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The non-chloroplastic α-glucan phosphorylase (EC 2.4.1.1) from spinach leaves has been purified to homogeneity as revealed by dodecylsulfate gel electrophoresis. Both purification and separation from the chloroplastic phosphorylase were achieved by chromatography on Sepharose-bound dextrin. The chloroplastic phosphorylase did not bind to Sepharose-dextrin and was removed from the column by washing with buffer, as verified by polyacrylamide gel electrophoresis of the buffer eluate and by chromatography of a preparation from isolated intact chloroplasts. The non-chloroplastic phosphorylase did bind to a high extent to Sepharose-dextrin and could be eluted by a dextrin gradient. Based on dodecylsulfate gel electrophoresis and pyridoxal phosphate determination, a molecular weight of about 90,000 was found for the monomer. Molecular-weight determination by porosity density gradient electrophoresis and gel filtration on Sephadex G-200 suggested that the native enzyme is a dimer, as are other phosphorylases.

A technique is described which combines electrophoresis in pore gradient polyacrylamide gel with crossed electrophoresis in an agarose gel containing an immune serum. This technique enables the study of antigenic relationships between constituents which differ in their molecular size. An example is provided with different oligomeric forms of purified β-amylase.

The use of gel electrophoresis in studies of nucleic acid-protein (especially DNA-protein) interactions has yielded much qualitative and quantitative information about a variety of such systems. The reduction in mobility of complexes relative to free DNA allows isolation and characterization of the complexes as well as determination of thermodynamic and kinetic properties of the interactions. This article begins with a review of recent applications of the "gel retardation" assay, by way of introduction to experiments in two areas. In the first, a hypothesis is tested regarding whether a DNA molecule with sizable proteins bound very near to each end migrates through a polyacrylamide gel differently than does the corresponding complex having the proteins in the middle of the DNA fragment. The data show little mobility differences for these types of complexes, implying that both may move in a linear, "snakelike", manner through the gel. The experiments also provide results pertaining to questions of DNA bending caused by the binding of the E. coli catabolite activator protein (CAP) and RNA polymerase to the lactose promoter region. It appears that DNA bending by CAP at its wild type lac binding site is retained in complexes where RNA polymerase is bound simultaneously at the lac UV5 promoter.

An apparatus suitable for the recovery of proteins from polyacrylamide gels on a milligram scale by displacement electrophoresis (isotachophoresis) is described along with a buffer system that is suitable for this purpose with most proteins. The technique is illustrated by the recovery of a protein from a 15% polyacrylamide gel. The recovery was almost quantitative and the eluted protein showed little contamination upon quantitative amino acid analysis and automatic Edman degradation.

A simple method is described of preparing a series of stable polymers of Bence-Jones protein, of known molecular weight, for use as molecular weight markers for sodium dodecyi sulphate (SDS) — polyacrylamide gel electrophoresis.

Although polyacrylamide gel has no affinity to lithium dodecyl sulfate (LDS) at 25 degrees C, the gel maximally binds 17 mg of LDS per gram dry weight at 4 degrees C. When polyacrylamide gel electrophoresis is carried out at 4 degrees C in the presence of LDS instead of sodium dodecyl sulfate (SDS) using a continuous buffer system, migration of proteins with lower molecular weight is accelerated as a result of the deficiency of LDS in the frontal region of the gel. When the gel is saturated with LDS, electrophoresis in the presence of LDS at 4 degrees C shows a resolution higher than that of SDS-polyacrylamide gel electrophoresis at 25 degrees C.

Micelles of sodium dodecyl sulfate (SDS) are significantly retarded by the addition of a small amount of dodecyl alcohol to a sample solution in SDS-polyacrylamide gel electrophoresis. The phenomenon can be ascribed to the decrease in charge density due to the incorporation of dodecyl alcohol into SDS micelles. The effect is extended to SDS-protein polypeptide complexes when the amount of SDS micelles is insufficient to accomodate the dodecyl alcohol. A similar effect is likely to occur when SDS-polyacrylamide gel electrophoresis is applied to a sample containing lipophilic materials.

C33k polypeptide, which is a cytosol polypeptide with molecular weight of 33 000 and approximate pI value of 7.5, has three common electrophoretic phenotypes and is an abundant polypeptide in peripheral blood lymphocytes, fibroblasts and red blood cells. Family and population studies indicate that the three phenotypes of C33k polypeptide are determined by two common alleles at a single autosomal locus. The gene frequencies of the two common alleles were 0.642 and 0.358, respectively, in a Japanese population. Since esterase D has a subunit size and gene frequencies similar to those of C33k polypeptide, the phenotypes of C33k polypeptide and esterase D were compared in 18 families totaling 72 members. Perfect concordance of the phenotypes between C33k polypeptide and esterase D was observed in all 72 members. In addition, a gene dosage effect on the expression of the phenotype of C33k polypeptide was observed in the red blood cell lysate from a patient with partial 13q trisomy who was reported to have two doses of EsD1 and one dose of EsD2. These data indicate that the polymorphic C33k polypeptide is esterase D, which is assigned to chromosome 13q14. This finding is useful for the detection of proteins coding for by chromosome 13q14-linked genes in the studies on human gene mapping using somatic hybrid cell lines and two-D gel electrophoresis.

A method was developed for high resolution electrophoresis of proteins in linear gradient (3 to 30%) polyacrylamide gel rods in a neutral phosphate buffer containing 0.1% sodium dodecyl sulfate. Well-defined protein zones were observed and improved resolution was attained especially for low molecular weight proteins in preparations containing a variety of polypeptides, e. g. viruses that are often separated by continuous gel methods. Electropherograms of continuous (8%)and gradient (3 to 30%) gels were made of purified vesicular stomatitis virus, variola virus, Rickettsia rickett-sii, and alpha and beta chains of hemoglobin in order to demonstrate the resolution of the gradient system.

Proteins fractionated by electrophoresis on 18% polyacrylamide gels with low crosslinking can be directly visualized by ultraviolet light-induced fluorescence and can be recovered by electroelution.

This chapter discusses the analysis of DNA fragments after enzymatic cleavage. The method describes some basic principles of agarose gel electrophoresis performed on a larger scale. The larger-scale gel allows for longer electrophoresis and a better resolution of fragments and can also resolve fragments from preparative digests of plasmids for making DNA probes. These gels are well suited for Southern blotting. Their disadvantage compared to minigels is the longer electrophoretic time required. The time required to carry out the procedure for analyzing the DNA fragment is a minimum of four hours. The procedure requires a horizontal agarose gel electrophoresis apparatus, some well-forming combs, and gel mold. The chapter also lists the reagents that are required in this method, such as ethidium bromide, a 40 × TAE buffer, and a gel loading solution. RE digest of the DNA samples is prepared in advance, and progress of digestion with a minigel is also checked side by side.

Preparative scale polyacrylamide gel electrophoresis has been employed for isolating milligram quantities of globin messenger RNA from total RNA of rabbit reticulocyte ribosomes. Adherence to defined conditions for gel formulation, polymerization and electrophoresis have allowed recovery of globin messenger RNA within 24 hours electrophoresis time. The biologic activity of mRNA so isolated has been determined by assay in mRNA-dependent protein synthesis systems, and the products identified by standard procedures as rabbit globin. Application of preparative scale polyacrylamide gel electrophoresis for isolating other mRNAs as well as for resolving various similar molecular weight messenger RNAs (i. e., alpha-and beta-globin messages) is concluded to be feasible and is being investigated.

The electrophoresis step in the RNA fingerprinting procedure has been modified by replacing the large and expensive electrophoresis tank with a horizontal agarose gel electrophoresis box, a common piece of laboratory equipment. Two RNA molecules (67 and 298 nucleotides long) transcribed in vitro from a truncated human β-globin gene have been fingerprinted and analyzed by the modified technique. The resolution of the oligonucleotides in this procedure is comparable to that seen using the tank and is sufficient to separate relatively large oligonucleotides with similar base composition.

Publisher Summary This chapter discusses the detection and localization of single base changes by denaturing gradient gel electrophoresis. For most purposes, single-stranded deoxyribonucleic acid (DNA) probes 140–1000 bases in length are used, which allows about half of this number of base pairs to be screened for base changes in a single gel lane. The chapter discusses the use of single-stranded RNA probes. It describes the development of two new methods for detecting and localizing single base changes in cloned and genomic DNA. In both procedures, a single-stranded, radioactively labeled probe of wild-type sequence is annealed to cloned or genomic DNA. If the DNA under test carries a single base change, a hybrid double-stranded species containing a mismatch is formed. In the first procedure, either a ribonucleic acid (RNA) or a DNA probe is used, and the mismatched duplex is separated from the perfectly paired, wild type duplex by electrophoresis in a denaturing gradient gel. In the second procedure, the probe is composed of single-stranded RNA and the mismatch is cleaved by ribonuclease; the resulting cleaved products are examined by polyacrylamide gel electrophoresis and autoradiography. It is estimated that each method will detect approximately 50% of all possible substitutions, insertions, or deletions in a probed region. Although there is some overlap in the base changes that are detected with the two procedures, it is likely that the use of both methods will be complementary, resulting in the detection of a very large fraction of all possible substitutions and they should be particularly useful in mapping and diagnosing small mutations that result in genetic disease. This method has led to the detection of neutral polymorphisms for genetic linkage studies. The chapter lists all the equipment needed to prepare and run the gels and also describes the hybridization procedure for RNA probes.

Electrophoresis in vertical polyacrylamide gel slabs containing 8 m urea was used to examine the products of incomplete hydrolysis of bacteriophage f2 RNA generated by a number of nucleases. Oligonucleotide patterns were reproducible and characteristic of each nuclease; even closely related enzymes (i.e., with the same primary specificity) produced bands which were unique. Enzymes which were covalently bound to agarose generated band patterns which were indistinguishable from those produced by the soluble enzymes, suggesting their use in RNA sequence analysis. Agents which modify secondary structure in the RNA substrate significantly affected the oligonucleotide patterns resulting from the action of a given nuclease. The results indicate that both gross enzyme specificity and RNA secondary structure determine which bonds are hydrolyzed in early stages of the enzyme catalyzed breakdown of RNA. Secondary aspects of nuclease specificity are demonstrable but appear to be much less significant.

An improved procedure, "thermoblotting", is described for transferring proteins by diffusion from PhastGel Gradient media to an immobilizing matrix after horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis the gels were left on the separation bed of PhastSystem, the blotting matrix was applied and a transfer temperature was selected between 5-70 degrees C. An experimental series at fixed diffusion times showed that the transfer yield was significantly increased with temperature. The evaluation was done visually after staining of the blots with colloidal gold. An evaluation study comparing nitrocellulose, nylon, and polyvinylidenedifluoride of different pore sizes is also reported. Finally, the transfer efficiencies for 125I-labelled bovine serum albumin and soybean trypsin inhibitor were estimated using four different blotting procedures: two diffusion blotting techniques and two electrophoretic blotting techniques (tank vs. semi-dry).

Publisher Summary This chapter discusses the utilization of boronate-containing gels for the electrophoretic analysis of both ends of ribonucleic acid (RNA) molecules. It describes the development of an affinity electrophoretic method, which is based on the specific recognition of free ribose-type cis-diol functional groups, for avoiding traditional nucleolytic terminal analyses, which inevitably lead to the destruction of the starting material. A combination of this selectivity with enzymatic or chemical reactions specific for certain terminal phosphorylation patterns enables to distinguish among the different phosphorylation types in a single, nondestructive electrophoresis experiment. Conversely, enzymes specific for various terminal modifications may be assayed by this system using well-defined RNA termini as substrates. The technique of boronate affinity electrophoresis combines the use of two observations, which have previously been well characterized as separate entities. To combine the property of boronate/diol complex formation and the principle of affinity electrophoresis for studying the termini of RNA, a boronic acid is acylated with acryloylchloride (Scheme 1), and the resulting acrylamide derivative could be covalently incorporated into a polyacrylamide electrophoresis gel. The chapter describes several procedures including the synthesis of N-Acryloyl-3-aminophenylboronic Acid (APB). The method described in the chapter involves a straightforward chemical synthesis at moderate cost and the simple addition of a further component to the standard polyacrylamide gel electrophoresis mixtures. The examples illustrated in the chapter show the way the application of affinity electrophoresis can be used to analyze the termini of RNA in the field of nucleic acids.

Factor IX was isolated from human plasma by a six-step procedure employing adsorption to DE-52 cellulose in batches, aluminium hydroxide adsorption, affinity chromatography on heparin-coupled Sepharose, preparative polyacrylamide disc gel electrophoresis, immunosorbent absorption, and DE-52 cellulose chromatography. Determination of factor IX activity and antigen revealed a specific activity of about 290 U per absorbancy unit at 280 nm. Analytical polyacrylamide disc gel electrophoresis showed one major and three to four minor bands with lower electrophoretic mobility. Disc gel electrophoresis in the presence of 10 M urea gave a double major band and two additional minor bands with higher electrophoretic mobility. One major band was observed in SDS polyacrylamide gel electrophoresis, and no change in mobility took place after reduction.

Proteins were subjected to polyacrylamide gel electrophoresis at pH ∼ 4 in the presence of the cationic detergent HPCl. The relative electrophoretic mobilities of the proteins were correlated to their molecular weight.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this ‘three-dimensional’ polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.

A phospholipase A 2 (PLA 2 , EC 3.1.1.4) from rat lung has been isolated and characterized. PLA 2 was purified with ion-exchange and affinity chromatography and the purified enzyme was characterized with regard to pH optimum and calcium dependence. The isolated enzyme was also analyzed with two-dimensional gel electrophoresis and identified by Western immunoblots. The enzyme activity was found to be highest at pH 9.5–10.0, with a requirement for calcium, and the molecular mass was estimated to be 12 kDa by means of SDS-polyacrylamide gel electrophoresis. The two-dimensional gel electrophoresis analysis revealed two isoforms of PLA 2 with isoelectric points of 7.8 and 9.5. On DEAE-Sepharose, PLA 2 eluted as two peaks, one in the flow-through fraction and the other with increased salt concentration. Both peaks contained the same two PLA 2 isoforms, as judged by two-dimensional gel electrophoresis. These results demonstrate the presence in rat lung of two isoforms of a calcium-dependent 12 kDa PLA 2 with alkaline pH optimum. Using two-dimensional gel electrophoresis, this enzyme can be identified also in rat bronchoalveolar lavage fluid.

Polyacrylamide gel electrophoresis of Fluorescamine-modified peptides is a rapid (90 min), sensitive ( Fluorescamine-modified peptides, ranging in size from 3 to 215 amino acids, have been examined in a variety of buffer systems between pH 2.3 and 9.8 and gel concentrations varying between 4% and 16% acrylamide. Similar peptides were examined by high-voltage paper electrophoresis and detected by Fluorescamine. Peptides of low solubility have been examined in the presence of urea. The method reported here was used to resolve small peptides differing in size by a single amino acid, as well as peptides of the same size differing only by a sinle charge. It was successfully employed as a criterion for homogeneity of peptides in the course of purification for amino acid sequence analysis.

The zone dispersions of the reduced subunit of β-lactoglobulin B and its derivative with sodium dodecyl sulfate (SDS) were measured during polyacrylamide gel electrophoresis (PAGE) using the apparatus for continuous optical scanning at 280 nm. The ratio of apparent diffusion coefficients (D′) of the reduced subunit of β-lactoglobulin B (1.71 × 10−6 cm2/s) and of its SDS-derivative (7.1 × 10−7 cm2/s) was found to be 2.4 under the conditions of PAGE (pH 10.4, 0.015 ionic strength, 1°C, 4 mA/cm2 current density, 50 μg protein load, 10% T gel) used. This is nearly twice the value of 1.3 predicted, under the assumption of sphericity for these protein molecules, on the basis of the binding of 1.4 g of SDS per gram of protein. It is postulated that the increment in zone sharpness (decrease in apparent diffusion coefficient) over that predicted by SDS binding alone is a general property of SDS-proteins providing gel electrophoresis in SDS-containing buffers with a resolving power larger than that obtained in the absence of the detergent.

An investigator must take several factors into account for the process of staining and recording the location of bands after electrophoresis (Wilson, 1979, 1983). These should include (1) the selection and availability of an appropriate dye of satisfactory quality that will give adequate sensitivity; (2) the selection of a solvent that will provide a suitable dye concentration, solubility, and stability; (3) the formation of a stable dye—protein complex; (4) the easy removal of excess free dye for easy detection of stained proteins; and (5) storage of gels for further reference or for permanent recording by photography or analysis by computer, densitometry, or spectrophotometry.

A two dimensional preparative polyacrylamide gel electrophoresis system is described which has both high resolving power and high loading capacity (approximately 100 mg protein). The first dimension is electrophoresis toward the cathode at acid pH in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC), and the second dimension is electrophoresis toward the anode in the presence of sodium dodecyl sulfate. Proteins purified in this way have been sequenced and used successfully as antigens.

The catalytic subunits of adenosine 3′:5′ monophosphate-dependent protein kinase (ATP:protein transferase, E.C. 2.7.1.1.37) from the soluble and membrane fractions of swine kidney were purified to homogeneity by a new procedure and their structural, kinetic and immunological properties were compared. The specific activities of the purified enzymes were 2.35 and 2.6 µmol/min/mg of protein, with histone as the substrate. Both preparations contained a single polypeptide chain, and only one band was observed upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of both enzymes determined by gel electrophoresis was 42 000 ± 1000, and sedimentation equilibrium yielded a value of 41000 ± 800. Analysis by sedimentation velocity showed the presence of a single peak with and S20,w of 3.1 ± 0.2 for each preparation. The amino acid compositions are very similar, and each enzyme contains about one residue of cysteine which is essential for enzymatic activity. ATP and Mg2+ protect both enzymes from inhibition by thiol specific reagents to the same extent. The catalytic subunits have similar apparent K m's for protein substrates. The enzymes exhibit single completely confluent precipitin lines when examined by immunodiffusion and the particulate catalytic subunit competitively displaces the soluble 125I-catalytic subunit in homologous radioimmunoassays. The soluble and particulate 125I-catalytic subunits bind to the regulatory subunits in the washed plasma membranes with attendent loss of kinase activity, which could be reversed by cyclic AMP. The results of experiments with kidney cortex slices treated with parathyroid hormone, epinephrine or dibutyryl cyclic AMP showed the translocation of phosphotransferase activity from the cytosol to the particulate membrane fraction. Taken collectively, these observations suggest that only one form of the catalytic subunit of cyclic AMP-dependent protein kinase is present in swine kidney, and that it may exchange between the cytosol and membrane fractions in response to specific physiological signals.

A method for resolving an overlapped polypeptide pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis was described. The procedure was essentially a Gaussian fitting using the least squares method, and could resolve more than 20 overlapped components simultaneously. The applicability to overlapped and shouldered patterns was evaluated using practical electrophoretic data with varying amounts of mitochondrial samples. The relative contents of respective polypeptide components gave a good agreement regardless of the loaded amounts.

Sea urchin histones can be separated from ribosomal proteins by two-dimensional gel electrophoresis. Electrophoresis on Triton X-100/6 m urea gels in the first dimension results in preferential retardation of the histones, which then migrate more rapidly than ribosomal contaminants on SDS gel electrophoresis in the second dimension. The advantages and generality of the system are discussed.

The effect of electrophoresis conditions on the separation of low molecular weight RNAs has been studied. Electrophoresis at pH 5.8 or pH 8.3, presence or absence of 7 m urea, and heating all influence the relative migration of specific Escherichia coli tRNAs. Combinations of these electrophoresis conditions were used to find an improved 2-dimensional polyacrylamide gel system for separating low molecular weight RNAs. A first dimension, 16% gel with 7 m urea at pH 8.3 followed by an identical second dimension gel without urea gave the greatest separation of E. coli and rat liver cytoplasmic tRNAs. The rat liver tRNA was separated into approximately 45 spots. Eukaryotic tRNAs gave more complex patterns than E. coli tRNAs both in this new 2-dimensional system and in the previously reported system, which used a pH 5.8 gel in the first dimension gel and a pH 8.3 gel in the second dimension. The separation of tRNAs by polyacrylamide gel electrophoresis probably depends on conformational differeces and changes in the ionization of some bases. The use of this and other 2-dimensional gel systems for specific purposes is also discussed.

Two-dimensional gel electrophoresis with non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide gels in the second dimension has been used for the analysis of organ-specific proteins in maize. The method has been used to study the whole protein pattern of developing organs and adult leaves as well as protein patterns of in vitro translation. Examples of two-dimensional immunoblotting and in vitro translation of endosperm-specific proteins are also shown. Two-dimensional gel electrophoresis appears as an essential analytical step in the identification of organ-specific proteins and for the detection of the protein products related to organ-specific genes identified by other means., The authors acknowledge the Fnancial support of CSIC (grant No 500) and Plan PROA (CSIC-CDTI).

A novel cetyltrimethylammonium bromide-polyacrylamide gel electrophoresis system which is useful for the separation of native forms of proteins consistent with their molecular weights is reported here. Many proteins examined in this system demonstrated the same association patterns which have been shown by other techniques to exist under nondenaturing conditions. In addition, biological activity could be assayed directly in the gel after electrophoresis. Based on the peculiar characteristics of cetyltrimethylammonium bromide, a possible explanation which may account for the behavior of proteins in this system is presented.

A simple and specific detection method for multiple forms of cystine aminopeptidase (oxytocinase-isoenzymes) in pregnancy serum is described using S-benzyl-L-nitroanilide as substrate. The relative distribution of both cystine aminopeptidase isoenzymes at different stages of pregnancy is given.

From other chapters it will be evident that the molecular biologist often needs to separate the components of a complex mixture of DNA molecules either analytically or preparatively. Although it is sometimes possible to use density or affinity techniques to separate DNA molecules on the basis of their base compositions, such an approach cannot be used with complex mixtures or when, as is often the case, different DNAs have similar base compositions. Separations based on size differences are far more generally useful. The identification of restriction fragments containing a particular sequence of bases (e.g. a particular gene) depends on the separation of DNA fragments according to size (Chapter 15), as does the mapping of the position of such a sequence in the intact DNA. Separated fragments can be recovered and then replicated indefinitely by cloning (Chapters 11, 12 and 13). The sequencing of DNA also relies on the ability to resolve nucleic acids by size (Chapter 16).

The nonionic detergent Triton X-100 binds in varying proportions to specific ribosomal proteins and decreases the relative mobility of these proteins during electrophoresis. When Triton X-100 binds to these ribosomal proteins in the first-dimension gel, the resolution of the ribosomal proteins in the second-dimension gel pattern is greatly improved. Maximum binding of Triton X-100 to the ribosomal proteins is dependent on pH, urea concentration, and the complete reduction of cysteine and methionine. After first-dimension electrophoresis the Triton X-100 in the gel does not interfere with the binding of sodium dodecyl sulfate to the ribosomal proteins and the molecular weight of these proteins can still be estimated directly from the second-dimension slab gel.

A procedure using polyacrylamide gel electrophoresis has been devised for determining the nucleotide composititon of small amounts (50 μg) of RNA. The procedure permits analysis of the separated nucleotides in the gel matrix, thereby avoiding sample manipulation and allowing for greater reproducibility.

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 silverstaining 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.

A two-dimensional electrophoretic system has been developed for the separation of chloroplast thylakoid membrane proteins. This system incorporates nondenaturing polyacrylamide gel electrophoresis in the presence of the nonionic detergent dodecyl-β- d -maltoside in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Thylakoid membranes isolated from Spinacia oleracea were solubilized in 1.0% dodecyl-β- d -maltoside and separated in 4–7% linear acrylamide gradient tube gels which contained 0.05% dodecyl-β- d -maltoside. After electrophoresis, the tube gels were equilibrated with a sodium dodecyl sulfate-containing equilibration buffer and applied to a 12.5–20% acrylamide linear gradient gel. The Lammelli buffer system was used in both dimensions. The two-dimensional gels were analyzed by staining sequentially with 3,3′,5,5′-tetramethylbenzidine-H2O2, Coomassie blue, and silver staining. A number of protein components were identified on “Western blots” of these two-dimensional gels by immunological localization. Membrane protein complexes such as the light-harvesting chlorophyll a b protein complex, photosystem I, photosystem II, the cytochrome b 6 f complex and ribulose bisphosphate carboxylase appear to migrate as essentially intact complexes in the first dimension and appear as vertical series of resolved subunits in the second dimension. This technique complements isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis in providing additional information concerning the subunit composition of membrane protein complexes and may prove to be of general utility for studying the protein composition of other membrane systems.

Polyacrylamide gel electrophoresis has been used to study the heterogeneity of glucagon, and changes in composition resulting from thermal degradation. The same technique can be employed, as a limit test, to monitor the amounts of insulin and desamidoglucagon in glucagon intended for therapeutic use.

Isolation of nucleolar proteins was obtained by dissociation in the presence of urea – guanidine hydrochloride, followed by high-speed centrifugation to remove nucleic acids. At least 31 fractions of nucleolar proteins were detected by isoelectrofocusing gel electrophoresis in the pH range 3.5–10. Following two-dimensional gel electrophoresis on sodium dodecyl sulfate – polyacrylamide slab gels, more than 100 components of nucleolar proteins were identified. Two-thirds of nucleolar proteins were located in the pH range 5–8 following isoelectrofocusing. The molecular weights of these classes of proteins were shown to be mostly 30 000 – 70 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis.

A rapid, inexpensive method for the salt-free concentration of small quantities of proteins for analysis by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been developed. Proteins adsorbed to diatomaceous earth are subsequently removed using either sodium dodecyl sulfate or urea solubilization reagents for 2D-PAGE analysis. This procedure has been found to concentrate proteins having wide ranges of molecular weight and charge. It is also valuable for the concentration of large numbers of small samples from cells cultured in vitro.

Publisher Summary Because the functional specificity of plasma lipoproteins resides in major part in the apolipoprotein moieties associated with the lipoproteins, techniques that combine size analysis, such as gradient gel electrophoresis, with apolipoprotein identification, either directly by immunoblotting or indirectly by immunoaffinity chromatography, appear highly promising for investigation of lipoprotein structure and function. This chapter discusses the application of gradient gel electrophoresis primarily to the analysis of HDL and LDL subpopulations and indicates approaches for more detailed identification of these subpopulations. Gradient gel electrophoresis entails the migration of charged particles through a matrix composed of increasing concentrations of polyacrylamide gel. The effective pore size of the matrix is progressively reduced as the gel concentration increases, resulting in differential retardation of the migrating charged particles. Although protein molecular weight determination is possible by gradient gel electrophoresis by use of appropriate calibration standards, such determination of lipoprotein molecular weights is still only approximate and requires validation by use of lipoprotein species with molecular weights determined by other methods.

The incorporation of 6 m urea into polyacrylamide-agarose gels reduces the width of the bands formed upon electrophoresis by single-stranded RNA molecules in the 10 5 - to 10 6.5 -dalton size range. As a result the resolving power of gels is increased and molecules that differ in size by as little as 5% can be separated.

The resolving power of two-dimensional ultrathin-layer polyacrylamide gel electrophoresis with and without “stacking” was investigated. Side-by-side analysis shows that the use of a properly adjusted upper gel improves the resolution and reproducibility of this sensitive analytical method. The effects of various detergents (Nonidet-P40, Zwittergent, urea) on the ultrathin-layer poltacrylamide gel electrophoresis were also investigated. For this case, whole cell proteins of Pseudomonas aeruginosa and Staphylococcus aureus treated with different detergents were electrofocused in the presence of the same detergents.

Human urinary kallikrein (urokallikrein) has been purified by affinity chromatography with aprotinin coupled to CH-Sepharose and by gel filtration. The isolation procedure, which was performed under mild conditions, was completed in a 36-hr period and yielded an overall recovery of more than 75% and a purification of 1727-fold. Homogeneity of the urokallikrein was demonstrated by three criteria: the coincidence of the stained protein band and functional urokallikrein in duplicate gels after alkaline disc gel electrophoresis; the appearance of a single stained band of molecular weight 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis of reduced and unreduced enzyme; and the finding of a single amino-terminal residue, namely alanine after dansylation and acid hydrolysis of purified enzyme. The Km of urokallikrein on N alpha-p-tosyl-L-arginine methyl ester was 400 microM, and the Vmax was 194 mumol/min per mg of protein, which is higher than that observed with any previous preparations. The molecular weight of 48,700 determined on gel filtration and the molecular weight of 48,000 observed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis are in good agreement with the molecular weight of 48,213 calculated from the amino acid composition. The finding of a molecular weight higher than those previously reported, namely 27,000-43,500, the increased functional activity on tosylarginine methyl ester, and the detection of a single amino-terminal residue are consistent with the isolation of a more native protein by the procedure described in this paper.

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.

Publisher Summary This chapter describes the techniques for the recovery of DNA from gels. Agarose or polyacrylamide gel electrophoresis is widely used as a high resolution technique for fractionation of DNA molecules by size. The basic methods for recovery of DNA from gel slices are (1) electroelution, (2) elution by diffusion, (3) gel dissolution, and (4) extrusion of DNA by gel compression. Choice of a method depends on a number of factors such as the type of gel matrix, gel concentration, DNA size, scale of the procedure, and the level of gel contamination that can be tolerated in the recovered DNA solution. With agarose gels, contamination of recovered DNA with varying amounts of agarose is a certainty, and in many cases this interferes with subsequent enzymatic treatments or analysis. Methods of agarose removal include equilibrium density gradient centrifugation, extraction, and chromatographic separation. The chapter describes the methods of DNA recovery, the methods of purification and concentration of recovered DNA, and the choice of method.

A specially designed apparatus and conditions are described for the rapid analysis of ribosomal proteins by two-dimensional gel electrophoresis on a micro scale. The resolution of proteins in electropherograms is comparable to that obtained with other systems, but because of miniaturization, only 0.5 to 1 mug of each protein is required, and the entire procedure, including electrophoresis in both dimensions, and staining and destaining can be completed in 6 to 7 hours.