Isoelectric focusing of catalase from acatalasemic mouse and human blood, and cultured human skin fibroblasts

Hemolysates of normal, heterozygous hypocatalasemic and acatalasemic mice and of Japanese acatalasemic subjects were separated into three fractions, A, B and C, by DEAE-cellulose column chromatography, and pI values of A, B and C fractions were determined by isoelectric focusing. The pI value of catalase in the A, B and C fractions increased in the order of normal, hypocatalasemic and acatalasemic mouse blood. The results obtained from Japanese acatalasemic blood samples showed that the pI values of catalase in the A, B and C fractions were similar to those in normal blood. Catalase in Japanese acatalasemic cultured skin fibroblasts was also analyzed by isoelectric focusing. The pI values of catalase in the extract from the cultured skin acatalasemic fibroblasts was similar to that in normal fibroblasts.     

Preparative separation of immunoglobulins from bovine colostrum by continuous divergent-flow electrophoresis

Immunoglobulins in bovine colostrum were separated and fractionated from other proteins using the method and instrumentation developed in our laboratory. The proposed separation was based on bidirectional isotachophoresis/moving boundary electrophoresis with electrofocusing of the analytes in a pH gradient from 3.9 to 10.1. The preparative instrumentation included the trapezoidal non-woven fabric that served as separation space with divergent continuous flow. The defatted and casein precipitate-free colostrum supernatant was loaded directly into the instrument without any additional colostrum pre-preparation. Immunoglobulin G was fractionated from other immune proteins such as bovine serum albumin, β-lactoglobulin, and α-lactalbumin, and was continuously collected in separated fractions over 3 h. The fractions were further processed, and isolated immunoglobulin G in the liquid fractions was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by re-focusing in gel isoelectric focusing. Separated immunoglobulin G was detected in seven fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a gradually decreased concentration in the fractions. Re-focusing of the proteins in the fractions by gel isoelectric focusing revealed multiple separated zones of immunoglobulin G with the isoelectric point values covering the range from 5.4 to 7.2. Each fraction contained distinct zones with gradually increased isoelectric point values and decreased concentrations from fraction to fraction.     

Analysis of histones by on-line capillary zone electrophoresis-electrospray ionisation mass spectrometry

Clotting factor IX preparations from human plasma (pdFIX) have been characterized using electrophoretic methods like sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis. Factor IX prior to and after activation with factor XIa was separated by one- and two-dimensional polyacrylamide gel electrophoresis and on isoelectric focusing gels. The main differences between the band patterns of the two pdFIX preparations are due to their purity. Vitronectin was identified by immunological techniques as major accompanying plasma protein, separated from factor IX and characterized by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis.     

Two-dimensional electrophoresis of the total polypeptides in ripe red grape berries.

The total polypeptide composition of mature grape berries was analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis (isoelectric focusing in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension), followed by Coomassie Blue and nitrate silver staining, respectively. Adapted methods for total protein preparation of grapes and for two-dimensional gel electrophoretic separation of polypeptides are presented. The grape patterns presented up to 52 fractions with Mrs ranging from 15,000 to 110,000. The polypeptides displayed pIs from 4.6 to 7.3. A group of spots from Mr 28,000 to 83,000 and with a pI from 4.6 to 5.4 was strongly silver stained. The Mr 28,000 spot, pI 4.6, was revealed to be a complex of four fractions. Reproducible separations were obtained with the different carrier ampholyte mixtures tested.     

Cystic fibrosis proteins detected by electrophoretic techniques

For the detection of the cystic fibrosis protein (CFP) in serum of cystic fibrosis (CF) carriers, thin-layer polyacrylamide gel isoelectric focusing proved inappropriate as a diagnostic test, but was useful for screening fractions on purification of CFP by chromatofocusing on a Mono P column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis an Mr 12,000 protein (P12) was found in most CFP-positive sera, indicating good correlation between these two CF-associated proteins. Detection of the P12 protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was well reproducible and less delicate than IEF. The technique was also used to purify P12 from serum by two successive preparative electrophoresis steps in a 7.5-15% gradient and 15% homogeneous gel. The use of silver staining revealed that P12, which was present in all sera of CF patients and carriers with variable intensities, was also present in trace amounts in normal sera.     

Application of Neuhoff's optimized Coomassie brilliant blue G-250/ammonium sulfate/phosphoric acid protein staining to ultrathin polyacrylamide gels on polyester films

An optimized Coomassie staining procedure, utilizing Coomassie Brilliant Blue G-250 in phosphoric acid/ammonium sulfate, was applied to ultrathin-layer isoelectric focusing in 0.18 mm polyacrylamide gels, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 0.38 mm polyacrylamide gels, both backed to Gel-Fix polyester supporting films. After isoelectric focusing staining of gelatin and acidic proteins was better with the phosphoric acid/ammonium sulfate procedure than with conventional organic solvent methods. When applied to gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis the sensitivity of the phosphoric acid/ammonium sulfate method was equal to that on conventional staining but lower than on silver staining.     

Identification of rat liver glutathione S-transferase Yb subunits by partial N-terminal sequencing after electroblotting of proteins onto a polyvinylidene difluoride membrane from an analytical isoelectric focusing gel

Rat liver glutathione S-transferases were partially purified using S-hexyl glutathione affinity chromatography, followed by native isoelectric focusing employing a pH 7-11 or pH 3-10 gradient. Proteins were excised and eluted from the gel for determination of subunit composition using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In separate experiments, isoelectric focusing gels were equilibrated with a sodium dodecyl sulfate-containing buffer at high pH, and proteins on the gel were electroblotted onto a polyvinylidene difluoride membrane, utilizing graphite plates as electrodes. The membrane-bound proteins were visualized by Coomassie Brilliant Blue staining. The protein bands were then excised from the membrane and inserted into a gas phase sequenator for direct sequencing. N-Terminal sequences thus determined were compared with published cDNA sequences. The isoelectric points (pIs) and positions on the isoelectric focusing gel of Yb1Yb1, Yb1Yb2 and Yb2Yb2 subunits were determined. We have also located on the pH 3-10 focusing gel an N-terminal blocked glutathione S-transferase which has a molecular weight similar to Yb subunits.     

Preparative separation of poliovirus structural polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, copper staining and electroelution, and induction of monospecific antisera

Viral polypeptides were prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by copper staining and electroelution from gel slices. Poliovirus capsid polypeptide VP 1 isolated by this procedure induced monospecific antibodies in rabbits, i.e., antisera reacting only with the homologous polypeptide. Our results demonstrate the applicability of the described copper staining method as a rapid visualization step for preparing viral proteins after SDS-PAGE.     

Reassessment of commercially available molecular weight standards for peptide sodium dodecyl sulfate-polyacrylamide gel electrophoresis using electroblotting and microsequencing

Myoglobin CNBr peptides, constituting the commercially available molecular weight calibration kits for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were analyzed by microsequencing after electroblotting on polyvinylidene difluoride (Immobilon) membranes. An obvious disagreement was found between peptide identification and the data provided by the manufacturers. We observed 6 peptides from Mr 2500 to 17,000 corresponding, in increasing size order, to the 3 peptides resulting from the total CNBr digestion, to 2 incompletely cleaved peptides and to the intact myoglobin. Using a corrected calibration curve, a linear relationship was established from Mr 6000 to 43,000 and a second one for shorter peptides. This method of electrophoresis and electroblotting, easily adapted for peptides, is a powerful tool for peptide identification correlated with size determination. It is especially useful for CNBr-cleaved peptides.     

Deoxyribonuclease zymography adapted to the precast PhastGel electrophoresis media.

A set-up for casting fluorescent indicator agarose gels on ultrathin polyacrylamide microelectrophoresis gel media (Pharmacia PhastGel media) is described. The zymogram system allows a rapid and sensitive detection of deoxyribonuclease in various gel media, following isoelectric focusing, native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Identification of Tuber borchii Vittad. mycelium proteins separated by two-dimensional polyacrylamide gel electrophoresis using amino acid analysis and sequence tagging

This paper reports the first results in the proteome analysis of Tuber borchii Vittad. mycelium, an ectomycorrhizal fungus poorly defined genetically, but known for its generation of edible fruit bodies known as white truffles. Employing isoelectric focusing on immobilized pH gradients, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we obtained an electropherogram presenting over 800 spots within the window of isoelectric points (pI) 3.5-9 and a molecular mass of 10-200 kDa. Different reducing agents were tested in the sample preparation buffers, and the standard lysis buffer plus 2% w/v polyvinylpolypyrrolidone allowed the best solubilization and resolution of the proteins. The T. borchii proteins separated in micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and visualized by Coomassie staining. Twenty-three proteins were excised and analyzed by the combination of amino acid and N-terminal analysis. One protein was identified by matching its amino acid composition, estimated isoelectric point and molecular mass against the SWISS-PROT and EMBL databases. Four spots were successfully tagged by Edman microsequencing but no homologous sequences were found in databases.     

Diagnostic use of an analysis of urinary proteins by a practicable sodium dodecyl sulfate-electrophoresis method and rapid two-dimensional electrophoresis

Two methods suitable for routine clinical analyses of urinary proteins are presented and compared. The first is a horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique, suitable for simultaneous analysis of 20 native urinary samples. This method uses polyacrylamide gradient gels, prepared with a laboratory-built gel casting device. The second method is a rapid two-dimensional electrophoresis procedure, combining cellulose acetate electrophoresis and sodium dodecyl sulfate-electrophoresis. The first step uses a routine system (Chemetron), the second separation step followed by staining with Coomassie Brilliant Blue R is performed on the PhastSystem. The resulting two-dimensional patterns reveal urinary proteins distributed according to the 5-zone pattern of native proteins (albumin, alpha-1, alpha-2, beta, gamma-globulin) as well as to the logarithm of their molecular weights. Examples of (routine) diagnoses with a special interest in the monitoring of kidney transplant patients are shown.     

Preparative isoelectric focusing of reduced wheat gluten proteins

Proteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2-mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3-10 gradient containing 50% v/v 1-propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1-propanol was comparable to that obtained on two-dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.     

A new multiphasic buffer system for sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100,000-1000, and their detection with picomolar sensitivity.

A novel multiphasic buffer system for high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dansylated and nondansylated proteins/peptides in the relative molecular mass (Mr) range of 100,000-1000 is described. The system, based on Jovin's theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins/peptides within the above Mr range. The buffer system uses Bicine and sulfate as trailing and leading ion, respectively, and Bistris and Tris as counter ions in the stacking and separating phase, respectively. Through selection of two different counter ions--the characteristic feature of the present ionic system--the stacking limits of a multiphasic buffer system can be further widened, thus making it applicable to gel electrophoresis of a larger spectrum of rapidly migrating species, such as sodium dodecyl sulfate-proteins/peptides and nucleic acids, than has been possible previously. Highly sensitive detection methods for proteins as well as for polypeptides down to approximately Mr 1000 are described. Dansylated proteins/peptides were detected by their fluorescence either directly within the gel or following electroblotting into anion-exchange or polyvinylidene difluoride membranes. The latter procedure resulted in detection sensitivities of approximately 1 ng. Nondansylated proteins/peptides were either detected within the gel by colloidal Coomassie staining or by electroblotting into polyvinylidene difluoride membranes, followed by colloidal gold staining. Prior to both staining procedures the proteins/peptides were pretreated with glutardialdehyde in the presence of borate at near neutral pH values to generate protein/peptide polymers of poor solubility. For a given pH the efficiency of the latter procedure was significantly influenced by the nature of the buffer ion used in the fixation buffer. In contrast to conventional fixation procedures even small polypeptides (Mr 1000) were immobilized and approximately 15 ng and 0.75 ng could be detected after colloidal Coomassie and colloidal gold staining, respectively.     

The simplified technique of high resolution two-dimensional polyacrylamide gel electrophoresis: biomedical applications in health and disease

The application of our simplified technique of high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to human body fluids is reviewed. Serum/plasma protein changes associated with alcohol abuse, familial dyslipoproteinemia ("fish-eye" disease), and myocardial infarction are demonstrated. High resolution 2-D PAGE of amniotic fluid, cerebrospinal fluid, urine, and saliva is shown with reference to the work of others, and the detection of pink-violet staining "lumicarmines" in sweat and tear fluid is reported for the first time. General aspects relating to the methodology are discussed. These include sample preparation, the choice of electrophoresis conditions (denaturing or nondenaturing) and detection method (Coomassie Brilliant Blue or silver), and the effects of native protein pretreatment with sodium dodecyl sulfate prior to silver staining or isoelectric focusing gel shrinkage in glycerol prior to second-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Two-dimensional electrophoresis of membrane proteins: separation of myelin proteins

A method for two-dimensional electrophoretic separation of myelin proteins is presented. The first dimension consists of isoelectric focusing of lyophilized and delipidated membrane proteins, solubilized in a mixture of the nonionic detergent Triton X-100, the zwitterionic detergent CHAPS, 9 M urea and carrier ampholytes, and incorporated into a slab gel before separation. Subsequent discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed by moulding the isoelectric focusing slab gel with its supporting glass plate into the stacking gel. This method proved to give highly reproducible results since mechanical forces and thus the risk of stretching, folding or rupture of the isoelectric focusing slab gel is minimized. Furthermore, by immunoblotting, the positions of myelin-associated glycoprotein and 2',3'-cyclic nucleotide 3'-phosphodiesterase were established with specific antibodies.     

Fish species identification by peptide mapping of the myosin heavy chain.

Fish species of seafood made of washed fish flesh (e.g. imitation crab meat from surimi) were identified by peptide mapping of the myosin heavy chain (MHC). In the first step the MHC was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 7.5% T gel, followed by a second SDS-PAGE in a 15% T gel with proteolytic digestion of the MHC in the gel. The resulting peptides gave species specific patterns.     

Characterization of cationic acid phosphatase isozyme from rat liver mitochondria.

Acid phosphatase isozyme was highly purified from rat liver mitochondrial fraction. The enzyme showed an isoelectric point value of above 9.5 on isoelectric focusing, and the apparent molecular weight was estimated to be 32000 by Sephadex G-100 gel filtration or 16000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed the hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, thiamine pyrophosphate, inorganic pyrophosphate, and phosphoprotein such as casein and phosvitin, but not of several phosphomonoesters, except for p-nitrophenyl phosphate and o-phosphotyrosine. The enzyme was not inhibited by L-(+)-tartrate, and was significantly activated by Fe2+ and reducing agents such as ascorbic acid, L-cysteine,and dithiothreitol. The enzyme was found to be distributed in various rat tissues including liver, spleen, kidney, small intestine, lung, stomach, brain and heart, but not in skeletal muscle.     

Use of chromatofocusing for separation of beta-lactamases. IX. Analytical chromatofocusing for the separation of a chromosomal cephalosporinase from Proteus vulgaris 1028

Simultaneous purification and isoelectric point (pI) determination was carried out at analytical scale of the chromosomal cephalosporinase from the Proteus vulgaris 1028 strain. Comparison of the enzyme to the purification results with m-aminophenylboronic acid-agarose affinity chromatography with sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that minute amounts of accompanying proteins having identical pI values but different molecular masses were found in the chromatofocused preparation. The molecular mass of the enzyme was 24,000 dalton. The pI was found to be 8.3.     

Barley cultivar discrimination: I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and glycoprotein blotting.

Two different methods of detecting electroblotted glycoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Tris-buffer soluble barley seed proteins were examined for their applicability for barley cultivar discrimination. These are the highly specific, lectin-based concanavalin A/peroxidase method and the more general periodate/danyslhydrazine method. The results of the periodate/dansylhydrazine method enabled us to divide the 20 examined cultivars into three groups, whereas the more sensitive concanavalin A/peroxidase method revealed six different glycoprotein patterns. In comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the alcohol-soluble barley seed proteins (hordeins) gave nine different banding patterns. A combination of hordein electrophoresis together with glycoprotein staining by the concanavalin A/peroxidase method made it possible to classify the cultivars into twelve groups, the largest of which contained four cultivars. The qualitative expression of the glycoprotein patterns seemed to be independent of growth conditions, whereas the band intensities obviously were not. As a whole, glycoprotein blotting is a valuable supplement to sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hordeins in barley cultivar discrimination.