A convenient apparatus and tracking dye for anaerobic analytical polyacrylamide-gel electrophoresis

An anaerobic modification of conventional polyscrylamide-gel electrophoretic equipment is described. The modified apparatus has been applied to the separation of Azotobacter vinelandii nitrogenase components and should prove useful in the analysis of other O₂-sensitive proteins. Electrophoresis in reducing gels can be followed with a dithionite-resistant tracking dye, potassium gualazulene-1-sulfonate.     

An apparatus for submerged gel electrophoresis.

A novel apparatus for submerged gel electrophoresis is described in detail. It includes an upper buffer compartment, a lower buffer compartment, and a horizontal plate between the two compartments. The horizontal plate is a heat exchanger connected to an external heater/cooler. Buffer circulates between the two compartments through openings in the horizontal plate. In the upper compartment two separated openings are positioned on each side of the horizontal plate between the side walls and long vertical barriers. The barriers initially direct the flow of buffer and define the electric field on the sides of the upper compartment. The electric field is confined essentially into a rectangular box, defined on the ends by the end walls, on the sides by the barriers, on the bottom by the cooling plate, and on the top by the air. Since the volume of buffer is smaller in the electrode compartment than in the reservoir under the cooling plate, this design enables formation of a substantially uniform electric field without creating too high a current. To enhance uniformity of the electric field, anode and cathode consist each of two platinum wires positioned one above the other at a distance of 6 mm. The electrodes can be placed parallel to the sides and perpendicular to the buffer flow or parallel to the ends and the flow of buffer. The stream of buffer in the upper compartment is regulated by two dams, perpendicular to the long barriers, on each end of the horizontal plate.(ABSTRACT TRUNCATED AT 250 WORDS)     

An apparatus for simultaneous manual solid-phase synthesis of multiple peptide analogs

A new design of reaction vessel for simultaneous manual solid-phase synthesis of multiple peptide analogs is described. Simultaneous use of four of these vessels attached to a single rotary mixer has been successfully applied to synthesis of two sets of four decapeptide amide analogs. Efficient coupling was indicated by chemical determination at the end of each synthesis cycle and overall final yields of between 78 and 84% were obtained. The products obtained were of a high quality, as assessed by high-performance liquid chromatography and amino acid analysis. This system allows expeditious synthesis of multiple peptide analogs for structure-function studies with economical use of efficiently ventilated laboratory space.     

An apparatus for the simultaneous demineralization of fifty-four specimens.

An apparatus designed to demineralize 54 specimens simultaneously is described. A drum with built-in specimen holders rotates continuously through a bath of acid, allowing a free exchange of demineralizing fluid over the specimens. Individual specimens can be easily introduced or withdrawn from the apparatus without disturbing others.     

Dodecyl sulphate/polyacrylamide-gel electrophoresis at low pH values and low temperatures.

A simple method is described for dodecyl sulphate/polyacrylamide-gel electrophoresis of pH- and temperature-labile biological intermediates. The method is based on a catalyst system that works at temperatures of 2--4 degrees C and pH values of 2--4 and an appropriate buffer system containing Li+ or Tris [CH2OH--C(CH2OH)2--NH3+] instead of Na+. This system does not lead to the precipitation of 1% dodecyl sulphate.     

Fractionation of heparin-derived oligosaccharides by gradient polyacrylamide-gel electrophoresis.

Heparin-derived oligosaccharides, prepared by using flavobacterial heparinase, having a high degree of heterogeneity (sequence variability) were resolved into sharp well-defined bands by using polyacrylamide gel electrophoresis (PAGE). The use of a stacking gel and a high-density-pore-gradient resolving gel was primarily responsible for the success of this separation. Low-Mr standards of known structure and having a degree of polymerization (dp) 2-6 were used to establish that the separation on gradient PAGE was primarily dependent on molecular size. High-Mr oligosaccharides (dp 8-20) were prepared using strong-anion-exchange h.p.l.c. and were used to help characterize the gradient PAGE separation. Kinetic profiles were obtained for the depolymerization of heparin and heparan sulphate with heparinase and heparitinase respectively. The utility of this approach in sequencing oligosaccharides derived from glycosaminoglycans is discussed.     

Isolation of coconut storage proteins by polyacrylamide-gel electrophoresis

By combining sodium dodecyl sulfate (SDS)-gel electrophoresis with a new chilling technique for visualization of protein-SDS complexes in polyacrylamide gels, a process has been developed which will permit the isolation of milligram quantities of pure polypeptides. Using this technique, we have isolated two molecular weight classes of polypeptides from coconut storage globulins and determined the amino acid composition of each. When the two amino acid compositions were summed on a molar basis, the result agreed reasonably well with the amino acid composition of the starting material with the exception of cystine. Apparently, some contaminant from the polyacrylamide caused its destruction to be accelerated during hydrolysis.     

An analysis of the polypeptide composition of bovine heart mitochondrial NADH-ubiquinone oxidoreductase by two-dimensional polyacrylamide-gel electrophoresis.

Purified preparations of Complex I (NADH-ubiquinone oxidoreductase) from bovine heart mitochondria may be resolved into 26 polypeptides by two-dimensional analysis combining isoelectric focusing and polyacrylamide-gel electrophoresis in sodium dodecyl sulphate. Similar analyses of the fragments obtained from chaotropic resolution of the enzyme show that each of these fragments contains a distinct and non-overlapping set of polypeptides. Evidence that the polypeptides seen in the intact enzyme are true constituents comes from analyses of immunoprecipitates obtained by allowing Complex I or solubilized submitochondrial particles to react with antisera directed against the whole enzyme and a subfragment of the enzyme.     

Analysis of the heterogeneity of human collagens by two-dimensional polyacrylamide-gel electrophoresis.

The heterogeneity of the CNBr-cleavage peptides of human types I, II, III and V collagens were studied by using two-dimensional electrophoresis combining non-equilibrium pH-gradient-gel electrophoresis and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Specific 'maps' were produced by the peptides obtained from the chains of each type of collagen, and most peptides had at least three charged forms of the same molecular weight. Specific 'maps' were also produced by the peptides of types I, III and V collagens from insoluble dermis and the peptides of types I and V collagens from decalcified bone. The alpha 1(I) CB7 and alpha 1(I) CB8 and the alpha 2 CB4 peptides obtained from the type I collagens of these tissues contained the same number of charged components, but there was a relative increase in the more basic components in bone. Some aspects of the involvement of the alpha 1(I) CB6 and the alpha 1(III) CB9 peptides in cross-linkages were also studied. The recovery of the alpha 1(I) CB6 peptide from bone and dermis was decreased and the alpha 1(III) CB9 peptide was not detected in dermis. Additional peptides, which were probably cross-linked peptides involving the alpha 1(I) CB6 peptide, were also observed.     

Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of the human erythrocyte membrane.

A two-dimensional polyacrylamide gel electrophoresis technique has been developed, improving the analytical separation of some proteins and glycoproteins of the human erythrocyte membrane. Freshly prepared membranes are totally solubilized, subjected to dodecylsulfate--polyacrylamide gel electrophoresis in the first dimension, followed by electrophoresis in the second dimension, using a detergent-free polyacrylamide gradient gel. By this method the proteins of the human erythrocyte membrane could be resolved into a two-dimensional pattern, which has been shown to be highly reproducible with respect to various blood-groups and within one blood-group from specimen to specimen. The method enables especially the investigation of the hydrophobic and very likely integrated membrane proteins and glycoproteins. Thus, band III[Fairbanks, G., Steck, Th. & Wallach, D. F. H., Biochemistry, 10, 2606--2617 (1971)] could be shown to consist of five proteins, one of them being the major glycoprotein of the human erythrocyte membrand. The two spectrin bands differed considerably in their two-dimensional patterns. The value of the given method for the investigation of membrane defects, which may be linked with various diseases of human erythrocytes, could be demonstrated in the case of two patients suffering from congenital dyserythropoetic anaemia.     

Anomalous migration of leghaemoglobin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

The estimate of the molecular weight of leghaemoglobin by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis is about 20% too low. This is due to an anomalously high limiting relative mobility. Leghaemoglobin binds 1.4 g of sodium dodecyl sulphate/g of protein with a concomitant decrease in the helical content from 71-72% to 49-51%.     

Interchain disulfide bonds in immunoglobulins: analysis by two-dimensional polyacrylamide-gel electrophoresis.

The arrangement of interchain disulfide bonds in immunoglobulins may be determined in a single experiment by two-dimensional SDS-polyacrylamide-gel electrophoresis. The noncovalently associated subunits are first separated on a cylindrical acrylamide-gel by electrophoresis under dissociating conditions. The subunits are then subjected to reduction in situ followed by electrophoresis on a slab gel of acrylamide in a direction perpendicular to the first to separate the constituent polypeptide chains (heavy and/or light chains) of the individual subunits.     

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.     

The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis

1. Gels were prepared with recrystallized acrylamide and bisacrylamide. Electrophoresis was in tris-sodium acetate-EDTA buffer for 0.5 to 3hr. Gels were scanned at 280 or 265mmu. Techniques are described for slicing and radioactive counting. 2. The mobility of RNA was inversely related to the sedimentation coefficient and varied with gel concentration. Electrophoresis in 2.2-2.6% gels gives a fractionation similar to density-gradient centrifugation. It shows the two ribosomal RNA components, the 45s precursor, transfer RNA and minor components. In 5% and 7.5% gels, 4s and 5s RNA separated and ribosomal RNA was excluded. 3. The resolution is greater and more detailed than by centrifugation, and many samples can be analysed simultaneously and rapidly.