An exponential gradient marker for use with minigel polyacrylamide electrophoresis systems

Minigel electrophoresis has reduced the required sample size and cost of running polyacrylamide gels without loss of resolution. The many problems incurred in the minigel system when pouring multiple gradient gels are eliminated by pouring individual gels with the described economical, exponential gradient marker, which can accurately deal with the small gel volumes. The advantages of exponential gradients are discussed.     

A rapid and sensitive potentiometric assay for monoamine oxidase using an ammonia-selective electrode

A convenient method to store polyacrylamide gel slabs is described. The method involves sealing the wet gels between transparent plastic sheets using a kitchen-type bag sealer. The sealed gels are suitable for long-term storage, photography, and densitometry and can be used in autoradiography.     

The combined determination of indolyl-3-acetic and abscissic acid in plant materials

A method for isoelectric focusing of antibodies in agarose gels with ampholytes synthesized in the laboratory from pentaethylenehexamine is presented. The ampholytes are easy to prepare, give results comparable to those with commercial ampholytes, and are much less expensive. Substituting agrarose bonded to plastic film for the polyacrylamide gels on glass plates commonly used offers many advantages and enhances the usefulness of isoelectric focusing as a tool for studying antibody molecules.     

Quantitative electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels onto positively charged nylon membranes

A method for the reproducible and quantitative electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a single sheet of either Zetabind or Gene Screen Plus membranes is presented. This procedure uses commercially available equipment and includes three crucial parameters: the omission of methanol from the transfer buffer, the use of thin (0.75-mm) resolving gels, and a newly developed protocol for pretreatment of the polyacrylamide gel after electrophoresis and before electroblotting. This combination of parameters yields a blot that both qualitatively and quantitatively reflects the proteins in the original polyacrylamide gel.     

A vertical submarine electrophoresis apparatus for polyacrylamide minigels

A vertical submarine electrophoresis apparatus for use with minislab polyacrylamide gels is described. The design allows polyacrylamide gels to be run with the same ease and convenience that agarose gels are run with horizontal submarine apparatuses. The vertical submarine features a single buffer chamber with a restriction between the upper and the lower portions of the chamber. Acrylamide gels, cast between 9 X 10-cm glass slides, are inserted into the restriction and are completely immersed in buffer. Thus, current flows primarily through the gel itself, but some current flows through the buffer in the restriction surrounding the gel. Because water-tight separation of buffer chambers is not necessary, time-consuming and/or expensive procedures such as sealing with agarose or using fragile notched glass plates are eliminated. The apparatus can be set up to run a gel in less than 30 s. It is versatile in that gels of varying thickness (0.5, 0.8, 1.5, and 3 mm) can be run on a single apparatus. The apparatus has been used for sodium dodecyl sulfate gels, low ionic strength native gels for nucleoprotein complexes, and composite acrylamide-agarose gels.     

Highly simplified analytical or preparative slab gel electrophoresis

A simple, rapid, and efficient procedure has been developed for performing analytical or preparative slab gel electrophoresis using only common laboratory materials which can be assembled without special tools or equipment. From one to four gel slabs of variable size can be made between glass plates embedded inside a watertight, supple plastic bag which is then used as the upper electrode buffer chamber. This technique has been applied, with different electrophoretic systems, for both the analysis and isolation of serum proteins and rat liver histones.     

A simple and cost-effective solid-phase protein nano-assay using polyacrylamide-coated glass plates

A new solid-phase protein nano-assay is suggested for simple and sensitive estimation of protein content in sample buffers (a 1-μl sample is sufficient for analysis). The assay is different from conventional “on-filter” assays in that it uses inexpensive fully transparent polyacrylamide gel (PAAG)-coated glass plates as solid support and, thus, combines the convenience of “on-membrane” staining with the sensitivity and ease of documentation of “in-gel” staining (and, therefore, is especially suited for standard lab gel documentation systems). The PAAG plates assay is compatible with all dyes for in-gel protein staining. Depending on the sensitivity of the staining protocol, the assay can be used in macro-, micro-, and nano-assay formats. We also describe a low-cost two-component colloidal Coomassie brilliant blue G-250 (CBB G-250) staining protocol for fast quantitative visualization of proteins spotted on a PAAG plate (the detection limit is up to 2 ng of proteins even when using a Nikon CoolPix digital camera and white light transilluminator instead of a gel scanner). The suggested colloidal CBB G-250 protocol could also be used for visualizing nano-amounts of proteins in polyacrylamide gels. The PAAG plate assay could be useful for proteomic applications and, in general, for all cases where a fast, sensitive, and easily documentable cost-effective solid-phase protein assay is required.     

Visualization by chilling of protein bands in polyacrylamide gels containing 8 urea: Preparation and quantitation of foot-and-mouth disease virus capsid proteins

Protein bands become visible in polyacrylamide gels containing 8 urea after chilling the gels in air for 5 to 10 min at −70°C. Urea appears to crystallize preferentially as opaque bands in regions of the gel where protein reduces the amount of free water available as solvent for the urea molecules. Thus detected, the gel sections containing protein bands from foot-and-mouth disease virus can be immediately cut out, and their proteins obtained by electrophoretic elution or extraction procedures. Analysis of the proteins for purity and concentration is then carried out by electrophoresing measured aliquots on analytical gels, staining with Coomassie brilliant blue, scanning the gels for absorbance at 600 nm, and converting peak areas to micrograms of protein using Folin phenol standard curves determined for each purified capsid protein. The most basic capsid protein and its in virion proteolytic-cleavage products stain metachromatically.     

A simplified electrophoretic system for determining molecular weights of proteins.

Electrophoresis of 31 different proteins in commercially prepared polyacrylamide gradient gels, Gradipore, yields a linear relationship between a hypothetical limiting pore size (the reciprocal of a limiting gel concentration, GL) and the cube root of the mol.wt., over the range 13 500-9000 000. A regression analysis of these data reveals that 98.6% of all variability in 1/GL is explained by the molecular weight, and this degree of accuracy compares favourably with existing methods for the determination of molecular weight by retardation of mobility in polyacrylamide. This new procedure has the additional advantages that molecular-weight standards can be obtained from readily available body fluids or tissue extracts by localizing enzymes and other proteins by standard histochemical methods, and that the same electrophoretic system can be used in determining molecular weights as is used in routine surveys of populations for individual and species variation in protein heterogeneity.     

Selection of chemical spacers to improve isoelectric focusing resolving power: implications for use in two-dimensional electrophoresis

Presented here is a straightforward and inexpensive method for expanding isoelectric focusing pH gradients relative to the gradients that are formed by commercially available narrow range ampholytes. This method requires no special equipment or techniques and is applicable to isoelectric focusing in acrylamide gels, in Sephadex, and in agarose. The utility of separators in improving the resolving power of two-dimensional polyacrylamide gel electrophoresis is demonstrated using proteins from the exocytotic trichocyst organelle of Paramecium tetraurelia. The mode of action of separators is briefly described.     

A simple preparative polyacrylamide disc gel electrophoresis apparatus: purification of three branched-chain amino acid binding proteins from Escherichia coli

The equipment used for preparative polyacrylamide gel electrophoresis has been either difficult to construct or costly if purchased commercially. An inexpensive preparative acrylamide gel apparatus and peristaltic pump are described in this paper which are easy to use and may be constructed from readily available materials. The construction of the preparative gel apparatus requires no special machining or glass blowing.This report describes the use of the disc gel apparatus in the final purification step of three binding proteins which appear to be involved in the transport of the branched-chain amino acids in Escherichia coli. Two of these proteins have been described previously (1–4). The apparatus has also been successfully used in a number of other laboratories for the purification of a variety of other proteins (5–9).     

Immobilization of acrylamide-modified oligonucleotides by co-polymerization.

A flexible chemistry for solid phase attachment of oligonucleotides is described. Oligonucleotides bearing 5'-terminal acrylamide modifications efficiently co-polymerize with acrylamide monomers to form thermally stable DNA-containing polyacrylamide co-polymers. Co-polymerization attachment is specific for the terminal acrylamide group. Stable probe-containing layers are easily fabricated on supports bearing exposed acrylic groups, including plastic microtiter plates and silanized glass. Attachment can be accomplished using standard polyacrylamide gel recipes and polymerization techniques. Supports having a high surface density of hybridizable oligonucleotide (approximately 200 fmol/mm2) can be produced.     

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.     

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.     

Preparative Scale,High Resolution Purification of Low Molecular Weight DNA Fragments

Abstract

A commercially available continuous electro elution system has been used to separate and purify low molecular weight DNA fragments from polyacrylamide gels. This method has several advantages over previously reported methods for the recovery of DNA fragments from polyacrylamide gels. This technique, which gives very high recovery rates (80–95%), can be carried out on a relatively large scale and in a way that is not labour intensive. Data are presented for the purification of DNA fragments with molecular weights in the range 1–4 ± 10 (200–700 base-pairs), although the method is also applicable to larger molecular weight DNA fragments, RNAs and proteins.     

Isoelectric focusing with reduced cathodic drift and migration into the anode chamber

An apparatus has been developed to reduce cathodic drift and migration into the anode chamber in vertical gel rod isoelectric focusing (IEF). In contrast to commercially available apparatuses, this apparatus can easily handle many more gels at one time, and the length, diameter and shape of its gel can be arbitrarily changed. In addition, high concentrations of detergent can be used to dissolve the protein samples, and removal of the gel cylinders from the glass tubes is easy.     

Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.

We describe the use of polyacrylamide gel electrophoresis to estimate chain lengths of double- and single-stranded DNA molecules in the size range 20-1000 base pairs (or nucleotides). Double-stranded DNA molecules of known length produced either by organic synthesis or by restriction endonuclease digestion of viral DNAs were used as standards. The relative electrophoretic mobilities of these standards were examined on both nondenaturing (aqueous) polyacrylamide gels and on denaturing gels containing 7 M urea or 98% formamide. Electrophoretic mobility of DNA is a linear function of the log of molecular weight if appropriate conditions are used, although exceptions are noted. Chain lengths can be conveniently estimated by using as standards bacteriophage gamma DNA restriction fragments or commercially available tracking dyes.     

Detergent-mediated destaining of coomassie brilliant blue-stained SDS polyacrylamide gels

A simple and one-step detergent-mediated destaining procedure for SDS Polyacrylamide gels for proteins is described. Suspension (5%, w/v) of a commercially available household detergent, Vim Ultra, has been found to be very efficient in destaining polyacrylamide gels without interfering with the resolution of proteins. As compared to the routinely used solvent (methanol-acetic acid-water)-mediated destaining procedure, the present method is economical and user-friendly.     

Drying and storage of polyacrylamide slab gels: a simple procedure

A simple procedure for drying and storing of polyacrylamide slab gels is described. A polyacrylamide slab gel is fixed in acetic acid plus glycerol and then sandwiched between a gel bond plastic sheet and a dialysis membrane in the presence of a minute amount of gelatin and dried on the benchtop at room temperature. The fixed gel can be stored indefinitely.     

Visualization by chilling of protein bands in polyacrylamide gels containing 8 M urea: preparation and quantitation of foot-and-mouth disease virus capsid proteins

Protein bands become visible in polyacrylamide gels containing 8 m urea after chilling the gels in air for 5 to 10 min at ?70°C. Urea appears to crystallize preferentially as opaque bands in regions of the gel where protein reduces the amount of free water available as solvent for the urea molecules. Thus detected, the gel sections containing protein bands from foot-and-mouth disease virus can be immediately cut out, and their proteins obtained by electrophoretic elution or extraction procedures. Analysis of the proteins for purity and concentration is then carried out by electrophoresing measured aliquots on analytical gels, staining with Coomassie brilliant blue, scanning the gels for absorbance at 600 nm, and converting peak areas to micrograms of protein using Folin phenol standard curves determined for each purified capsid protein. The most basic capsid protein and its in virion proteolytic-cleavage products stain metachromatically.