bluuebunny/crossref_metadata_2025 huggingface dataset:
Two‐dimensional polyacrylamide gel electrophoresis reveals differences between osteoblast and fibroblast extracellular proteins
Authors: Hankey David P. , Nicholas R. M. , Hughes Anne E.
Published: May 2005

Normal human skin fibroblast primary cell lines secrete over 50 proteins into culture medium. These have been mapped previously using two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) and this technique has now been used to investigate extracellular protein secretion by human osteoblasts in vitro. We report the mapping of a number of consistent markers specific to the osteoblast. In particular, one protein chain with posttranslational modifications was found to be unique to the osteoblast extracellular protein map. The absence of the N‐ and O‐glycoforms of collagenase from the osteoblast profile in this study concurs with findings reported using the immunoprecipitation functional assay and Northern blot analysis. The use of 2‐D PAGE in phenotypic assessment provides a more complete analysis than the standard range of single‐parameter tests for osteobiasts. Mapping of extracellular and cellular proteins in addition to bone matrix protein analysis will allow a comprehensive analysis of normal osteoblast function. This technique may also be applied to the study of osteoblasts in relation to bone disease and in assessing the phenotypic shift within a normal osteoblast culture.
Gel electrophoretic analysis of chitosan hydrolysis products
Authors: Audy Patrice , Asselin Alain
Published: May 2005

Enzymatic hydrolysis of commercial crustacean chitosan by barley chitosanases was analyzed by subjecting chitosan to electrophoresis in a 10% w/v polyacrylamide slab gel in the presence of 7 M urea and 5.5% v/v acetic acid. Chitosan migrated as a polycation. Chitosan was stained with Coomassie Brilliant Blue R‐250 or visualized by ultraviolet transillumination after staining with Calcofluor White M2R. Some chitosan molecules were retarded by gel electrophoresis while small chitosan molecules migrated at the bottom of a 10% w/v polyacrylamide gel. Such analysis revealed that 96 h were necessary to convert all chitosan to oligosaccharides under our assay conditions. Chitosan oligosaccharides generated by enzymatic or chemical hydrolysis were further analyzed by electrophoresis in a 33% w/v polyacrylamide gel containing urea and acetic acid. Coomassie Brilliant Blue R‐250 was found to be better than Calcofluor White M2R for staining chitosan oligosaccharides. Chitosan oligomers of four residues (tetramers) or more were easily resolved in such a polyacrylamide gel system. To our knowledge, this is the first report of a gel electrophoretic separation of chitosan and its oligosaccharides.
Analysis of carbohydrate‐mediated heterogeneity and characterization of N‐linked oligosaccharides of glycoproteins by high performance capillary electrophoresis
Authors: Taverna Myriam , Baillet Arlette , Biou Daniel , Schlüter Michael , Werner Rolf , Ferrier Danielle
Published: May 2005

Capillary zone electrophoresis (CZE) has been investigated as an alternative method to analyze the carbohydrate moieties of glycoproteins. Carbohydrate‐mediated microheterogeneity of the recombinant plasminogen activator (rt‐PA) was examined. The glycoprotein was resolved in multiple electrophoretic species using CZE but the separation was complicated by adsorption of the molecules to the wall of the capillary. The influence of several parameters, such as pH, molarity of the buffer and addition of a cationic additive, on the separation of glycopeptides was investigated. High resolution and reproducible separations of rt‐PA glycopeptides carrying hybrid and complex type chains were obtained using either a 100 mM phosphate buffer, pH 6.6, or a 100 mM Tricine buffer, pH 8.2, containing 1.25 mM of putrescine. N‐Oligosaccharides from fetuin, t‐PA and α1‐acid glycoprotein were separated within 20 min on the basis of both their sialic acid content and their structure. The use of an oligosaccharide fingerprinting technique, such as the present one, could have many applications in biotechnology to assess, for example, the consistency of production of a glycoprotein or for analytical glycoprotein chemistry.
Specific polypeptide markers in chronic B cell malignancies detected by two‐dimensional gel electrophoresis
Authors: Talebian‐Ziai Simin , Azgui Zahia , Valensi Françoise , Sigaux François , Charron Dominique
Published: May 2005

Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) analysis of silver stained polypeptides was used to discriminate the cell protein profiles in chronic B cell malignancies. We identified 5 distinct polypeptides (H1 to H5) present in hairy cell leukemia (HCL) (14 cases) and absent in most chronic lymphocytic leukemia (CLL) and normal B lymphocytes. Two polypeptides, H2 and H5, were absolutely specific of HCL. The 2‐D PAGE profiles found in 3 splenic lymphoma with villous lymphocytes (SLVL) and 2 HCL variants (HCL‐V) were similar to HCL with a unique additional peptide present in 3/3 SLVL and 1/2 HCL‐V. When HCL and SLVL were assessed for the CLL stage‐specific 2‐D PAGE patterns, HCL was related to stage A/B while SLVL was related to stage C. In conclusion, 2‐D PAGE analysis of the molecular pattern of B lymphocytes provides a new means to recognize the leukemic origin of the sample and distinguish HCL from CLL and SLVL.
A new technique for detecting oxytocinase activity in electrophoresis gels
Authors: Roy Ashim C. , Saha Nilmani , Tan Suan M. , Kamarul Farida Z. , Ratnam Shan S.
Published: May 2005

A sensitive staining method for the detection of oxytocinase (EC 3.4.11.3) activity in electrophoresis gels has been described. The method is based on the enzymatic release of p‐nitroaniline (PNA) from two specific synthetic oxytocinase sub‐strates, S‐benzyl‐L‐cysteine‐p‐nitroanilide (BCN) and L‐leucine‐p‐nitroanilide (LN), respectively. The PNA is then diazotized with sodium nitrite and subsequently coupled to a chromogen, N‐(1‐naphtyl)‐ethylenediamine dihydrochloride (NED) to produce a deep pink/magenta colored azo‐dye at the site of oxytocinase activity.
Tissue‐specific pattern of nonhistone high mobility group proteins in various organs of the chicken
Authors: Pedrini Michael , Grunicke Hans , Csordas Adam
Published: May 2005

Electrophoretic analysis of tissue‐specific differences of nonhistone high mobility group (HMG) proteins from nuclei of various organs of the chicken revealed that in organs with a higher proportion of replicating cells (thymus, Bursa Fabricii, spleen) the relative amount of HMG‐17 is considerably higher than that of HMG‐14; however, in transcriptionally active organs with a very small proportion of replicating cells (glandular stomach, liver) HMG‐14 and HMG‐17 are present at roughly equal and low amounts. In glandular stomach, liver and spleen, the relative contents of both HMG‐1 and HMG‐2 are markedly lower than in thymus and Bursa Fabricii. Moreover, the total amount of HMG proteins is higher in those organs which contain replicating lymphocytes.
Distinction between supercoiled and linear DNA in transverse agarose pore gradient gel electrophoresis
Authors: Wheeler David , Lin Jia‐Hwei , Chrambach Andreas
Published: May 2005

Four species of linear DNA and the first four members of a linking series, generated by treatment of plasmid DNA (PUC19, 2.7 kb) with mitochondrial topoisomerase, were differentiated by transverse agarose pore gradient gel electrophoresis. The experimental curves of migration distance vs. agarose concentration (Ferguson curves) of supercoiled DNA exhibit a steeper trajectory than those of linear DNA of the same size range. As a consequence, the four supercoiled species exhibit an increase in apparent size (relative to linear DNA standards) with increasing agarose concentration. Both the crossing of the Ferguson curves with those of linear standards as well as the apparent size increase with agarose concentration can serve to detect supercoiled plasmid‐sized DNA in mixtures with linear DNA.
Rapid high resolution two‐dimensional electrophoresis of human sperm proteins
Authors: Kritsas John J. , Schopperle William M. , De Wolf William C. , Morgentaler Abraham
Published: May 2005

An improved protocol has been developed for two‐dimensional gel analysis of human sperm proteins through the application of recent technical advances. Advantages of this protocol consist of increased resolving power, reduced processing time, enhanced reproducibility of staining patterns, and applicability to small quantities of protein. Technical improvements include determination of optimal protein loading (20 micrograms/gel), development of a reliable tube gel casting system, and application of minigel technology. In this study over 500 proteins were resolved with molecular weights ranging from 12 000 to 105 000 and isoelectric points from 5.0 to 8.5. This is a three‐fold improvement in resolution over earlier results. A single operator was able to generate two‐dimensional gels of multiple samples in less than one day. This rapid high resolution technique should facilitate further investigation of human sperm proteins under normal and pathologic conditions.
Two‐dimensional electrophoresis of cat sera: Protein identification by cross reacting antibodies against human serum proteins
Authors: Miller Ingrid , Gemeiner Manfred
Published: May 2005

After two‐dimensional electrophoresis of cat serum, individual proteins were identified by means of cross reacting antibodies against the human protein homologues. Testing by cross reactivities seemed the method of choice because immunoreagents for cat proteins are not easily available.
Using Coomassie Blue to stabilize H2O2‐guaiacol stained peroxidases on polyacrylamide gels
Authors: Fieldes Mary Ann
Published: May 2005

Shimoni and Reuveni [1] have reported a procedure which uses Coomassie Brilliant Blue R‐250 staining and destaining to stabilize H2O2‐guaiacol stained peroxidases on polyacrylamide gels and at the same time detects nonspecific protein bands. The procedure has the advantage that the otherwise orange and unstable peroxidase bands are visualized as blue bands and it, therefore, facilitates laser densitometry of electrophoretically separated peroxidases. In the modification of the procedure reported here, the Coomassie staining reagent was adjusted and the destaining step was eliminated. The modified procedure has added advantages: it simplifies the procedure, increases the consistency of the results across gels prevents or reduces the staining of nonspecific proteins, and still gives excellent resolution of stable bands which can be used to quantify peroxidase activity.
Separation of cresols using coelectroosmotic capillary electrophoresis
Authors: Masselter Sonja Marie , Zemann Andreas Josef , Bobleter Ortwin
Published: May 2005

A new method of capillary electrophoresis was established to separate the three cresol isomers. Cresols are of importance due to their use as insecticides, precursors of various dye intermediates, resins, fire‐resistant fluids and other purposes. Reversion of the electroosmotic flow was carried out by a polycationic ammonium compound. The separations were carried out at high pH values (pH 12) to obtain complete dissociation of the cresols and short times of analysis (less than 5 min) through fast migration of the cresol towards the anode. Various aliphatic alcohols were added to improve peak shape and resolution.
Characterization of the electrophoretic properties of nucleosome core particles by transverse polyacrylamide pore gradient gel electrophoresis
Authors: Orbán Lászlo , Garner Mark M. , Wheeler David , Tietz Dietmar , Chrambach Andreas
Published: May 2005

Transverse pore gradient gel electrophoresis, previously applied to bent DNA, has extended the usefulness of the gel retardation assay in two ways: (i) by differentiating between different DNA conformations; (ii) by providing information regarding the physical properties of DNA. In the present study, similarly extended information is obtained with regard to a well‐characterized DNA‐protein complex, the chicken erythrocyte nucleosome core particle. (i) The winding of DNA around the protein core constrains the DNA which renders its Ferguson curve (migration distance vs. gel concentration) similar to that of kinetoplast DNA, i.e. it intersects sharply with the Ferguson curves of linear DNA standards. By contrast, the deproteinized nucleosome DNA exhibits a Ferguson curve similar to linear standards of the same lenght. (ii) Interpretation of the Ferguson curve based on a mathematical model shows that the nucleosome exhibits a linear Ferguson plot [log(mobility) vs. gel concentration]. This is similar to and characteristic of spherical proteins, contrasting with the concave plot typical for linear and bent DNA. (iii) The effective size of the nucleosome, evaluated in terms of an “equivalent sphere” (i.e. a hypothetical spherical particle with a radius, Res, having the same electrophoretic mobility as DNA for a particular set of experimental conditions), remains invariant across the gel concentration range of 3–9 % T. This is similar to proteins and bacteriophages and contrasts with the progressive decline of Res with increasing gel concentration observed for linear DNA and the deproteinized nucleosomal DNA.
Analysis of plasmid‐DNA and cell protein of recombinant Escherichia coli using capillary gel electrophoresis
Authors: Hebenbrock Kirstin , Schügerl Karl , Freitag Ruth
Published: May 2005

Plasmid DNA prepared from cultivation samples of recombinant Escherichia coli was analyzed by capillary gel electrophoresis. We used this method to control the genetic stability during a fed‐batch culture. The plasmid DNA and a standard DNA mixture with molecular weights in the size range of 3000–22 000 base pairs (bp) were analyzed in capillaries that were filled with solutions of non‐cross‐linked polyacrylamide. With this method the plasmid DNA from recombinant E. coli cultivation samples was analyzed within 30 min. Separation parameters such as gel concentration, capillary length and current were optimized for that purpose. The method was modified to allow the analysis of proteins. Crude cell lysate samples could be screened for the protein pattern within 15 min in sodium dodecyl sulfate‐polyacrylamide filled capillaries. The method was also used to verify product purity after the down‐stream process.
A traveling‐wave micropump for aqueous solutions: Comparison of 1 g and μg results
Authors: Müller Torsten , Arnold W. Michael , Schnelle Thomas , Hagedorn Rolf , Fuhr Günter , Zimmermann Ulrich
Published: May 2005

The operation of a micro‐chip‐based fluid circulator based on the interaction of a high‐frequency traveling wave with a thermally generated inhomogeneity in an aqueous medium is investigated. The profiles of the electric field, the thermal gradient and the driving force within the device are derived numerically, and the dependence upon applied voltage under different experimental conditions is derived. The importance of convection in the operation of the device is assessed by operating it in various orientations, as well as under microgravity, and also by investigating the reversal and turn‐on characteristics. The pump showed the unexpected ability to trap microparticles present in the driven fluid.
Capillary electrophoresis quantitation of l–L–folinic acid in the presence of its inactive d–L–form
Authors: Cellai Luciano , Desiderio Claudia , Filippetti Raffaella , Fanali Salvatore
Published: May 2005

The diastereomers d‐L and l‐L‐folinic acid were separated by capillary electrophoresis, using heptakis (2,3‐di‐O‐methyl)‐β‐cyclodextrin as a chiral active component in the background electrolyte. The method proved suitable for the quantitation of these compounds in one commercial pharmaceutical preparation.
Simultaneous determination of native and subunit molecular weights of proteins by pore limit electrophoresis and restricted use of sodium dodecyl sulfate
Authors: Tyagi Rakesh Kumar , Babu Batchu Ramesh , Datta Kasturi
Published: May 2005

In the present communication, we describe a method for the determination of both the native and subunit molecular weight of a protein by a single pore limit electrophoretic run. When native proteins and sodium dodecyl sulfate (SDS)‐heat denatured proteins were electrophoresed in a 4–28% gradient‐polyacrylamide gel, in the presence of 0.02% SDS in running buffer, their respective molecular weights (native and subunit) could be determined by comparing the migration distances of unknown and standard proteins. The presence of SDS up to 0.02% in the running buffer did not dissociate the native proteins into their subunits. The linearity in distribution of native and SDS‐polyacrylamide gel electrophoresis (PAGE) standard marker proteins used for the molecular weight determination further showed the efficacy of the present technique.
Lack of inter‐species reactivity between antigens and antibodies is overcome by protease treatment of Western blots
Authors: Pan Jie , Pfeiffer David C. , Reimer Corinne L. , Crawford Bruce J. , Vogl A. Wayne , Auersperg Nelly
Published: May 2005

The analysis of proteins by Western blotting is frequently limited by the inability of antibodies to recognize antigenic epitopes in proteins of different species. In the present study, we investigated the influence of mild protease digestion on the reactivity of nitrocellulose‐blotted proteins and microscopic sections with antibodies produced against analogous proteins of different species. The proteins were partially purified, electrophoresed and blotted by standard procedures. They were then incubated with either trypsin or pepsin, at concentrations ranging from 0.5 to 80 μg/mL, for different time periods prior to reaction with the first antibody. After incubation with the second antibody, the bands were visualized by chemiluminescence. The following antibody/antigen pairs produced no signal by conventional methods but resulted in the appropriate bands following protease treatment: (i) monoclonal antibody (Mab) to human keratin #18 / rat keratin; (ii) Mab to starfish extracellular matrix/ mouse laminin; (iii) rabbit antiserum to human platelet myosin II/ratfish nonmuscle myosin II. In the latter system, protease treatment also revealed previously hidden epitopes in microscopic sections. Appropriate controls demonstrated that the antibodies retained their specificity after the protease treatment of the preparations. Optimal conditions varied and had to be defined for each protein under study. The results suggest that protease treatment of immunoblots reduces the lack of inter‐species reactivity between antibodies and antigens.
Immunoaffinity analysis of cross‐reacting allergens by protein blotting
Authors: Donovan Gregory R. , Baldo Brian A.
Published: May 2005

IgE antibodies from sera having reactivity against ryegrass pollen protein allergens, wheat endosperm protein allergens and also several other cereal protein allergens were adsorbed with either ryegrass pollen or the wheat/globulin fraction immobilised on solid phases and subsequently eluted with low pH buffer. The eluted antibodies were reacted with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) blots of the different allergens. Antibodies adsorbed and subsequently eluted from the two allergen sources recognised different spectra of proteins in the ryegrass pollen and cereal allergen sources and indicated the degree of immunological cross‐reactivity. Intra‐species cross‐reactivity of IgE antibodies was demonstrated employing similar methods to those used for the pollen and cereal allergens by using a recombinant allergen from the venom of the ant Myrmecia pilosula as the immunoadsorbant protein on the solid phase.
Calculating paternity probabilities from DNA multilocus fingerprints in some cases of deficiency
Authors: Yassouridis Alexander , Epplen Jörg T.
Published: May 2005

Paternity determination via DNA multilocus fingerprints in normal trio cases, namely when the fingerprints of the trio mother, child and putative father are available, is performed on a routine basis. But in cases of deficiency, where the DNA fingerprint of mother or putative father is missing, there exists no systematic, analytical way for paternity calculations, even in cases where DNA fingerprints of other persons related to the missing individual are available. The aim of this paper is to establish a mathematical, analytical background, that enables paternity calculations in cases of deficiency if complementary information can be obtained from the DNA fingerprints of at least one of the parents of the missing individual. Using the basic outline of our previously described procedure, the formulas for paternity calculations also allow mutations.
The fractional volume available to prolate spheroids in a network of randomly oriented fibers obtained by computer modeling: Correlation with the Ogston equation
Authors: Wheeler David , Chrambach Andreas
Published: May 2005

Computer modeling was used to measure the fractional volumes available to prolate spheroid objects in a random, inert network of fibers. The data fit the Ogston equation exactly when the object was a sphere (axial ratio = 1). When the axial ratio was increased from 1 to 9, the Ogston equation was still obeyed if the fiber concentration is multiplied by a factor, A, which increases linearly in proportion to the axial ratio. The factor A allows one to adjust the retardation coefficient derived from gel electrophoresis, KR, for spherical objects to that of prolate spheroids with axial ratios from 1 to 9. Potentially, the same adjustment of KR is possible for objects of other shapes.
Isotachophoresis of proteins in sucrose density gradients
Authors: Acevedo Fernando
Published: May 2005

The separation of proteins from human serum by isotachophoresis in sucrose density gradients, with mixtures of discrete amphoteric substances as spacers, is described. Open columns and columns with a dialysis membrane to hold the sucrose gradients were used. A simple algorithm based on the Kohlrausch function was used to calculate the amount of each spacer. The pH gradients generated in open columns were found to be in agreement with the calculations. The load was up to two gram proteins. The analysis of the fractions obtained after the separation showed a distribution of components similar to as analytical isotachophoresis. It is concluded that sucrose density gradients are suitable as supporting media for the preparative separation of proteins by isotachophoresis. The high resolution attained and the possibility of scaling‐up the separation systems are major advantages of this system. In addition, the sample is easily and completely recoverable.
Characterization of the gene for apolipoprotein E5‐Frankfurt (Gln81 ‐> Lys, Cys112 ‐> Arg) by polymerase chain reaction, restriction isotyping, and temperature gradient gel electrophoresis
Authors: Ruzicka Viktor , Mäz Winfried , Russ Andreas , Fisher Eva , Mondorf Werner , Groß Werner
Published: May 2005

A new apolipoprotein (apo) E variant, apoE5‐Frankfurt, was identified in a 43‐year‐old male with moderate hypercholesterolemia. On isoelectric focusing in an immobilized pH gradient, apoE5‐Frankfurt migrated to a position more cathodic than apoE4 (Cys112 ‐> Arg). On sodium dodecyl sulfate‐gel electrophoresis, its apparent molecular weight could not be distinguished from that of the three common apoE isoforms (E2, E3 and E4). Restriction isotyping with CfoI (HhaI) showed that apoE5‐Frankfurt had arginine in positions 112 and 158 of the mature protein, suggesting that the mutation accounting for the additional positive charge had occurred in an ϵ4 allele. The third and the fourth exon of the apoE gene were amplified using the polymerase chain reaction and analyzed by temperature gradient gel electrophoresis. This suggested that there were two mutations in the fourth exon of the mutant allele. Cloning and sequencing disclosed that, apart from the exchange of arginine for cysteine in position 112, a C to A substitution replaced glutamine (CAA) in position 81 by lysine (AAA).
Differences in the isoelectric focusing patterns of serum and cerebrospinal fluid transthyretin
Authors: Szilágyi A. Katalin , Ha Nguyen Thu , Szetényi Judit , Puskás Éva
Published: May 2005

Transthyretin isolated by polyacrylamide gel electrophoresis from human serum and cerebrospinal fluid, dissociated into its subunits, was subjected to isoelectric focusing in polyacrylamide gels containing 8 mole/L urea. The isoelectric focusing multi‐component patterns of serum and cerebrospinal fluid transthyretin differ in a characteristic way, having only one main protein zone in common. Double diffusion immunotest and immunoblotting revealed the immunological identity of serum and cerebrospinal fluid transthyretin and of the main components separated by isoelectric focusing. The different isoelectric focusing zones can be consistently explained when they are ascribed to structurally identical transthyretin subunits associated with different ligands specifically occurring in either serum or cerebrospinal fluid. Only the protein zone located at the same pI, in serum and cerebrospinal fluid transthyretin patterns may be assigned to ligand‐free subunits. Thus, the typical differences in isoelectric focusing patterns may point to different carrier functions of transthyretin in serum and cerebrospinal fluid.
A method for simultaneous determination of groupspecific component (GC) and alpha2HS‐glycoprotein (AHSG) by ultrathin‐layer isoelectric focusing and immunoblotting
Authors: Kuchheuser Werner , Krause Dieter
Published: May 2005

A method for simultaneous determination of group‐specific component and alpha2‐HS‐glycoprotein by ultrathin‐layer isoelectric focusing and immunoblotting is described. The advantages of this combined technique are the saving of chemicals and time. The band patterns of both marker proteins were always reproducible.
Human erythrocyte pyrimidine 5′‐nucleotidase isozymes: Effect of sulfhydryl reagents and electrophoretic discrimination
Authors: Manco Licínio , Amorim António
Published: May 2005

The electrophoretic behavior of human red cell pyrimidine 5′ ‐nucleotidase iso zymes (UMPH1 and UMPH2) was studied on starch gels with and without treatment with thiol reagents. It was found that at least one reactive sulfhy‐dryl group occurs in the UMPH1 isozyme but not in the UMPH2 isozyme. An electrophoretic system is described that allows the discrimination of UMPH1 and UMPH2 isozymes.
Disulfide linked dimers of apolipoprotein D in urine
Authors: Blanco‐Vaca Francisco , Pownall Henry J.
Published: May 2005

Apolipoprotein (apo) D is a glycoprotein that contains at least one free cysteine. This allows the formation of disulfide linked dimers of apoD, a phenomenon that could interfere with the study of the isoforms of apoD. Consequently, it is important to consider the effects of hetero‐ and homodimer formation on the molecular heterogeneity of apoD as well as on the evaluation of the specificity of antibodies to this glycoprotein. The identification of apoD in urine has provided a potential new marker of tubular proteinuria [1]. Thus, we have studied the specificity of our polyclonal antibodies to apoD against the proteins present in normal urine, and at the same time, the existence of dimeric species of apoD linked by disulfide bonds in urine. The specimens were obtained from apparently healthy individuals and analyzed by Western blot. The results showed that apoD in urine exists as a mixture of monomers and dimers, the latter having apparent molecular weights different from those occurring in plasma. Only monomeric apoD was observed under reducing conditions, proving the monospecificity of the polyclonal apoD antibodies.
The rat liver epithelial (RLE) cell nuclear protein database
Authors: Wirth Peter J. , Luo Lin‐Di , Benjamin Timothy , Hoang Tanya N. , Olson Arthur D. , Parmelee David C.
Published: May 2005

The master two‐dimensional computer database of rat liver epithelial (RLE) cellular proteins (Wirth et al., Electrophoresis 1991, 12, 931–954) has been expanded to include detailed information concerning 1100 nucleoplasmic (cytosolic) and 850 particulate associated [35S]methionine labeled as well as 215 nucleoplasmic and 269 particulate associated [32P]orthophosphate labeled RLE nuclear polypeptides, respectively. The RLE nuclear protein database developed using the Elsie 5 gel analysis system contains both qualitative and quantitative annotations including polypeptide identification number, protein name (if known), molecular weight and pI information, quantitation and polypeptide spot shape, subcellular location, as well as specific information regarding transformation (chemical and spontaneous) and growth‐related characteristics. Microsequencing of polypeptides directly from two‐dimensional (2‐D) blotted membranes has recently been established in our laboratory and provides a highly efficient and rapid means of polypeptide identification in the absence of specific antibodies. At present the RLE protein database is still in the developmental stage and is continually being updated as additional information is obtained. Nonetheless, it is anticipated that knowledge obtained concerning the identification and characterization of specific transformation and/or growth regulatory proteins in the RLE in vitro cell system will not only have direct application to other rodent and human 2‐D protein databases currently under development but will also complement them.
Plasma and red blood cell protein maps: Update 1993
Authors: Golaz Olivier , Hughes Graham J. , Frutiger Séverine , Paquet Nicole , Bairoch Amos , Pasquali Christian , Sanchez Jean‐Charles , Tissot Jean‐D. , Appel Ron D. , Walzer Claude , Balant Luc , Hochstrasser Denis F.
Published: May 2005

This publication updates the reference plasma and red blood cell protein maps obtained with immobilized pH gradients. Seventeen polypeptide spots or chains were partially characterized by direct N‐terminal sequencing or by sequencing of peptides obtained from enzymatic digestion. Additional new polypeptides and previously known proteins are listed in a table and/or labeled on the protein maps, thus providing the 1993 update of the human plasma and red blood cell two‐dimensional gel SWISS‐2DPAGE database. SWISS‐2DPAGE and the SWISS‐PROT protein sequence databases are closely linked together through the use of common accession numbers.
Peculiarities in electrophoretic behavior of different serum albumins
Authors: Miller Ingrid , Gemeiner Manfred
Published: May 2005

Behavior of serum albumins of different species was compared with regard to electrophoretic mobility under native conditions, molecular mass and isoelectric point in presence or absence of denaturing agents. In addition to serum samples from human, horse, cow, pig, cat, dog, sheep, goat, chicken, rabbit, mouse, and rat, commercially available standards or albumin preparations obtained by chromatographic procedures were analyzed. Feline and canine albumins showed marked differences in all investigated methods. Our experiments also revealed several factors influencing albumin patterns, especially during isoelectric focusing in immobilized pH gradients or in the presence of carrier ampholytes.
An efficient disk based data structure for rapid searching of quantitative two‐dimensional gel databases
Authors: Lemkin Peter F. , Wu Yecheng , Upton Kyle
Published: May 2005

Fast access of two‐dimensional (2‐D) gel quantitative databases is important for rapid searching for protein differences between sets of 2‐D gels from an experiment. The GELLAB‐II system organizes corresponding spots from the gels in the database into reference or “Rspot” sets. These Rspot numeric names index fixed regions in the paged composite gel database file. This is adequate for an existing database, but has several problems. (i) Building the initial database requires guessing how much disk space to pre‐allocate for each corresponding spot (i.e. spots from different gels). If it ever runs out of preallocated space during this process, it must expand the size of each corresponding set of spots copying the old database data into the new in‐place on the disk. (ii) When adding new gels or editing the database, if a new spot is created, the system may also go into this expansion mode. The time spent and wasted disk space can be appreciable – depending on the size of the database (order of 100 gel database). (iii) Because each set of corresponding spots is the same size, we waste space in most spot sets since they do not requuire the additional space a few spot sets require which contain additional fragmented spots. We present a new low‐level disk object‐based structure and algorithm paged indexed buckets (PIB), which optimizes disk space usage while having similar retrieval speed to the original method.
Consecutive silver staining and autoradiography of 35S and 32P‐labeled cellular proteins: Application for the analysis of signal transducing pathways
Authors: Luo Lin‐di , Wirth Peter J.
Published: May 2005

The methodology for the simultaneous analysis of protein synthesis concomitant with protein phosphorylation/dephosphorylation is described. The technique consists of metabolic labeling of rat liver epithelial (RLE) cells with [32P]orthophosphate and [35S]methionine, performing two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) of the mixed samples, followed by silver staining and subsequent autoradiography of the dried silver stained 2‐D PAGE electrophoretograms using two films placed back‐to‐back. The first film, which is positioned in direct contact with the dried silver‐stained gel, visualized both exposure to 35S and 32P while the second film recorded exposure to only 32P due to the differential energy levels of the two isotopes. The juxtapositioning of the silver‐stained images with the two autoradiographic film images permits the unambiguous mapping of the phosphorylated polypeptides back to their corresponding silver‐stained and methionine‐labeled counterparts. This strategy provides quantitative information utilizing both silver staining (measure of constitutive levels of protein expression) and metabolic labeling to measure rates of protein synthesis and/or degradation and phosphorylation and/or dephosphorylation using [35S]methionine and [32P]orthophosphate, respectively. We have utilized this methodology for the in vitro analysis of transforming growth factor type β1 (TGF‐β1)‐mediated signal transduction in RLE cells and have identified three nuclear polypeptides, 1 (pI4.95/Mr 97 kDa), 2 (5.00/85 kDa) and 3 (4.90/84 kDa) whose phosphorylation status is rapidly and transiently modulated by TGF‐β1. The methodology described should have wide applications in studies where it is desirous to measure protein synthesis and/or degradation concomitant with signal transduction pathways involving protein phosphorylation.
A comparative two‐dimensional gel protein database of the intact and regenerating newt limbs
Authors: Tsonis Panagiotis A.
Published: May 2005

In this paper we describe a two‐dimensional gel database of the regenerating newt limb. Protein synthesis was compared in the intact limb, in the 1‐week regenerating limb, representing the dedifferentiation stage, and in the 2‐week regenerating limb, representing the formation of the blastema. This comparative database provided data on differential expression of about 800 proteins during the process of limb regeneration. In addition, a map has been generated for these proteins for future guidance in characterizing further new, unknown proteins. The overall expression patterns of the proteins indicated that the dedifferentiation stage was marked by down‐regulation of most proteins, while the blastema formation was marked by the appearance of many new proteins. The potential use of such a database in isolating factors involved during limb regeneration is discussed.
Comparisons of protein changes in human and rodent hepatocytes induced by the rat‐specific carcinogen, methapyrilene
Authors: Richardson Frank C. , Strom Stephen C. , Copple Deborah M. , Bendele Raymond A. , Probst Gregory S. , Leigh Anderson N.
Published: May 2005

There is a growing concern that the rodent biossay may not always serve as an appropriate model to assess the carcinogenic risk for humans exposed to certain compounds. Mechanistic research that examines the effects of a compound in rodent and man could help in the interpretation of bioassay results. This paper reports a novel use of two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) technology to assess similarities and differences in the response of rodents and humans to the rat‐specific hepatocarcinogen, methapyrilene (MP). A sequential examination of rodent and human hepatic proteins was conducted following in vivo exposure of rats and mice and in vitro exposure of rat, mouse, and human hepatocytes to MP. Results showed that covalent modifications observed in rats and mice in vivo were duplicated both qualitatively and quantitatively in the corresponding in vitro systems and that these modifications correlated with carcinogenic susceptibility. Covalent modifications in human hepatocytes were minimal despite exposure to concentrations of MP that were 6‐fold higher than those used in rodent hepatocytes. These studies suggest that in the case of MP the rat is not the most appropriate model for assessing the human situation. Furthermore, these data show that in vitro‐in vivo comparisons based on 2‐D PAGE may provide adjunctive information for extrapolating rodent toxicity/bioassay results to human risk assessment.
Membrane electrophoresis of DNA
Authors: Heller Christoph , Beck Stephan
Published: May 2005

We have separated double stranded DNA fragments by electrophoresis using a membrane instead of a gel as separating matrix. Such a membrane, containing separated DNA fragments, is suitable for subsequent hybridization analysis. The method combines electrophoretic separation and transfer onto a solid support and therefore eliminates the need for a transfer or blotting step.
Typing of the 3′ hypervariable region of the apolipoprotein B gene: Approaches, pitfalls, and applications
Authors: März Winfried , Ruzicka Viktor , Fisher Eva , Russ Andreas P. , Schneider Wolfgang , Groß Werner
Published: May 2005

Apolipoprotein B‐100 is the principal protein component of lipoproteins with very low, intermediate, and low density. The interaction of apoB‐100 with low density lipoprotein (LDL) receptors is responsible for the uptake of LDL into cells. An AT‐rich hypervariable region is located adjacent to the 3′ end of the apoB gene. It consists of a variable number of tandemly repeated sequences (VNTR). Two approaches were used to analyze this polymorphism. In both, the region harboring the VNTR was amplified with the polymerase chain reaction (PCR). In the first method, fluorescently labeled primers were used in the PCR reactions and products were separated in agarose gels by means of an automated fluorescent fragment analyzer. In the second method, PCR products were analyzed in denaturing polyacrylamide gels and detected with silver staining. Even in the highly sophisticated automated system, agarose gel electrophoresis did not always enable unequivocal assignment of VNTR alleles. In contrast, denaturing polyacrylamide gel electrophoresis made it possible to distinguish the 15 bp differences between the VNTR alleles in a precise and simple manner. The VNTR polymorphism was typed in 234 individuals. Among these were 136 patients with coronary artery disease and 74 healthy controls. Thirteen alleles could be distinguished. The allele containing 49 repeats (VNTR‐49) was found in 9.2% of the coronary artery disease patients and in 4.7% of the controls. Thus, the VNTR‐49 allele increases relative coronary risk by about twofold. It is concluded that the apoB VNTR polymorphism is a potentially useful genetic marker. Since agarose gel electrophoresis may lead to ambiguous results, we prefer typing by denaturing polyacrylamide gel electrophoresis. This has to be accounted for, especially if the apoB VNTR polymorphism is applied to forensic studies.
Amplification of a Y‐chromosomal DNA sequence by the polymerase chain reaction for documentation of residual recipient cells in small samples from bone marrow, peripheral blood and cerebrospinal fluid after bone marrow transplantation
Authors: Suttorp Meinolf , Sprenger Christine , Dreger Peter , Sievers Erika , Claviez Alexander , Löffler Helmut , Schaub Jürgen , Schmitz Norbert
Published: May 2005

The long arm of the human Y‐chromosome contains about 800 to 5000 copies of the tandemly repeated DNA sequence DYZ1. A major part of the repeating unit (pHY10)has been cloned and sequenced [1]. Primers were designed to match a part of this repeat sequence for the amplification of a 154 bp fragment spanning the Eco RI restriction site of the unit [2]. Typical dilution experiments showed that this polymerase chain reaction (PCR) method allows the detection of 5 to 10 male cells among 100000 female cells [3], or in 500 μL of cerebrospinal fluid containing only one cell per μL. In addition, the quality of the DNA used for the amplification reaction is less critical, thus allowing analysis of long‐term stored samples such as bone marrow smears or dried blood stains spotted onto filter paper, which might contain partially degraded DNA [4]. We applied this technique to detect residual host cells in the clinical setting of human sex‐mismatched bone marrow transplantation (BMT). Fourteen patients, receiving transplantations because of leukemias could be supervised so far. Throughout the whole period of monitoring (days + 14 until +911 post BMT; median: 160 days), residual host cells were detected in all but three patients. Persistence of host cells in the early phase post‐BMT was mostly transient and probably due to long‐term surviving host T‐lym‐phocytes. Reappearance of host cells several months after BMT is highly suspicious of relapse from the underlying malignancy. Due to its high sensitivity, PCR is a valuable tool in monitoring the switch from recipient to donor cell population. In addition, the documentation of persisting or reappearing recipient cells could be associated with clinical relapse [5].
Agarose electrophoresis of DNA in discontinuous buffers, using a horizontal slab apparatus and a buffer system with improved properties
Authors: Zsolnai Attila , Orbán Lászlo , Chrambach Andreas
Published: May 2005

Using a horizontal slab apparatus with a buffer in the reservoirs at the level of the gel (“sea‐level electrophoresis”), the retrograde discontinuous buffer system reported by Wiltfang et al. for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of proteins was applied to DNA electrophoresis. This application yielded the advantages of an increased displacement rate of the moving boundary front and a decrease in the concentration of the counterion base in the resolving phase, which yielded reduced relative mobility values at equivalent gel concentrations and practicable low buffer concentrations. The change of relative mobilities (Rf) with a variation of field strength is decreased compared to that of the migration rate in the continuous Tris‐boric‐acid‐EDTA (TBE) buffer and thus the robustness of the system is improved, as well as the efficiency of separation. The system of Wiltfang et al. has in common with previously described discontinuous DNA systems, that it is able to stack DNA from dilute samples and is insensitive to sample components with lower net mobilities than DNA, such as acetate. However, the variance of Rf at constant current density in the discontinuous buffer system is not improved over that of the migration rate at constant field strength in the continuous TBE buffer.
On the use of dimensionless parameters in acid‐base theory. II. The molar buffer capacities of bivalent weak acids and bases
Authors: Rilbe Harry
Published: May 2005

It is shown that stoichiometric rather than thermodynamic dissociation constants can and should be used in the buffer capacity theory. The mean valence of bivalent weak acids and bases is deduced in a form with dimensionless parameters. The expressions for the molar buffer capacities of bivalent weak acids and bases are identical with that for a bivalent ampholyte, and the theory for the latter given earlier is thus valid in all details for bivalent weak acids and bases.
Pattern variations of polyclonal and monoclonal immunoglobulins of different isotypes analyzed by high‐resolution two‐dimensional electrophoresis
Authors: Tissot Jean‐Daniel , Hochstrasser Denis F. , Spertini François , Schifferli Jürg A. , Schneider Philippe
Published: May 2005

High‐resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was used to analyze serum samples and purified immunoglobulins (Ig) obtained from “normal” individuals and from patients diagnosed with monoclonal gammopathies (MG) (n = 47; 5 IgA, 15 IgM, 15 IgG, 4 biclonal IgG, 1 IgD, 7 Bence Jones proteins). Polyclonal and monoclonal heavy (H) chains were located at different restricted gel positions according to their isotype. Monoclonal H chains appeared as sets of spots characterized by charge (pI) and size (Mr) microheterogeneity. Most of the monoclonal gamma chains were not seen on the gels (12/15). Supplementary polypeptides of 45–48 kDa were detected in serum samples containing monoclonal IgM, but were not seen in MG of other isotypes. However, these polypeptides were not specifically associated with monoclonal IgM because they were also found on protein maps of purified polyclonal IgM. Polyclonal light (L) chains appeared as cloudy bands containing several zones of higher density, whereas monoclonal L chains were usually resolved as single sharp spots. In 6 samples, monoclonal L chains were not seen, and in 9 samples, they appeared as two or more spots, characterized by different pI and/or Mr. In one sample obtained from a patient with a biclonal gammopathy, the L chains were resolved as 4 different spots. Our results confirm that 2‐D PAGE is an excellent tool to study Ig. Analysis of the L chain region of the gels was particularly informative. Several monoclonal L chains exhibited heterogeneous two‐dimensional spot patterns, suggesting that “subtile” clonal mutations of B‐cell lineage and/or posttranslational modifications were involved in their production.
Subtyping of alkylated human orosomucoid: Evidence for a duplicated gene, ORM1*F2S
Authors: Rocha Jorge , Amorim António , Luckenbach Christine , Kömpf Jost , Ritter Horst
Published: May 2005

Isoelectric focusing of human orosomucoid (ORM) was studied following different sample treatment. It is shown that: (i) alkylation with iodoacetamide leads to a drastic change in the isoelectric point (pI) of both ORM1 F2 and ORM2 A gene products and greatly improves the discrimination between ORM1 F1 and ORM1 F2; (ii) previous reduction of the molecule with dithiothreitol partially inhibits the pI transitions with resultant artifactual ORM1 F1F2S patterns that correspond in most cases to F2S phenotypes. With the technique now described, the persistence of three ORM1 gene products was found in only one individual and the segregation analysis is consistent with the existence of a rare ORM1*F2S haplotype.
Reusing Cellogel strips after visualization of electrophoretically separated isozymes
Authors: Destro‐Bisol Giovanni
Published: May 2005

A simple procedure which allows reusing electrophoresed and stained Cellogel strips is described. The method is based on washing with H2O and absolute methanol and allows reusing the strips two or three times, thus considerably reducing the costs.