Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.     

Agarose-acrylamide gradient gel electrophoresis of proteins

A new agarose-acrylamide gradient slab gel electrophoresis system is described. The preparation of this new gel has been facilitated by the use of agarose with a relatively low gelation temperature. Fractionation of marker proteins and crosslinked proteins from a subcellular cytoskeletal preparation on agarose-acrylamide gradient gels is compared to that found using other acrylamide gel electrophoresis systems.     

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.     

Polyacrylamide slab gel electrophoresis: an improved design

An apparatus for polyacrylamide slab gel electrophoresis is decribed which combines all parts into one integral unit. It eliminates several steps in the process of sealing, pouring, and setting the gels. Construction is easy with modest workshop facilities and the design easily adapted to suit most requirements. The apparatus provides a high degree of versatility and is suitable for use with many slab gel electrophoretic techniques.     

一步法电泳分析肌联蛋白亚型和肌球蛋白重链

目的为研究超大分子量肌小节蛋白肌联蛋白(titin)的生理病理功能,在一次电泳过程中同时分离titin各亚型和中分子量肌小节蛋白肌球蛋白重链(myosin heavy chain,MHC)。方法使用16cm×18cm垂直电泳系统,在电泳板下1/3灌注10g/L SDS-PAGE胶,上2/3灌注60g/L SDS-琼脂糖(SDS-VAGE)胶。低温8℃下持续电泳5h,在电泳板上层以SDS-VAGE胶电泳分离titin亚型,下层以SDS-PAGE胶电泳分离MHC。电泳后VAGE胶使用银染法标记titin各亚型,PAGE胶使用考马斯亮蓝染色法标记MHC。结果 titin各亚型得到有效的分离,目标蛋白条带显示清晰,与其分子量大小一一对应,分离效果明确。结论一步法垂直电泳系统可应用于超大分子量蛋白的电泳,同时可分离多个分子量差距大的蛋白,提高蛋白电泳实验效率。     

Rapid microsatellite analysis using discontinuous polyacrylamide gel electrophoresis.

A method for screening large numbers of samples for microsatellites using discontinuous, non-denaturing polyacrylamide gels and rapid fluorescent gel staining is described. Disc electrophoresis on slab gels provides high-resolution of PCR products. It is useful for collecting population data once microsatellite loci have been characterized.     

Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility and polypeptide resolution.

The development of a dedicated two-dimensional gel electrophoresis system is described that provides superior performance in terms of high resolving power and enhanced gel-to-gel reproducibility. Isoelectric focusing is performed in a 1-mm capillary tube with a 0.08-mm thread, optimized for this application, incorporated along its length prior to polymerization of the gel matrix. The isoelectric focusing gel is 4% T, 2.6% C to minimize sieving of proteins and promote adhesion of the gel to the thread. The thread incorporated in the isoelectric focusing matrix prevents gel stretching and breakage during its application to the second dimension. An optimum ampholyte pH range has been defined based on 1600 polypeptides present in a transformed fibroblast cell lysate and verified using a variety of other cell types. The length of time required to complete an electrophoretic separation in the second dimension was found to depend on buffer conductivity establishing the importance of high quality electrophoresis grade reagents devoid of contaminating salts. To ensure reproducibility of electrophoretic separations, it is critical to maintain a strict control of temperature during the second dimension separation. This prevents altered migration of some polypeptides relative to neighboring polypeptides that have constant Rfs over a broad temperature range. It was also determined that to obtain the maximum information from a complex protein mixture it is critical to use a large format 22- x 22-cm two-dimensional electrophoretic system. Using the optimized two-dimensional electrophoretic system and computerized gel analysis, it was determined that molecular weight estimates of polypeptides differed by approximately 350 daltons between gels, while isoelectric point estimates differed by approximately 0.03 pH units between gels. Using the two-dimensional electrophoresis system described, approximately 1000 polypeptides can be routinely detected from silver-stained 10% polyacrylamide gels or 1600 polypeptides from autoradiographs of 35S-methionine-labeled polypeptides.     

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.     

Two-dimensional agarose gel electrophoresis without gel manipulation

The apparatus and procedure to perform two-dimensional agarose gel electrophoresis without manipulating the gel used for the first electrophoresis (first-dimension gel) have been developed. The procedure is less complex, less damaging to first-dimension gels, and more precise than procedures that require manipulation of the first-dimension gel. When combined with gel-embedding techniques, the procedure presented can be used to perform the second electrophoresis in a gel different from the first-dimension gel. A first-dimension gel too dilute to be manipulated and a more concentrated gel for the second electrophoresis have been used to separate DNA open circles from a mixture of variable-length linear DNAs.     

SDS microslab linear gradient polyacrylamide gel electrophoresis

An improved sodium dodecyl sulfate (SDS) microslab linear gradient polyacrylamide gel electrophoresis (PAGE) technique has been developed. Several important features present in this microslab SDS-PAGE system include (1) high resolution and sensitivity; (2) rapid electrophoresis, staining, and destaining; (3) high reproducibility; and (4) low cost of construction and operation. Several gels are east at once between unmodified commercially available microslides separated by 0.5-mm thick Teflon spacers. The total time from start of electrophoresis to completion of destaining spans 2 hr. Gels are dried between transparent cellophane membranes in 1 hr and can be easily scanned with a microdensitometer. As little as 20 ng of a purified protein stained with Coomassie blue is detectable.     

A multiple high-resolution mini two-dimensional polyacrylamide gel electrophoresis system: imaging two-dimensional gels using a cooled charge-coupled device after staining with silver or labeling with fluorophore.

A multiple mini two-dimensional electrophoretic method which results in three two-dimensional protein spot patterns being positioned side by side in an individual gel has been developed. Preparation time has been minimized by employing disposable capillary tubes for the isoelectric focusing gels and reducing the number of second-dimensional gels required. Commercially available vertical slab units were used for the second-dimensional electrophoresis. The protein spot patterns were visualized either by staining the second-dimensional gel with silver or fluorescently labeling the focused proteins while present in the isoelectric focusing gel and subsequently electrophoresing them into the second-dimensional gel. The fluorescently labeled second-dimensional gel was imaged while still present in the glass mold immediately following electrophoresis. Two fluorophores were compared: 2-methoxy-2,4-diphenyl-3(2H)-furanone and 5-(4,6-dichlorotriazin-2-yl)aminofluorescein hydrochloride. A rapid imaging system based on a cooled charge-coupled device was used to view both the silver-stained and fluorescently labeled two-dimensional spot patterns. The sensitivity of detection of protein spots in the mini two-dimensional gels was similar for the two types of fluorescently labeled gels and the silver-stained gels.     

Use of "submarine" gel electrophoresis for running multiple two-dimensional protein gels

An apparatus commonly used for the electrophoresis of submerged agarose gels was used to separate proteins in the second dimension, after isoelectric focusing in the first dimension. Multiple second-dimension gels were stacked one above the other and run horizontally, submerged in the sodium dodecyl sulfate-containing Laemmli buffer system. The reproducibility of the gels run under these conditions is remarkable and eliminates the need for individual vertical electrophoresis units for routine analysis. The units for submerged horizontal gel electrophoresis are easily made or are inexpensively available commercially.     

Analytical techniques for cell fractions. XXII. Two-dimensional analysis of serum and tissue proteins: multiple gradient-slab gel electrophoresis

Two-dimensional electrophoresis of proteins and protein subunits, employing isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate in the second, yields the highest resolutions currently available. In this paper separations in the second dimension are considered (the so called DALT system). Methods for multiple-parallel casting of gradient gels in slab gel holders are described. The problem of electrical isolation of the ends of the slabs together with continuous cooling of both surfaces of the slab gel holder along their entire length has been achieved by running the gels in a horizontal direction in a three-compartment tank with the holders inserted in insulating septa. In the system described, 10 slabs are run simultaneously. This, however, is not the upper limit of the number of slabs which can be conveniently run in parallel.     

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.     

Quantitative aspects of mercurial-agarose gel electrophoresis

Single-stranded nucleic acids are capable of extensive intramolecular base pairing as well as intermolecular aggregation. Consequently, electrophoretic studies of single-stranded nucleic acids are most effective when conducted under denaturing conditions. A number of techniques are available for nucleic acid denaturing gel electrophoresis (1–3). In this paper we describe certain quantitative features of one of these techniques, mercurial-agarose gel electrophoresis (4–7). Specifically, we address the questions of resolution and base composition dependence and we introduce a new mercurial for agarose gel electrophoresis, p-chloromercuriphenyl-sulfonic acid.In a previous publication we demonstrated that methylmercury was an effective denaturant in an agarose gel (4). The mechanism of denaturation is presumably the disruption of hydrogen bonding by the reversible binding of methylmercury to uridine and guanosine imino nitrogens. At saturating mercurial concentrations accurate molecular weights can be determined, free of conformation effects. The presence of methylmercury has no observable effect on the mechanical properties of the gel. Hence the denaturing power of the gel can be readily varied. The strength and rigidity of agarose gels make them considerably easier to handle than acrylamide gels, and the large pore size ensures a system compatible with high molecular weight RNA.     

Polyacrylamide gel electrophoresis on micro slabs

An electrophoretic technique using micro polyacrylamide flat gels is described and its usefulness demonstrated. The gels are vertically cast and electrophoresed in slab form (75 × 18 × 0.75 mm) in closed thin glass cells (cuvets) made from detachable microscope slides. The main features of the method are: requirement of small sample quantities (0.1–20 μg contained in <1–5 μl solution), simultaneous analysis of several samples in a single gel, relatively brief running periods, easy removal of the gel for rapid staining due to the two-piece gel mold, little pattern diffusion, convenient optical evaluation, drying, autoradiography and other contact print methods, and easy application of immunodiffusion techniques. Continuous gradient gels can be prepared. The advantage and complications of the technique are discussed and certain applications in biochemistry, clinical chemistry, and medicine are pointed out.     

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.     

One-step casting of Laemmli discontinued sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel

A modified Laemmli sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protocol is described. The new method saves 30 min for gel casting without loss of the resolution power of Laemmli gel. In this method, both the upper and lower gels can be cast at the same time because the lower gel contains 10% glycerol, which generates higher density in the lower gel than in the upper gel.     

Two-dimensional SDS-polyacrylamide gel electrophoresis of heat-modifiable outer-membrane proteins.

An examination has been made of the effect which temperature of solubilization has upon the subsequent migration in SDS-polyacrylamide gel electrophoresis of proteins from the cell envelopes of Escherichia coli K12 and Neisseria sicca ATCC 9913. Conventional electrophoresis in tubes revealed substantial differences in the staining patterns of gels, depending upon whether the envelope samples were solubilized at 37 degrees C or 100 degrees C; in the case of N. sicca at least 6 of 13 discernible bands displayed heat-modifiable behavior. The relationship of the bands produced by each of the two temperatures was investigated by a two-dimensional electrophoresis procedure, in which a sample was solubilized at 37 degrees C and run in a usual cylindrical gel; the entire gel was then resolubilized at 100 degrees C, and laid along an acrylamide slab for electrophoresis in the second dimension. It was found that "free endotoxin" of both organisms examined contained the same major proteins as the total envelope fraction, and that these free endotoxin proteins showed the same heat-modifiable properties as when present in total envelopes.     

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.