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.     

A versatile apparatus for polyacrylamide and agarose gel electrophoresis in Plexiglas slab gel molds

A simple vertical slab gel electrophoresis apparatus for analytical, preparative, and two-dimensional electrophoresis is described. The use of permanently sealed Plexiglas acrylic plastic slab gel molds which need to be sealed only at the bottom during gel formation, rather than the glass plate sandwich used in most previous designs, virtually eliminates leakage during gel formation and, in addition, permits the continuous monitoring with ultraviolet light of proteins and nucleic acids labeled with fluorescent dyes during electrophoresis. Results obtainable with this apparatus are equivalent to those achieved in other apparati which are more expensive to fabricate or purchase.     

Apparatus for discontinuous electrophoresis in polyacrylamide gel slabs

Construction of an inexpensive slab discontinuous electrophoresis apparatus is described. Using this apparatus 23 human serum proteins were resolved and the gel could be scanned with a standard densitometer to yield a trace with discrete peaks or pronounced shoulders for each protein band. The advantages of a single homogeneous slab for comparative studies are indicated.     

Separation of apolipoprotein B species by agarose-acrylamide gel electrophoresis

Human apolipoprotein (apo) B has been recognized to exist in two different forms designated apoB-100 and apoB-48. The two apoB forms are usually separated by NaDodSO4 gel electrophoresis with a low percentage polyacrylamide gel in a tube gel apparatus. However, the matrix of this low percentage gel is relatively weak, and one can separate the two forms of apoB in a slab gel apparatus only if one utilizes a gradient polyacrylamide gel or a higher percentage polyacrylamide gel which results in a poorer separation of the protein bands. We have developed an agarose-acrylamide gel electrophoretic method to separate the two major apoB forms. The gel is a mixture of 0.5% agarose and 2% acrylamide. The agarose-acrylamide method is fast, has the advantage of being able to be used on an analytical or preparative scale in a vertical slab gel apparatus, and the gel is of sufficient strength to be used in immunoblotting and/or radioautography.     

Modifications and additions to the ISO-DALT system for two-dimensional gel electrophoresis

Modifications of ISO-DALT devices that further enhance the efficiency and reproducibility of two-dimensional mapping of proteins are described. The principal changes in ISO system devices include the introduction of a gel casting trough with a removable panel to permit the removal of excess gel without introducing air into the electrofocusing gels and the introduction of an upper electrode compartment with a separate watertight septum for each electrofocusing tube to permit tube removal for cleaning and replacement. The principal changes in DALT system devices include the use of modified powder funnels to introduce acrylamide solutions into the slab gel gradient former without aeration; the introduction of a flexible outlet system for the gradient former to facilitate the removal of air bubbles; the introduction of an inexpensive two-part mixing chamber to permit disassembly for cleaning; the use of split gel holders to eliminate deformation and breakage of electrofocusing gels during loading onto slab gels; the introduction of an inexpensive integrated slab gel casting/rotating apparatus; and the introduction of a simple, water-cooled slab gel electrophoresis apparatus to reduce the volume of running buffer used in electrophoresis.     

Separation of the two high molecular weight spectrin bands by a newly designed preparative slab electrophoresis technique.

Constructions and operation of an inexpensive preparative slab gel electrophoresis apparatus is described. A slab is made from two wide glass plates with symmetrical windows cut out from both sides. Coating the plates with a silane reagent allows good adhesion of a low concentration acrylamide sodium dodecyl sulfate gel. The migration of extracted chromatographed and fluorescent spectrin mixture is monitored with a uv light. At the end of the run, the slab is turned upside down and the material cluted upward in the small space formed by a dialyzing membrane placed in between the slab and the upper buffer reservoir. Using this apparatus and technique, the two heavy molecular weight spectrin bands can be purified. Advantages of this new system are discussed.     

An apparatus for thin layer vertical polyacrylamide gel electrophoresis with discontinuous buffer and gel system

A vertical polyacrylamide gel slab electrophoresis apparatus with a discontinuous gel and buffer system and a running gel of 1 mm in thickness was devised. Using this apparatus, which employs stacking and sieving effects, sharp bands comparable to those of disc electrophoresis were obtained.Furthermore, a new application using detection with ultraviolet method was introduced for isozyme study.     

An electroblotting apparatus for multiple replica technique and identification of human serum proteins on micro two-dimensional gels

A new apparatus for electrophoretic transfer of proteins from micro polyacrylamide slab gels has been developed. The apparatus enabled the easy changing of nitrocellulose sheets and was suited for obtaining multiple blots from a gel. Electrophoretic conditions were determined so that all of the blots obtained sequentially from one slab gel were successfully used to visualize specific proteins irrespective of their molecular weight. Combining the transfer technique with the technique of parallel micro two-dimensional electrophoresis, 20 blots could be obtained within 1 h of electroblotting time. The locations of 28 human serum proteins were determined simultaneously on these blots using commercial specific antisera.     

A compact apparatus for vertical gel slab isoelectric focusing: resolution of rabbit anti-pneumococcal polysaccharide antibodies.

A compact apparatus for analytical and preparative vertical gel slab isoelectric focusing is described. The apparatus was extremely versatile, easy to use, and comparatively inexpensive. The apparatus was useful to compare the electrofocusing patterns of anti-pneumococcal polysaccharide antibodies from several different restricted and partially restricted rabbit responders.     

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.     

Design and construction of a simple and inexpensive slab gel electrophoresis apparatus

A very simple and inexpensive slab gel electrophoresis apparatus is described. This integral design reduces the leakage, cost, and size limitations frequently encountered in the construction and use of currently available apparatuses. An additional refinement eliminates the need for notching one member of the usual pair of glass plates used as gel slab molds. The apparatuses, in which linear, gradient, and two-dimensional gels have been routinely run, can be built in a wide variety of sizes and shapes for either analytical or preparative purposes. Several gel apparatuses can be clamped together and run simultaneously from a single power source. Ease of construction permits more than a dozen apparatuses of this design to be built in the space of a day or two by unskilled personnel.     

Electrophoretic mobility of high-molecular-weight, double-stranded DNA on agarose gels.

A new theoretical model for the migration of high-molecular-weight, double-stranded DNA on agarose gels is presented. This leads to the prediction that under certain conditions of electrophoresis, a linear relationship will exist between the molecular weight of a DNA molecule, raised to the (-2/3) power, and its electrophoretic mobility. Agarose gel electrophoresis of the fragments of bacteriophage lambda DNA produced by several restriction endonucleases confirms this relationship, and establishes some of the limits on its linearity. For this work, a polyacrylamide slab gel apparatus was modified for use with agarose gels. This apparatus has several advantages over others commercially available for agarose gel electrophoresis, including the abilities to run a larger number of samples at one time, to use lower-concentration gels, and to maintain better temperature stability across the width of the gel. The validation of the relationship developed here between molecular weight and electrophoretic mobility should make this a useful method for determining the molecular weights of DNA fragments.     

Anaerobic multiphasic gel electrophoresis of the molybdoproteins in extracts of Clostridium pasteurianum

Simple procedures using multiphasic buffer systems for anaerobic electrophoresis have been devised to identify oxygen-labile metalloproteins. An anaerobic slab gel apparatus was developed with cooling and design for anaerobic conditions. Included is a procedure to remove sample wells after stacking proteins in a crude extract, to prevent streaking (background) caused by continuous leakage of "nonstacked protein" from the sample wells. Identification of eleven Mo zones in extracts of Clostridium pasteurianum demonstrates the usefulness of the technique in identifying radiolabeled oxygen-labile proteins in cell-free crude lysates.     

Purification of individual proteinase isozymes from Bacteroides nodosus by use of polyacrylamide gel electrophoresis,a fluorogenic substrate detection system,and a simple electroelution apparatus

A method is described for the purification from Bacteroides nodosus of five individual proteinase isozymes which could not be purified by column chromatography techniques. The isozymes were separated by horizontal slab polyacrylamide gel electrophoresis. Their exact location within the gel was determined with a fluorescein-casein substrate, and they were extracted from the gel by a simple electroelution apparatus. In a typical purification, microgram quantities of three individual isozymes were recovered free of other isozyme activities. The other two isozymes were each contaminated (<5%) with another isozyme activity. Occasionally, all the individual isozymes were recovered in pure form. The molecular weights were 78,000, 82,000, 88,000, 96,000, and 107,000.     

Changes in glycerol solubility and amino acid incorporation of a protein possessing the characteristics of tubulin during first cleavage in the sea urchin embryo

A method is described for the quantitation of the pool size of a tubulin like protein during synchronized cell division in the sea urchin Lytechinus variegatus. The method involves the use of a thin SDS slab polyacrylamide gel system in which tubulin can be quantitated in the submicrogram range. Employing a microtubule stabilization buffer, the intact tubule fraction was removed and the soluble tubulin pool was quantitated with this gel system. Amino acid incorporation into this protein was also quantitated. The resulting specific activity values and values for the amount of tubulin-like protein present in the pool fraction suggested that the tubulin pool decreases at fertilization and then remains constant through the first cell cycle. Tubulin synthesis, however, steadily increased after fertilization and then decreased dramatically just prior to mitotic apparatus formation. No change in tubulin pool size was observed at the time of peak mitotic apparatus formation. These results are discussed in terms of the regulation of microtubule function.     

Recovery of native proteins from preparative electrophoresis gel slices by reverse polarity elution

A technique for high yield recovery of native, biologically active proteins from preparative polyacrylamide gel slices by reverse polarity elution is described. No apparatus other than the standard slab gel electrophoresis system is required. Several proteins have been recovered in biologically active form at a 90% yield, in quantities ranging from 0.4 mg to 4.2 mg. The method is effective with both small (9,000 dalton) and large (186,000 dalton) polypeptides. Both simple and complex proteins are recovered intact. For example, the copper-zinc and manganese superoxide dismutases from crude soybean extracts are active upon recovery. Similarly, the vitamin D-dependent calcium binding proteins from rat kidney and intestine are isolated by this method in homogeneous, active form.     

A method for separating DNA fragments by electrophoresis in polyacrylamide concentration gradient slab gels

Electrophoresis on slab gels containing a linear gradient of polyacrylamide concentration has been used to separate DNA fragments obtained by restriction of viral DNAs. A simple method of preparing gradient gels using a sucrose density-gradient mixer and preexisting slab gel apparatus is described. DNA fragments of molecular weights 7 × 10⁴–14 × 10⁶ have been fractionated on gels of 3.5–7.5% and 2.5–7.5% acrylamide concentration. In addition to the wide range of fragment sizes which may be run on a single gel, a further advantage of the system is that much sharper bands are obtained compared to conventional constant concentration gels, thus improving resolution.In the molecular-weight range below 5 × 10⁶, for bands whose terminal velocities in the polyacrylamide concentration gradient approach zero, an approximately linear relationship holds between the logarithms of the molecular weights of the fragments and the logarithms of the distances they have migrated in the gel. Thus, by choosing a suitable upper limit to the concentration gradient, the gel system provides a method for estimating approximate molecular weights of unknown DNA fragments, by comparing their mobilities to known standards.     

Preparative immobiline isoelectric focusing of plasma apolipoproteins on vertical slab gels

An effective preparative isoelectric focusing method has been developed using the LKB Immobiline system in a vertical slab gel apparatus. Advantages of this procedure are ease of sample application, excellent resolution, and the direct visualization of focused bands. Narrow pH gradients have been used to separate apolipoprotein E3 isoforms (pH gradient 4.9-5.9) and to resolve the apolipoprotein C mixture (pH gradient 4.0-5.0). Recoveries ranged from 40 to 70%. The method should be valuable for protein and isoform purification.     

乳酸脱氢酶与酯酶同工酶同板染色法

介绍一种在同一块凝胶板上染乳酸脱氢酶(LDH)与酯酶(EST)的染色方法. 该同板染色法利用两种同工酶显色反应互不干扰和颜色不同的特点, 先染LDH, 后染EST, 可以在同一块胶板上得到两种同工酶清晰的酶带, 每一种酶的酶带与单板染色的酶带完全一样. 这种染色法, 能节省同工酶分析所需的试剂、时间和经费, 也便于样品的鉴定与比较, 是一种经济有效的方法. 此方法, 同样适用于苹果酸脱氢酶(MDH)与酯酶等同工酶的同板染色.