Further development of a versatile system for preparative electrophoresis in acrylamide gel

Modifications to a preparative scale acrylamide gel electrophoresis apparatus enable better resolution to be obtained by reducing band distortion due to heating effects. A scaled-up (14-cm diameter) version of the apparatus is described, and also a system for automatic recycling of material only partially purified after one passage through the gel.     

Continuous preparative gel electrophoresis

An apparatus is described for continuous electrophoresis in polyacrylamide gel. Experiments may be run for 10 days or longer. Protein loads may be 1 g per day or more, and there are no obvious obstacles to scaling up. A revised classification of electrophoretic processes is required.     

A method for measuring activities of acid phosphatases separated by acrylamide gel electrophoresis

Methods for measuring the activities of acid phosphatases with the same substrate, alpha-naphthyl acid phosphate, both before and after acrylamide gel electrophoresis are described. The gel assay, which involves elution of the precipitated dye complex, can be used to measure the length of linear reaction rates of the enzymes separated in gels. It is possible with the use of these methods both to determine the effect of electrophoresis on the activity of acid phosphatases and to correlate the amount of precipitated dye with the area under the peak on a densitometric tracing of the same band.     

Purification of RNAase II by preparative polyacrylamide gel electrophoresis

Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease is activated by K⁺ and Mg²⁺ and hydrolyses poly(A) to 5′-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913–922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacrylamide gels gives a single band. A molecular weight of 76 000 ± 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 ± 2000, 33 000 ± 2000, and 26 000 ± 1000. The enzyme thus appears to consist of three dissimilar subunits.     

Purification of RNAase II by preparative polyacrylamide gel electrophoresis.

Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease is activated by K+ and Mg2+ and hydrolyses poly(A) to 5'-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913--922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacrylamide gels gives a single band. A molecular weight of 76 000 +/- 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 +/- 2000, 33 000 +/- 2000, and 26 000 +/- 1000. The enzyme thus appears to consist of three dissimilar subunits.     

High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis

Separation of milligram amounts of heparin oligosaccharides ranging in degree of polymerization from 4 to 32 is achieved within 6 h using continuous elution polyacrylamide gel electrophoresis (CE-PAGE) on commercially available equipment. The purity and structural integrity of CE-PAGE-separated oligosaccharides are confirmed by strong anion exchange high-pressure liquid chromatography, electrospray ionization Fourier transform mass spectrometry, and two-dimensional nuclear magnetic resonance spectroscopy. The described method is straightforward and time-efficient, affording size-homogeneous oligosaccharides that can be used in sequencing, protein binding, and other structure-function relationship studies.     

Apparatus and method for preparative gel electrophoresis

A new apparatus for preparative gel electrophoresis with continuous elution which includes a miniaturized electrode and elution chamber system is described. The design provides high resolution, high yield, applicability for small and large amounts of peptide material, and easy operation. Furthermore, the apparatus enables a very accurate gel column or gel gradient to be formed. A method for preparative gel electrophoresis in sodium dodecyl sulfate which allows the purification of peptides and proteins without concurrently modifying tryptophane residues or blocking N-terminal α-amino groups is also described.     

The identification of tRNA isoacceptors fractionated by polyacrylamide gel electrophoresis

A method is described by which specific tRNA isoacceptors may be identified in small amounts of bulk tRNA. The strategy relies on the retention of aminoacyl-tRNA by CNBr-Sepharose through covalent coupling of the alpha-NH2 group of the amino acid to the matrix. After removing unbound material by thorough washing, the bound specific isoacceptors are released by cleavage of the labile aminoacyl-tRNA ester bond through mild alkaline treatment. The product is analysed by two-dimensional gel electrophoresis and the spots obtained may be correlated with the pattern from bulk tRNA. Optimum sensitivity is achieved by combining the method with the recently introduced silver staining technique (Igloi, G.L. (1983) Anal. Biochem. 134, 184-188) for tRNA.