Polyacrylamide gel electrophoresis: recovery of non-stained and stained proteins from gel slices

Following electrophoresis or isoelectric focusing in gels of polyacrylamide the protein band of interest is cut out and placed above a sucrose gradient column, containing carrier ampholytes (Pharmalyte). By electrophoresis, isoelectric focusing or displacement electrophoresis the proteins migrate out of the gel slice and into the isoelectric focusing column for concentration and further purification. From this column, the proteins can be withdrawn and their isoelectric points determined. Even after staining with Coomassie Brilliant Blue at least some proteins can be recovered by this technique and used for further analyses, for instance amino acid determinations. The focusing in a pH gradient by carrier ampholytes can be replaced by an electrophoresis in a conductivity gradient column. However, in comparison with isoelectric focusing, this concentration technique has the drawback of not permitting further purification of the eluted protein.     

Low-cost two-dimensional gel densitometry

A major obstacle to full utilization of the powerful technique of two-dimensional (2-D) gel electrophoresis is the expense and complexity of quantifying the results. Using an analog-to-digital converter already present in the widely available Commodore 64 or Commodore 128 microcomputer, we have developed a 2-D gel densitometer (GELSCAN) which adds only $20.00 to the cost of the Commodore system (currently around $700.00). The system is designed to work with autoradiograms of 2-D gels. Spots of interest are identified visually and then positioned manually over a light source. A pinhole photoelectric sensor mounted in a hand-held, Plexiglas holder, or "mouse," is briefly rubbed over each spot. Maximum density of the spot is determined and its value is converted to counts per minute via an internal calibration curve which corrects for the nonlinear response of film to radiation. Local spot backgrounds can be subtracted and values can be normalized between gels to adjust for variation in amount of radioactivity applied or in exposure time. Reproducibility is excellent and the technique has some practical as well as theoretical advantages over other more complicated approaches to 2-D gel densitometry. In addition, the GELSCAN system can also be used for scanning individual bands in 1-D gels, quantitation of "dot-blot" autoradiograms and other tasks involving transmission densitometry.     

High concentration polyacrylamide gel electrophoresis

A simple technique is described for easy removal of high concentration polyacrylamide gels after electrophoresis from glass tubes without breaking them.     

High-density small-volume gel loading directly from capillary tubes

A technique has been developed for high lane density loading of small-volume DNA samples in a horizontal agarose gel. This technique has been investigated with a simple hand-held tool that is made to couple to sample output from a new capillary-based sample automation system. The approach consists of piercing the gel with pressurized sample capillaries and relieving the pressure shortly before withdrawal. The pressurization prevents the capillary from aspirating the gel buffer and keeps the sample at the tip of the capillary, so that it may be sucked into the gel during withdrawal. This method is shown to be adequate for a wide range of DNA ladders and PCR-based screening. In addition to allowing smaller lanes and a higher lane density than is achievable with traditional well-forming techniques, it relaxes the need for well formation and the alignment of the sample loader with those wells, providing an easy, efficient means of loading agarose gels.     

Isoelectric focusing of alkaline phosphatases in agarose gel

An isoelectric focusing technique in agarose gel is presented which is suitable for alkaline phosphatases from both serum and tissue sources. An anomaly in the literature about isoelectric focusing of serum alkaline phosphatase from liver origin is discussed and a possible explanation is proposed. The presented technique is used to demonstrate some differences in behaviour of serum liver and bone isoenzymes towards neuraminidase treatment.     

Capillary affinity gel electrophoresis

Capillary affinity gel electrophoresis is a new technique for the recognition of the specific DNA base and/or sequence. This technology is also applicable to the characterization of binding properties of DNA-based drugs, chiral separation, and the selective separation of antibody mimetics using imprinted polymers. This article reviews the present state of studies on the capillary affinity gel electrophoresis, including the principle, theory, methods, and applications of this technology. The great potential of capillary affinity gel electrophoresis for the detection of the mutation onDNA is illustrated.     

Characterization of fish acid proteases by substrate–gel electrophoresis

Several analytical techniques based upon the use of substrate–polyacrylamide gel electrophoresis were evaluated to achieve characterization of aspartate proteases in fish stomach. Since aspartate proteases of fish are more stable at high pH than mammalian pepsins, the most accurate technique for activity assessment is electrophoresis at neutral pH and revealing of such activity at low pH with hemoglobin as substrate. The technique is suitable for characterization of proteases and in comparative assessment of acid protease activity in different sparids.     

Two-dimensional gel electrophoresis in proteomics: a tutorial

Two-dimensional electrophoresis of proteins has preceded, and accompanied, the birth of proteomics. Although it is no longer the only experimental scheme used in modern proteomics, it still has distinct features and advantages. The purpose of this tutorial paper is to guide the reader through the history of the field, then through the main steps of the process, from sample preparation to in-gel detection of proteins, commenting the constraints and caveats of the technique. Then the limitations and positive features of two-dimensional electrophoresis are discussed (e.g. its unique ability to separate complete proteins and its easy interfacing with immunoblotting techniques), so that the optimal type of applications of this technique in current and future proteomics can be perceived. This is illustrated by a detailed example taken from the literature and commented in detail. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 2).     

Dopa stain for precipitins in gel

Incubations for 10 hr in 0.01 M 3,4-dihydroxylphenylalanine (dopa), pH 7.2, stained precipitin proteins in agar gel. The staining technique is simple and is relatively selective for the precipitins tested and, therefore, does not require elution of unbound proteins from the gel. The stain is permanent and permits definitive photography.     

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.     

Analysis of RNA structure by ultraviolet crosslinking and denaturation gel electrophoresis

Electrophoresis in polyacrylamide gels containing both formamide and urea is a high-resolution technique for the analysis of crosslinked RNA species. Combined with a specific crosslinking agent like uv irradiation, it allows a rapid fingerprint of structural differences between RNA forms. The technique reveals significant differences in the pattern of uv crosslinking of free Escherichia coli 16 S ribosomal RNA compared with the RNA in active or inactive 30 S subunits. Ultraviolet photocrosslinks seen only in the 30 S particle are likely to be tertiary structure contacts.     

Pulsed field gel electrophoresis: Theory,instruments and application

Pulsed field gel electrophoresis (PFGE) is a technique for the fractionation of high-molecular-weight DNA ranging from 10 kb to 10 Mb by electrophoresis in agarose gel with an electric field that alternates (pulsates) in two directions. This technology plays a key role in modern genomics, as it allows manipulations with DNA of whole chromosomes or their large fragments. In this review, we discuss (1) the theory behind PFGE; (2) different instruments based on the principle of pulsed field, as well as their advantages and limitations; (3) factors affecting the DNA mobility in PFGE gel; and (4) practical applications of the technique.     

The biased reptation model of DNA gel electrophoresis: mobility vs molecular size and gel concentration

The biased reptation model provides a good framework for interpreting the results of continuous field DNA electrophoresis experiments performed in agarose gels. Here we discuss the main features of the mobility-molecular size and mobility-gel concentration diagrams as obtained from new extensive computer simulations of the model. Our aim is to suggest a global and coherent picture of this widely used yet poorly understood experimental technique, and to point out the areas where a systematic experimental study is still needed.     

Multiresolution image registration for two-dimensional gel electrophoresis

In proteomic research, two-dimensional electrophoresis (2-D) is an important tool for investigating differential patterns of qualitative and quantitative protein expression. The strength of the technique is due to its unrivalled power of being able to separate simultaneously thousands of proteins. The key to the comparison of 2-D protein profiles, however, lies in the use of a fast and robust image matching process which is essential to the subsequent quantification procedure. To satisfy the growing demand for a robust and fully automatic method of matching 2-D gel protein separation profiles, we describe in this paper a novel registration technique based on image intensity distribution rather than selected features. The method uses a multiresolution representation of the gel profiles and exploits the fact that coarse approximations to the optimal matching can be extracted efficiently from low-resolution images. This permits the removal of misalignments at different scales in a systematic manner and the strength of the new method has been confirmed by a double blind trial of 111 2-D gel pairs. The proposed method requires neither landmarks nor an a priori image alignment, and takes about five seconds for processing a typical gel pair on a standard personal computer.     

BBC microcomputer controlled field inversion gel electrophoresis

Agarose gel electrophoresis to separate DNA molecules is a widelyused technique in molecular biology but there is an upper limitto the sizes that can be resolved. Pulsed field techniques haveextended this limit but require expensive equipment. Here wedescribe a home-made control unit to interface conventionalelectrophoresis equipment to a BBC microcomputer for the purposesof field inversion gel electrophoresis. Received on October 6, 1987; accepted on November 10, 1987     

Preparative agarose gel electrophoresis for the purification of small organelles and particles

A novel preparative method of quantitative flatbed agarose gel electrophoresis has been used to separate a number of small subcellular structures, such as ribosomes, coated vesicles, smooth vesicles, and ferritin. The technique utilizes continuous elution of a second, electrophoretically "downstream," well in the agarose gel. The elution occurs concurrently with the electrophoresis, so essentially no additional time is required for the recovery of the structures. The technique is nondestructive, relatively simple and inexpensive, and can be used by modifying any nonsubmerged horizontal agarose gel system. The preparative separation of small organelles and subcellular structures according to their charge allows the purification of small structures previously difficult to isolate by conventional techniques. Two novel structures purified by this technique are described: a short intermediate filament-like species consisting of a single polypeptide of Mr 142,000, and an ovoid species (70 X 35 nm) whose protein composition is dominated by a polypeptide of Mr 104,000.     

Two-dimensional gel electrophoresis image registration using block-matching techniques and deformation models

Block-matching techniques have been widely used in the task of estimating displacement in medical images, and they represent the best approach in scenes with deformable structures such as tissues, fluids, and gels. In this article, a new iterative block-matching technique—based on successive deformation, search, fitting, filtering, and interpolation stages—is proposed to measure elastic displacements in two-dimensional polyacrylamide gel electrophoresis (2D–PAGE) images. The proposed technique uses different deformation models in the task of correlating proteins in real 2D electrophoresis gel images, obtaining an accuracy of 96.6% and improving the results obtained with other techniques. This technique represents a general solution, being easy to adapt to different 2D deformable cases and providing an experimental reference for block-matching algorithms.     

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.     

DNA microarray probe preparation by gel isolation nested PCR

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.