Flat-bed Gel Research Articles

Analysis of plasmid profiles of Aeromonas salmonicida isolates by pulsed field gel electrophoresis

Plasmid constitutions of Aeromonas salmonicida isolates were characterised by flat-bed and pulsed field gel electrophoresis. Resolution of plasmids by pulsed field gel electrophoresis was greater and more consistent than that achieved by flat-bed gel electrophoresis. The number of plasmids separated by pulsed field gel electrophoresis varied between A. salmonicida isolates, with five being the most common number present in the isolates used in this study. Plasmid profiles were diverse and the reproducibility of the distances migrated facilitated the use of principal components analysis for the characterisation of the isolates. Isolates were grouped according to the number of plasmids supported. Further principal components analysis of groups of isolates supporting five and seven plasmids showed a spatial separation of plasmids based upon distance migrated. Principal components analysis of plasmid profiles and antimicrobial minimum inhibitory concentrations could not be correlated suggesting that resistance to antimicrobial agents is not associated with either one plasmid or a particular plasmid constitution.

Analysis of plasmid profiles of Aeromonas salmonicida isolates by pulsed field gel electrophoresis

Plasmid constitutions of Aeromonas salmonicida isolates were characterised by flat-bed and pulsed field gel electrophoresis. Resolution of plasmids by pulsed field gel electrophoresis was greater and more consistent than that achieved by flat-bed gel electrophoresis. The number of plasmids separated by pulsed field gel electrophoresis varied between A. salmonicida isolates, with five being the most common number present in the isolates used in this study. Plasmid profiles were diverse and the reproducibility of the distances migrated facilitated the use of principal components analysis for the characterisation of the isolates. Isolates were grouped according to the number of plasmids supported. Further principal components analysis of groups of isolates supporting five and seven plasmids showed a spatial separation of plasmids based upon distance migrated. Principal components analysis of plasmid profiles and antimicrobial minimum inhibitory concentrations could not be correlated suggesting that resistance to antimicrobial agents is not associated with either one plasmid or a particular plasmid constitution.

Analysis of Dissociation and Unfolding of Oligomeric Proteins Using a Flat Bed Gel Electrophoresis at High Pressure

A slab gel electrophoresis apparatus with the ability to operate over a pressure range of 10−3 to 2 kbar is described. The system presented here is an improvement of a previous apparatus (A. A. Paladini, J. L. Silva, and G. Weber, Anal. Biochem. 161, 358-364, 1987). It consists of a flat bed gel, with a significantly enlarged buffer reservoir, which eliminates the requirement of high concentrations of running buffers, and at the same time allows shorter runs, leading to enhanced resolution and reproducibility. The application of the method to the dissociation of the tetramer glycogen phosphorylase a as a function of hydrostatic pressure is described. The flat geometry of the apparatus allows for the first time the analysis of the stability of oligomers and their constituent subunits to chemical denaturation by urea gradient electrophoresis gels at high pressure. Dimeric hexokinase shows a reversible cooperative unfolding transition with a midpoint at 3.8 M urea. In contrast, the monomers unfold at very low urea concentration (<1.0 M). The observed differences in stability validates oligomerization as an important stabilizing element of the protein structure.

T-kininogenase activity of the rat submandibular gland is predominantly due to the kallikrein-like serine protease antigen γ

T-kininogen, the major kininogen in rat plasma, releases Ile-Ser-bradykinin (T-kinin) when incubated with trypsin, but is not a substrate for tissue kallikrein. Enzymes able to release T-kinins from T-kininogen have been found in the rat submandibular gland, but precise identification of these enzymes and their possible relationship to kallikrein-like enzymes has not been established. We studied T-kininogenase activity in fractionated submandibular gland homogenate. The main T-kininogen catalytic enzyme was purified and characterized, and found to be identical to antigen gamma, a kallikrein-like enzyme which we have previously characterized. Of other identified kallikrein-like enzymes only tonin showed weak T-kininogenase activity, which was about 0.25% of that of antigen gamma. No other T-kininogen catalytic enzymes were observed. Antigen gamma released a kinin which was identified as T-kinin by reverse-phase h.p.l.c. The T-kininogenase activity of antigen gamma had a Km of 29 +/- 4 microM and a kcat/Km of 140 M-1.s-1, and was comparable with its high and low molecular mass-kininogenase activity (7.4 and 10 micrograms of kinin/h per mg respectively). In contrast, tissue kallikrein released 0.2 and 42,200 micrograms of kinin/h per mg respectively. Thus antigen gamma is a weak kininogenase. The isoelectric point of antigen gamma, but not its molecular mass, differed from that of other kallikrein-like enzymes. Isoelectrofocusing in flat-bed gels combined with immunostaining was therefore a convenient method for identification. The kallikrein-like nature of antigen gamma was demonstrated by its immunological similarity to tissue kallikrein and tonin and by 91% and 87% amino acid sequence similarity with tonin and kallikrein respectively (67 amino acids sequenced). Complete identity was also not observed with other sequenced kallikrein genes, mRNAs or proteins.

Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate—polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies

A method for the preparative high-yield electroelution of proteins from sodium dodecyl sulphate (SDS) polyacrylamide gel strips was established. The method consisted of SDS-polyacrylamide gel electrophoresis, detection of proteins with sodium acetate and electrophoretic elution at 200 V for 3 h by utilizing a horizontal flat-bed gel electrophoresis apparatus. Standard proteins with molecular masses of 14–66 kilodalton (cytochrome c, aldolase, ovalbumin and bovine serum albumin) were recovered with an average yield of 73.6 ± 2.3%. A membrane-bound protein, rat skeletal muscle Ca 2+-ATPase (100 kilodalton) was also well recovered (over 60%). This method was applicable to the purification of proteins required for N-terminal amino acid sequencing and to raise antibodies.

An improved method combining two electrophoretic procedures: Application to the separation of lens α‐crystallin isoforms

Polypeptides having different net electric charges and very similar molecular weights, visualized as one single band in sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE), can be readily analyzed by an improved method combining two electrophoretic procedures. The methodology consists of the identification and isolation of selected protein bands from SDS-PAGE, their equilibration in an isoelectric focusing (IEF) sample buffer, and their casting and separation in an IEF flat-bed gel. This method requires no extra equipment, is highly reproducible, is suitable for quantitative and comparative studies, and is especially useful in the case of small samples. As a particular example, we analyze here the subunit composition of alpha-crystallins of young and embryonic quail lenses.

Immunological Detection of the Egf-Like Domain of the Core Proteins of Large Proteoglycans from Human and Baboon Cartilage

Recent data from the literature have shown that cDNA clones for the carboxyterminal domain of the core protein of large proteoglycan monomers from human cartilage contain an EGF-like domain, which appears to undergo alternative splicing. In the present study we have found that articular proteoglycans from human and baboon separated on agarose flat-bed gels and blotted onto nitrocellulose react with a rabbit antiserum to mouse EGF. In addition both forms of the proteoglycans (band I and band II) seen on these gels are reactive. Reactivity is seen with proteoglycans extracted from human articular cartilage of various ages (fetal, newborn, young and aged) and with proteoglycans extracted from cartilage of thanatophoric dysplasia and homozygous achondroplasia. Reactivity is dependent on prior digestion of the nitrocellulose blot with Chase ABC, suggesting masking of epitope by chondroitin sulfate. Reactivity of the EGF antiserum with cartilage proteoglycan core protein was also demonstrated in an ELISA system with core protein as coating antigen. The reactivity appears to reside in a tryptic peptide generated from Chase/keratanase digested core protein. The immunoreactive species migrates as a 68 KDa species on gradient gels. Immunological detection and quantitative analysis of the EGF-like domain could be useful for analysis of various proteoglycan samples.

Electrophoretic techniques in element species analysis. Comparison between flow-through and flat-bed techniques

A comparison of electrophoretic techniques for the separation of metal-protein complexes from food extracts is described. A preparative flow-through electrophoresis system with continuous elution of the fractionated substances and an analytical flat-bed gel electrophoresis technique with off-line electro-elution are optimized with regard to this separation problem. The metal-protein complexes are extracted from four flour samples using Tris-glycine buffer (pH 8.3). For the separation, polyacrylamide gels of 14% T and 3% C are prepared and Tris-HCl (pH 8.9) is used as an electrophoresis buffer. For a soy bean flour, not only the separation of protein fractions is achieved but also the metal distribution patterns that are determined by flame atomic absorption spectrometry are given. The results show that the use of the flow-through technique is limited to special fields of application, whereas the flat-bed electrophoresis with subsequent electro-elution of metal-protein complexes is a useful technique in element species analysis.

Preparative isoelectric focusing in ampholine electrofocusing columns versus immobiline polyacrylamide gel for the purification of biologically active leukoregulin

Preparative vertical and rotating horizontal (Rotofor) ampholine column and immobiline flat bed polyacrylamide gel isoelectric focusing were evaluated for the isolation of the biologically active acidic form of leukoregulin, a 50,000-Da glycoprotein lymphokine with tumor growth inhibitory activity. Leukoregulin secreted by normal human lymphocytes was concentrated by 10,000 nominal molecular weight size exclusion ultrafiltration and by DEAE anion exchange chromatography using step elution with 0.02 m Tris-HCl: 0.1 m NaCl, pH 7.4. Preparative isoelectric focusing was carried out in a 110-ml vertical column containing 1% ampholines in a pH 4–6 gradient at 15 W constant power for 16–18 h, in a Rotofor 55-ml horizontal column containing 2% ampholines in a pH 4–6 gradient at 12W constant power for 4–6 h, or in an immobiline pH 4.5–6.5 gradient within a 5% polyacrylamide 120 × 110 × 5-mm flat bed gel at 3 W constant power for 16–18 h. Recovery of biologically active leukoregulin from the vertical and horizontal ampholine columns was similar. The pH 4.9–5.2 fractions from the Rotofor ampholine column contained 4–7% and the fractions from the immobiline gel contained 4% of the leukoregulin activity applied to the electrofocusing column or gel, respectively. Analytical immobiline isoelectric focusing of the leukoregulin in the pH 4.9–5.2 fractions from the Rotofor column demonstrated that a single silver staining band with a p I of 5.1 can be obtained by this rapid method of preparative isoelectric focusing.

Hybrid isoelectric focusing: Adsorption of proteins onto immobilized pH gradient matrices and desorption by carrier ampholytes

AbstractComparing the separation patterns achieved by hybrid isoelectric focusing in immobilized pH gradients of 1 and 3 meqv/pH/L buffering power, it is demonstrated that a significant proportion of sample protein is lost in gels with the higher buffering power. A buffering power of 1 meqv/pH/L was found to be the lowest acceptable limit for flat bed gel hybrid isoelectric focusing still ensuring adequate control of the immobilized pH gradient. Protein losses were manifested as diminished overall staining intensity, disappearance of individual zones in dilution series, tailing effects and residual material at the application site, all of the above effects being more pronounced for larger than smaller proteins. Adsorption of proteins to charged binding sites of the immobilized pH gradient offers a plausible explanation for the observed phenomena. The visible effects of adsorption could be reduced by the addition of carrier ampholytes, but not by urea or detergents like Triton X‐100, 3‐(3‐chol‐amidopropyl)‐dimethylammonio‐l‐propanesulfonate (CHAPS) or octyl β‐thio‐glucoside, indicating ionic interactions between the immobilized charged groups, carrier ampholytes and proteins. The adsorption phenomena may strongly affect the quantification of proteins in immobilized pH gradients.

Collagen deficiency and ruptured cerebral aneurysms. A clinical and biochemical study.

Skin and temporal arterial biopsies were obtained from 17 patients undergoing surgery for ruptured cerebral aneurysm, and specimens were taken from six age- and sex-matched control surgical patients. Radioactively labeled and control tissue collagen patterns were studied by interrupted polyacrylamide gel electrophoresis (PAGE), using the trisborate buffer system or by carboxymethyl cellulose (CMC) chromatography. Type III/I collagen ratios were then measured from autoradiographs of the radioactively labeled samples using the Joyce Loebl gel scanner adapted for flat bed gels. In the case of the CMC labeled material, the ratios were measured by the ratios of the summed radioactively labeled alpha 1(III), alpha 2(II), and alpha 2(I) peaks. Eleven of the 17 patients were Type III collagen-deficient while all of the six control patients had normal collagen ratios. The implications of these findings are discussed.

Antigens of Alternaria: I. Isolation and partial characterization of a basic peptide allergen

A basic peptide allergen has been isolated from Alternaria extracts. Flat-bed gel preparative isoelectric focusing followed by dialysis and lyophilization allowed for concentration of substantial quantities of this heretofore unrecognized allergen. Rocket immunoelectrophoresis and autoradiography (using a 2 × concentrated, IgE serum fraction from Alternaria patient sera and 125 I-anti human IgE), a RAST inhibition assay, and skin testing of Alternaria-sensitive patients were used to demonstrate the allergenicity of this fraction. The allergenicity of this protein was sufficient to give 50% RAST inhibition at a concentration of 100 μg/ml using standard RAST procedures. The immunoelectrophoresis autoradiography also indicated allergenicity. Skin tests indicated that over 90% of patients hypersensitive to Alternaria crude extracts were also hypersensitive to the basic allergen fraction.

Allergens of Alternaria: further characterization of a basic allergen fraction.

A basic allergen fraction isolated from Alternaria extracts by preparative flat-bed gel isoelectric focusing (as previously reported) was subjected to further characterization. The fraction was chromatographed on a Sephadex G-25 column and exhibited two major peaks, the first of which was allergenic and the second of which was nonallergenic but caused a precipitate to form when added to a mixture of sodium dodecyl sulfate (SDS) (1%) and trichloroacetic acid (5%). SDS-PAGE indicated that the allergen component of the basic fraction migrated at about the same rate as insulin (5.8 kdaltons). Both G-25 peaks were carbohydrate rich with a protein:carbohydrate ratio (by weight) of 1:4.5 and 1:5.8 for peak 1 and peak 2 respectively. Heat (100 degrees C for 10 min) and enzyme digestion (trypsin, alpha-chymotrypsin and pepsin) did not reduce the RAST inhibition values for peak 1 or the original basic fraction. The basic fraction did not bind human IgE specific for birch pollen indicating that the RAST inhibition and IgE (Alternaria specific) binding to radioimmunoelectrophoresis plates was not due to nonspecific binding of IgE. We conclude that the allergenicity of the basic fraction is due to a small molecule that is predominantly carbohydrate in nature and is a major allergen of Alternaria.

Improving the quality of protein crystals through purification by isoelectric focusing.

Preparative flat-bed gel isoelectric focusing has reduced or eliminated observed heterogeneity in three different proteins, thereby greatly improving the quality of crystals grown from each of them. This improvement is manifested both in crystal size and in increased measurability of diffraction data. Isoelectric focusing is a general and nondestructive technique that may facilitate the crystallographic study of proteins which are biologically interesting, but which, for reasons of microheterogeneity, do not yield diffraction quality crystals.

Characterization of cellulase complexes from Trichoderma reesei QM 9414 and its mutants by means of analytical isoelectrofocusing in polyacrylamide gels

Crude cellulolytic enzyme complexes from Trichoderma reesei QM 9414 and its high-cellulase producing mutants were analyzed by means of analytical isoelectric focusing in polyacrylamide gel cylinders and in flat bed gels. Variations in the peptide composition of individual cellulase preparations as well as in the composition and relative abundance of endo-β- d-1,4-glucanase and β-glucosidase components in the individual cellulase preparations were detected.

Pteridines as nonretinal regulators of light-dependent melatonin biosynthesis.

6-Formylpterin was purified by paper chromatography, and bovine pineal hydroxyindole-O-methyltransferase (S-adenosyl-L-methionine:N-acetylserotonin O-methyl-transferase; EC 2.1.1.4) was purified by flat-bed gel electrofocusing. Fractions of the methyltransferase with isoelectric points at pH 5.0, 5.7, and 5.2 were tested in the presence of 6-formylpterin, and decreased activities were found. Because 6-formylpterin is one of the photolytic products of folic acid and decreased serum folic acid has been reported in light-exposed men, we suggest that pteridines may be involved in nonretinal effects of light on pineal melatonin biosynthesis. It may be that this metabolic pathway is an evolutionary relic of primitive light responses of pigmentation mechanism.

Localization of specific glycoproteins secreted by the rabbit and hamster epididymis.

To investigate the origin and fate of a number of specific epididymal proteins, the luminal contents from the cauda epididymides of sexually mature New Zealand White rabbits and Syrian hamsters were collected by retrograde perfusion. Epididymal fluid, separated from spermatozoa by centrifugation over a 10% Ficoll solution, was then subjected to isoelectric focussing on polyacrylamide flat bed gels. Three epididymal-specific proteins from the rabbit, pI 3.45, 4.15 and 4.65 and two from the hamster, pI 3.40 and 4.15 were identified as glycoproteins by their low isoelectric point and their affinity for concanavalin A. These fractions were isolated and implanted s.c. in guinea pigs and rabbits, respectively, to produce monospecific antisera as verified by immunodiffusion and immunoelectrophoresis. Localization of glycoprotein antigen within the epididymis was performed on longitudinal sections of snap-frozen epididymis using the unlabeled antibody enzyme method. Both rabbit and hamster glycoproteins were secreted mainly by epididymal epithelial cells in the caput and proximal corpus region. However, the initial segments and testis were devoid of antigen. Differences were also apparent in the staining pattern of individual glycoproteins. In the rabbit, glycoproteins were first associated with spermatozoa in the distal caput and proximal corpus epididymidis while glycoproteins in the hamster epididymis were first associated with spermatozoa in the proximal caput region. Reaction product to glycoproteins in the cauda epididymidis of both species was present on spermatozoa and at the adluminal border of epithelial cells. The significance of these results with regard to changes in the surface composition of the sperm plasmalemma during maturation is discussed.

Analysis of the iron-binding sites of transferrin by isoelectric focussing.

1. Human transferrin was labelled with ferric nitrilotriacetate (FeNTA) at one of its two metal binding sites by variation of the pH. 2. Four transferrin forms, transferrin, transferrin(Fe) (A-site), transferrin(Fe) (B-site) and transferrin(2Fe) could be separated on flat bed gels by isoelectric focussing. 3. Incubation time, temperature and medium play an important role in the specificity of the binding of Fe. In NTA-pH-buffer, at 0.5 Fe-saturation, the A-site was preferentially labelled at pH 7--8, the B-site at a pH 8--9. 4. Under physiological conditions iron from the B-site has the tendency to move to the A-site.

Host-Pathogen Interactions

The invertase present in the culture fluid of races 1, 2, and 3 of Phytophthora megasperma Drechs. var. sojae A. A. Hildebrand (Pms) were purified until they gave but a single band, whether stained for protein or carbohydrate, after isoelectric focusing in flat bed gels. The sugar compositions of multiple preparations of the purified invertases from each race of this fungal pathogen were determined by quantitative gas chromatography of their alditol acetates. The invertases are composed of about 25% carbohydrate. Mannose and glucosamine make up more than 97% of the carbohydrate portions of the invertases of all three Pms races analyzed, but the ratio of mannose to glucosamine is clearly not the same in each race. The glycosyl linkage compositions of the glucosamine-containing mannans of multiple preparations of the Pms invertases were determined by GC-MS analysis of the partially methylated alditol acetate derivatives. The results of these analyses demonstrate clear quantitative differences between the glycosyl components of the different Pms races. The existence of race-specific carbohydrate structures in the differentially virulent Pms races suggests that these carbohydrates may be involved in determining the specificity of hostpathogen interactions.

Flat bed gel isoelectric focusing of neuraminidases

Technical details and preliminary results are described for a new flat bed gel isoelectric focusing method to study charge heterogeneity in neuraminidases. The method combines simplicity and high resolving power with the capacity to analyse multiple samples in a single experiment. Isoelectric points are obtained which agree favourably with those gained by other methods.