Rapid polyacrylamide slab drying using a microwave gel dryer

A new gel dryer which uses microwave energy instead of radiant heat to dry slab electrophoresis gels has been designed. The use of microwaves results in substantial decreases in drying time. The potential utility of this instrument is discussed.     

Purification of tetanus toxin and its peptide components by preparative polyacrylamide gel electrophoresis

A discontinuous preparative gel electrophoresis system has been devised and used successfully to separate the different tetanus toxin forms and fragments into highly purified preparations. A major feature of the system is the interaction of toxin, a suitable reducing agent and a critical concentration of denaturant during electrophoresis. With this procedure, filtrate (nicked) toxin has been separated into two distinct, but closely related molecular species. They appear to be nicked close to but on either side of an interchain disulfide bond, yielding heavy and light chains. The heavy- and light-chain components of each form of nicked toxin have been prepared and characterized. The system was also used to prepare extract (unnicked) toxin to a degree of purity not previously achieved in this laboratory. Nicked and unnicked toxin as well as the two forms of both heavy and light chain can consistently be prepared in sufficient purity and quantity to allow extensive biological, chemical, and physical characterization of each.     

Isolation of outer membrane proteins of Escherichia coli and their characterization on polyacrylamide gel

Proteins from the outer membrane of Escherichia coli were studied on a ureadodecyl sulfate polyacrylamide gel by electrophoresis. A polyacrylamide gel containing sodium dodecyl sulfate and urea gave an excellent resolution of outer membrane proteins. Seventeen protein bands were reproducibly observed on a gel. By use of Sephadex G-200, DEAE-cellulose and polyacrylamide gel, eight proteins were purified to near homogeneity. Five of them were found to be heat-modifiable proteins. The behavior of these purified proteins was studied on a polyacrylamide gel under three different electrophoretic conditions, which had been used for the analysis of cell envelope proteins. Thus correspondence was made between these purified proteins and envelope proteins reported by other investigators.     

Analysis of polyacrylamide gel electrophoresis through the PIQUANT image recognition system

Polyacrylamide gels, with separated components which can be made visible by fluorescent or colored dyes and thereafter photographed, can be analyzed at high speed and resolution by a computerized image-recognition device (PIQUANT). The system provides relative mobilities and relative distributions of separated components of multiple samples at the rate of about 1 sec/sample and with greater resolution than can be attained by conventional methods.     

Simultaneous staining of proteins during polyacrylamide gel electrophoresis in acidic gels by countermigration of Coomassie brilliant blue R-250

A method for the simultaneous staining of proteins during polyacrylamide gel electrophoresis with Coomassie brilliant blue R-250 at pH 2.5 is described. Calf thymus whole histone and cytochrome c were stained by this method and the results obtained were similar to that obtained by staining after electrophoresis.     

Sequential elution of proteins in small volumes by preparative polyacrylamide gel electrophoresis

A simple flexible method for separation of proteins by polyacrylamide gel electrophoresis and sequential elution into dialysis bags has been devised. The system was applied to isolation of three glycoproteins from the peritoneal fluid of mice bearing Ehrlich ascites tumor.     

Extraction of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from protease-rich plant tissues

A method of extracting proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from plant tissues with high protease activity was described. It resolved protein bands in highmolecular-weight regions of the gel and replaced commonly used procedures which showed severe degradation of proteins, even in the presence of protease inhibitors.     

Abnormal bovine erythrocyte membrane proteins and glycoproteins during and after infection with Anaplasma marginale

The erythrocyte membrane proteins from normal and Anaplasma-infected bovine blood have been compared. Two distinct new polypeptides were present in membranes from acutely infected cells. The glycoprotein pattern was also altered: in addition to the three main bands observed in normal cells, there were four new bands present which were glycosylated. The normally found membrane glycolypeptide (250000 D) was missing. The role of these protein alterations in relation to the infectious process is discussed.     

Simplified techniques for elution of proteins from polyacrylamide gel, staining, destaining, and isoelectric focusing

A simple and rapid electrophoretic method for the simultaneous elution of proteins in high yields from polyacrylamide gel slabs is described. The apparatus is simple and easily constructed. The method involves vertical elution from a horizontally placed gel across its thickness into buffer-soaked polyurethane foam. Even the slow-moving, high-molecular-weight protein ferritin is eluted in 1 h. The technique can also be used for rapid destaining as well as for simultaneous staining and destaining of the polyacrylamide gel which are completed in 15 and 30 min, respectively. Polyurethane foam strips have also been used to simplify the preparative isoelectric focusing technique and the subsequent elution of protein bands, obviating the need for Ultrodex, fractionating grid, sample applicator, and elution columns.     

Controlled deaeration of polymerization mixtures in polyacrylamide gel electrophoresis

The inhibition by oxygen of the polymerization reaction yielding cross-linked polyacrylamide makes it necessary to control the oxygen concentration in the polymerization reaction mixture. In addition, polymerization within a convenlently short time (e.g., 30 min) is frequently not possible at acid to neutral pH values, in the presence of reactants such as buffers containing phenyl groups, and at low temperatures, unless the oxygen tension over the polymerization mixture is reduced. A simple device was constructed which allows for deacration of polymerization mixtures by an oil pump at low, controlled air pressures at the desired preset levels, and for a set time, by activation of a single switch.     

Anomalous behavior of bovine alpha s1- and beta-caseins on gel electrophoresis in sodium dodecyl sulfate buffers

Electrophoresis in the presence of sodium dodecyl sulfate (SDS) provides a relatively simple means of determining molecular weights of proteins. This technique relies on the validity of a correlation between some function of Mr and the mobility of the protein through the gel matrix. However, bovine caseins (especially alpha s1-casein) have lower mobilities than expected on the basis of their known Mr. The binding of SDS to both alpha s1-casein (Mr 23,600) and beta-casein (Mr 24,000) reached a maximum at the slightly low value of 1.3 g SDS/g protein. Gel-filtration chromatography showed, however, that the alpha s1-casein:SDS complex was larger than the beta-casein:SDS complex at pH 6.8 or 7.0, but that they were similar in size at pH 2.9 or 3.0. Circular dichroism spectra indicated that the low helical structure content of both alpha s1- and beta-casein increased with the addition of SDS and/or decreasing the pH to 1.5. 13C NMR results showed that SDS bound to alpha s1- and beta-casein in the same way as it did to bovine serum albumin. Either esterification or dephosphorylation followed by amidation of alpha s1-casein increased its mobility in SDS-gel electrophoresis, but neither modification affected beta-casein mobility. These and other results indicate that the low electrophoretic velocity of alpha s1-casein in SDS-gel electrophoresis results from its unexpectedly large hydrodynamic size. This is caused by localized high negative charges on certain segments of alpha s1-casein, which would induce a considerable amount of inter- and intrasegmental electrostatic repulsion, leading to an expanded or extended structure for portions of the alpha s1-casein molecule in the presence of SDS. It is clear that the conformation, and hence the equivalent radius, of an SDS:protein complex is determined by the sequence of amino acids in the protein and that, a priori, it cannot be anticipated that the electrophoretic mobility of such a complex will bear more than a casual relationship to the Mr of the protein.     

The use of agar for gel electrophoresis of DNA

Agar can be used instead of agarose for electrophoresis of DNA. DNA restriction fragments migrate in proportion to the log of their molecular weights in the ranges studied. Bands of both restriction fragments and discrete small low molecular weight DNAs such as plasmids are sharp and clearly visible. The DNA can be Southern blotted with very low nonspecific background binding of radioactivity. Fragments can be removed from the gel and can be further restricted and ligated. Plasmid DNA retains its capacity to transform host bacterial cells. Agar is about $\text{1}\text{10}$ the cost of electrophoresis-grade agarose.     

A sensitive polyacrylamide disc gel method for detection of proteinases

To enable direct detection of proteinase activities subsequent to electrophoresis, a technique utilizing the incorporation or diffusion of protein substrates into polyacrylamide disc gels was developed. Denatured insoluble substrates, casein or hemoglobin, were added to acrylamide solutions prior to polymerization of the gel mixture. Alternatively, soluble protein substrates were diffused into gels after electrophoresis. In either case, an incubation period ensued at the pH optimum of the proteinases to allow for their detection. Classification of resolved proteinases was accomplished subsequent to electrophoresis by incubation of gels in media containing either synthetic substrates, as the naphthylamide derivatives, or specific inhibitors of the enzymes. Separation of purified trypsin from chymotrypsin, and proteinases in preparations of seminal plasma and mouse blastocysts homogenates demonstrated the efficacy of the method at the submicrogram enzyme level.     

An efficient two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis method for simultaneous peptide mapping of protein contained in a mixture

A method for simultaneous peptide mapping of polypeptides contained in a mixture is presented. The polypeptides were first separated by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The strip of gel containing these unstained polypeptide bands was subsequently embedded perpendicular to the direction of electrophoresis in the stacking gel of a second gel. The proteolytic enzymes, loaded on top of the second gel, were brought in contact with the substrates through moving boundary electrophoresis. The peptides thus generated were then resolved by electrophoresis in a gradient gel. A polychromatic silver staining method added an extra dimension to the identification and characterization of the peptides in the maps obtained in that specific peptides got specific colors. Moreover, the sensitivity of this method was illustrated by the demonstration that original quantities in the submicrogram range of nonradioactive proteins (exemplified here by the structural proteins of densonucleosis virus) largely sufficed for satisfactory maps. Other advantages of this procedure over current methods included (i) the elimination of the purification step (and consequently virtually no loss or contamination), (ii) that only the strict minimum of material (necessary for the ultimate visualization of the maps) had to be used, (iii) that no special two-dimensional electrophoresis equipment was needed, and (iv) the consistency, speed, and simplicity of the method.     

Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter

Zeta-bind, a positively charged nylon membrane, was tested as an immobilizing matrix for the electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels. It was found that Zeta-bind has a considerably greater capacity than does nitrocellulose for protein binding. Because of this property, more efficient elution of proteins from gels can be used (by omitting methanol from transfer buffers). The procedure described is more amenable to quantitation than usual nitrocellulose-based transfer. Antibody or lectin overlay techniques are also more sensitive on Zeta-bind than on nitrocellulose.     

Activity staining of nucleolytic enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis: Use of aqueous isopropanol to remove detergent from gels

The sensitivity with which RNase and DNase activity can be detected after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) varies widely, depending upon the particular SDS preparation used for electrophoresis. (See also [10.], Anal. Biochem. 100, 357–363.) Sensitivity of detection is greatly increased by using buffered 25% isopropanol, rather than buffer alone, to wash detergent from gels after electrophoresis. Thus it is routinely possible to detect bovine pancreatic RNase A at the picogram level. Use of isopropanol improved activity staining of RNases with each of the 10 SDS preparations examined, including one containing 32% tetradecyl sulfate and 4% hexadecyl sulfate, and reduced the variability from preparation to preparation observed when buffer alone was used to remove SDS. Other water-organic cosolvent binary mixtures can be used but none shows advantages over aqueous isopropanol when sensitivity of detection as well as availability and cost of organic solvent are considered.     

Separation of branched from linear DNA by two-dimensional gel electrophoresis

A general method for separating branched DNA molecules, such as replication forks and recombination intermediates, from linear forms has been developed. Using as a model a stable X-shaped molecule constructed in vitro, it was found that this branched form migrated more slowly during agarose gel electrophoresis than did a linear form of the same mass. Higher agarose concentrations and higher electrophoretic voltages enhanced the extent of retardation. These properties provided the basis for an electrophoretic method of separating branched from linear molecules by variation of agarose concentration and voltage over two dimensions. In the first dimension, concentration and voltage were low; in the second, both parameters were increased, thereby forcing X-shaped molecules to migrate to positions distinct from a diagonal arc of linear molecules. In addition, two-dimensional electrophoresis was capable of separating X-shaped forms of different mass from each other, as well as from linear molecules.