) cross-sectional-area) preparative disk gel electrophoresis device, based on the design of M. Hayakawa et al. (Anal. Biochem. 288 (2001) 168), in which the elution buffer is driven by an electroosmotic buffer flow through the membrane into the elution chamber from the anode chamber was constructed. We have found that the dialysis membranes employed provide suitable flow rates for the elution buffer, similar to those of an earlier 3.6-cm-diameter device, resulting in the prevention of excess eluate dilution. The efficiency of this device was demonstrated by the fractionation of a bovine serum albumin (BSA) Cohn V fraction into monomer, dimer, and oligomer components using nondenaturing polyacrylamide gel electrophoresis (native-PAGE). The maximum protein concentration of the eluate achieved was 133 mg/ml of BSA monomer, which required a dilution of the eluate for subsequent analytical PAGE performance. As a practical example, the two-dimensional fractionation of soluble dipeptidyl peptidase IV (sDPP IV) from 50 ml fetal bovine serum (3.20 g protein) per gel is presented. The sDPP IV enzyme protein was recovered in a relatively short time, utilizing a 6.5% T native-PAGE and subsequential sodium dodecyl sulfate-PAGE system. This device enhances the possibility of continuous electrophoretic fractionation of complex protein mixtures on a preparative scale.

Extraction and isolation of individual ribosomal proteins from Escherichia coli.

We have described a new method for the quantitative separation of ribosomal proteins and ribosomal ribonucleic acid. A procedure for the preparation of individual ribosomal proteins by polyacrylamide gel electrophoresis is also described. By the use of gels with smaller pores, at least four of the electrophoretic components from the 30S ribosome can be split into additional protein fractions. By the methods described here, it is possible to isolate in high purity at least 15 different proteins from the 30S ribosome of Escherichia coli.

Electrophoretic analysis of ribosomal and viral ribonucleic acids with a simple technique for slicing low-concentration polyacrylamide gels.

The electrophoretic mobilities of ribosomal ribonucleic acids (RNA) from cultured mammalian (HeLa, Vero, MDBK), avian (chick embryo), and bacterial (Escherichia coli) cells, and RNA species extracted from selected viruses (Sindbis, polio, tobacco mosaic, Sendai) were compared, employing a simple, inexpensive technique for slicing low-concentration polyacrylamide gels. The procedure provides for rapid fractionation of gels used for characterization of RNA, incorporating extrusion and serial sectioning of frozen gels. Among 28S ribosomal RNA species, Vero and MDBK were indistinguishable, whereas HeLA RNA had a slightly lower mobility (higher apparent molecular weight) and chick RNA had a higher mobility (lower apparent molecular weight). The 18S ribosomal RNA species of the three mammalian sources were indistinguishable, but chick 18S RNA had a slightly lower apparent molecular weight. The inverse relation between mobility and log-molecular weight among the ribosomal and viral RNA species, though not highly precise, demonstrates the applicability of the technique to the study of molecular weights of viral RNA species.