bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrophoretic and isoelectric focusing analysis of human recombinant alpha2‐HS glycoprotein produced in insect cells: Analysis of the post‐translational events
Authors: Kalabay Laszlo , Mathur Savita , Bobin Sophie , Arnaud Philippe
Published: May 2005

Alpha2‐HS glycoprotein (AHSG) is a human serum glycoprotein synthesized by liver cells. It is a natural inhibitor of the insulin receptor tyrosine kinase activity. We produced this protein in insect cells by using a recombinant baculovirus expressing the whole coding sequence of the protein. By analyzing AHSG on isoelectric focusing and on sodium dodecyl sulfate (SDS) gels, followed by immunoblot, AHSG produced in insect cells was found to be phosphorylated and to possess the connecting peptide between the A and the B chains. The same features were found in the protein produced by Hep3B, a human liver cell line that synthesizes AHSG. By contrast, no phosphorylation could be detected in AHSG present in normal human plasma, and the connecting peptide was clipped. As the protein produced in insect cells is active on insulin receptors, in contrast to the plasma protein, our results suggest that the biological activity of the protein may be associated with its single chain form together with its phosphorylation.
Theoretical permutation gel electrophoretic analysis of a curved DNA fragment located in circular permutation
Authors: Nair T. Murlidharan , Madhusudan K. , Nagaraja V. , Kulkarni B. D. , Majumdar H. K. , Singh Rajan
Published: May 2005

Using the theoretical model for DNA curvature, we analyzed a set of fragments with a curved insert located in circular permutation. The theoretical permutation analysis of each of the cyclically located fragments reveals the presence of a shifting molecular bend locus. The delineation of the molecular bend locus associated with the fragments obtained by a second permutation helps in providing an explanation for the differential mobility behavior of the fragments.
Enhanced field strength and resolution in gel electrophoresis upon substitution of buffer by histidine at its isoelectric point
Authors: Chen Nong , Chrambach Andreas
Published: May 2005

Gel electrophoresis in isoelectric buffers, recently introduced by R. Westermeier and H. Schickle (Electrophoresis '95, Paris, Abstract No. 3, 1995), was applied to the automated HPGE‐1000 apparatus in the expectation to be able to increase the field strength under the limiting conditions of heat dissipation capacity and voltage of that apparatus. A previous attempt to achieve that aim by reduction of gel thickness had not yielded more than a twofold increment in resolving power. Replacing 0.2 × Tris‐boric acid‐EDTA (TBE) buffer, conventionally applied in the apparatus at 15 V/cm, by 0.05 M histidine, pH 7.6 (close to the pI of 7.47), allows one to increase the field strength to 60 V/cm, thus providing a nearly fivefold increment in resolution under otherwise identical conditions (fluorescein carboxylate‐labeled conalbumin‐sodium dodecyl sulfate (SDS) and soybean trypsin inhibitor‐SDS samples, 10°C, 4% MetaPhor agarose). An additional decrease in band dispersion can be obtained by decreasing the starting zone width through buffer dilution in the sample phase.
Calculation of modified capacity factor in micellar electrokinetic chromatography
Authors: Idei Miklós , Kiss Eva , Kéri György
Published: May 2005

In addition to the expression, k′ = (tm−to)/to (1–tm/tmc), we propose the expression k″ = (tm−to)/(tmc−to) to calculate the capacity factor in micellar electrokinetic chromatography (MEKC), where tm, to, and tmc are the migration time of the analyte, the flow marker, and the micelles, respectively. The k′ and k″ values that were obtained from simulated data as well as from MEKC analysis of different peptides (in 100 mM sodium dodecyl sulfate/0.1 N sodium borate buffer at pH 11.0) were calculated and compared. The k″ value is equal to zero for an analyte remaining in the aqueous phase whereas it is equal to one for an analyte always staying in the micellar phase. By applying k″ a finite capacity factor can be obtained for an analyte, indicating its partition between the two moving phases (aqueous and micellar) even in those cases when tm equals tmc. The slope of the curve k″ as a function of tm is constant through the whole migration window and therefore peak compression does not occur when applying k″ to calculate the capacity factor. A given difference in k″ corresponds the same difference in migration times and this value does not depend on the position within the migration window. Since k″ is a normalized paramete it is easy to evaluate the significance of a given difference in capacity factor or to estimate the relative position of an analyte with a given capacity factor in the migration window by applying k″. Therefore, k″ seems to be an adequate parameter to calculate the capacity factor in MEKC and, similar to k′, it also refers to the hydrophobicity of the analyte.
Separation and isolation of subcellular‐size particles by electrophoresis in polymer solution using the commercial scanning apparatus
Authors: Chang Huang‐Tung , Zakharov Sergey F. , Chrambach Andreas
Published: May 2005

Electrophoresis of fluorescently labeled rat liver microsomes and polystyrene carboxylates of 10 and 30 nm radius was conducted in buffered 10–15% polyvinylpyrrolidone (Mr = 106) solutions, using a horizontal gel electrophoresis apparatus with intermittent scanning of fluorescence (HPGE‐1000, LabIntelligence). Banding, constant migration rates and Ferguson plots were obtained in these polymer solutions. The major microsome band detected by the automated scan was located visually on the gel by means of its fluorescein label and was isolated by volumetric withdrawal; recovery was monitored by scanning and ascertained to be near quantitative after three consecutive steps, in each of which 30 μL were withdrawn. This preparative method promises to be generally applicable to particles that are too large to enter into gels.
Protein degradation in human pure pancreatic juice analyzed by two‐dimensional gel electrophoresis
Authors: Furui Toshifumi , Ikeda Miyuki , Chao‐Ming Liao , Okita Kiwamu , Nakamura Kazuyuki
Published: May 2005

Two‐dimensional gel electrophoresis (2‐DE) was used to study protein degradation in human pure pancreatic juice (PPJ) which was collected at 5 min intervals for 20 min by selective endoscopic cannulation of the main pancreatic duct. In PPJ collected from healthy subjects no significant degradation was observed by incubating PPJ at 37°C up to 6 h. By further incubation for 24 h, glycoprotein‐1, procarboxypeptidase A‐1 and lipase were nearly completely degraded, while α‐amylase and procarboxypeptidase B‐1 were not degraded under these conditions; α‐amylase became labile in the presence of 1 mM ethylene diaminetetraacetic acid (EDTA) or 10 mM phenyl methyl sulfonyl fluoride (PMSF). Protein degradation was observed by 2‐DE of an initial fraction of PPJ collected from patients with chronic calcific pancreatitis (CCP). The 2‐DE patterns of subsequent fractions resembled those of PPJ from healthy subjects. The mixture of the last fraction with the initial fraction showed significant protein degradation, inhibited by adding aprotinin. Furthermore, the extent of protein degradation correlated with the dilatation of the main pancreatic duct as a consequence of intraductal stagnation of pancreatic juice. These findings demonstrate that protein degradation in PPJ is accelerated by intraductal activation of serine proteases in the case of patients with CCP. 2‐DE of PPJ from patients with CCP provides useful information for the evaluation of intraductal activation of zymogens and the progress of chronic pancreatitis.
Structural analysis and identification of gel‐purified proteins, available in the femtomole range, using a novel computer program for peptide sequence assignment, by matrix‐assisted laser desorption ionization — reflectron time‐of‐fligh — mass spectrometry
Authors: Gevaert Kris , Verschelde Jean‐Luc , Puype Magda , Van Damme Jozef , Goethals Marc , de Boeck Stefaan , Vandekerckhove Joël
Published: May 2005

A procedure is described for structural characterization and identification of proteins, purified by either one‐ or two‐dimensional gel electrophoresis in the low picomole to femtomole range. The purified proteins are first detected in the primary gels by the sensitive reverse staining procedure described by Fernandez‐Patron et al. (Anal. Biochem. 1995, 224, 203–211) and consecutively reeluted from combined gel pieces and concentrated in the tip of a Pasteur pipette in a secondary gel matrix consisting of either sodium dodecyl sulfatepolyacrylamide or agarose. The concentrated proteins are in‐matrix‐digested and the resulting peptides are separated by reverse‐phase high performance liquid chromatography (HPLC) combined with microsequencing or analyzed by matrix‐assisted laser desorption ionization — time of flight — mass spectrometry. Protein identification is based on sequence homology or on the peptide mass pattern. The matching peptide sequences can additionally be verified by matching their measured post‐source decay spectra with the calculated fragmentation patterns of the isobaric candidate peptides appearing on the search list. This is done by a computer program referred to as MassFrag, described in this paper. We demonstrate that it is possible to identify protein that are only available in the femtomole range and whose sequences are stored in nonredundant protein databases or nucleotide and expressed sequence tag databases.
Investigation on minor degraded derivatives of the recombinant hirudin variant HM2 from Hirudinaria manillensis isolated by isoelectric focusing in multicompartment electrolyzers
Authors: Bossi Alessandra , Righetti Pier Giorgio , Visco Carlo , Breme Umberto , Mauriello Maurizio , Valsasina Barbara , Orsini Gaetano , Wenisch Elisabeth
Published: May 2005

On isoelectric focusing in immobilized pH gradients (IPG) a preparation of recombinant hirudin from Hirudinaria manillensis, purified to homogeneity, was found to still contain a total of 5% minor components: three with higher pI values (pIs 4.10, 4.25 and 4.31), one with a lower pI value (pI 3.98) as compared with the main form (pI 4.03). Multicompartment electrolyzers with isoelectric membranes and micropreparative IPG gel slabs allowed the recovery of pure fractions of such minor components, which were further characterized by electrospray mass spectra, limited proteolysis, and sequence analysis. All four minor isoforms were found to be cleavage products of the parent, full‐length hirudin molecule (molecular mass 6797 Da), as follows: the pI 4.31 (5032 Da) had lost sixteen amino acids from the N‐terminus, the pI 4.25 (6212 Da) lacked five amino acids from the C‐terminus, the pI 4.10 (2980 Da) was a cleavage product at residue Cys37, and the pI 3.98 (6610 Da) lacked the dipeptide Val‐Ser at the N‐terminus. Combining the extreme resolving power of IPGs with the high accuracy of mass spectra was found to be an attractive strategy in decoding post‐synthetic modifications often encountered in r‐DNA proteins.
Protease‐catalyzed incorporation of 18O into peptide fragments and its application for protein sequencing by electrospray and matrix‐assisted laser desorption/ionization mass spectrometry
Authors: Schnölzer Martina , Jedrzejewski Paul , Lehmann Wolf D.
Published: May 2005

Proteins were digested in normal and highly 18O‐enriched water using proteases commonly employed for protein sequencing. The extent of 18O incorporation into the resulting peptide fragments was characterized by electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The endoproteinases trypsin, Lys‐C and Glu‐C incorporate two atoms of 18O, resulting in a mass shift of +4 D for the peptide fragments. This indicates that, following proteolytic cleavage, peptide products continue to interact with these proteases and undergo repeated binding/hydrolysis cycles, resulting in complete equilibration of both oxygens in the carboxy terminus of the fragments with oxygen from solvent water. In contrast, chymotrypsin and Asp‐N incorporate only one atom of 18O, resulting in a mass shift of +2 D, indicating that after the cleavage step these proteases do not accept the peptides as substrates. In addition, it was found that the proteases trypsin, Glu‐C, and Lys‐C exhibit minor or nontypical sequence specificities, resulting in unexpected peptide fragments. These fragments incorporate only one 18O atom, indicating that they do not undergo further binding/hydrolysis cycles with the enzyme. Thus, it is possible to discriminate between enzyme‐typical peptide fragments with mass shifts of +4 D and nontypical fragments with mass shifts of only +2 D. Based on these observations, protein digest strategies are described for the generation of 1:1 ion doublets spaced either by 2 or 4 D. In addition, the C‐terminus of a protein can be identified by the absence of an ion doublet in the corresponding peptide fragment. In protein sequencing by mass spectrometry, digest protocols generating ion doublets provide the most clear‐cut analytical results for the recognition of ion series in ESI‐MS/MS and MALDI post‐source decay (PSD) product ion specta. Only the mass spectrometric fragment ions of a C‐terminal series show ion doublets spaced either by 2 or 4 D, whereas the fragment ions belonging to an N‐terminal series remain unshifted. This assignment unequivocally reveals the direction of the identified sequence.
Intrinsic curvature of plasmid DNAs analyzed by polyacrylamide gel electrophoresis
Authors: Strutz Kurt , Stellwagen Nancy C.
Published: May 2005

The electrophoretic mobility of two small DNA plasmids, pUC19 and Litmus 28, linerarized by digestion with a variety of single‐cut restriction enzymes, has been studied. The permuted sequence isomers migrate with identical mobilities in agarose gels, as expected, but exhibit different mobilities in large‐pore polyacrylamide gels, suggesting that the parent plasmids contain sequencespecific sites of curvature and/or anisotropic flexibility. Both plasmids contain apparent bend centers near their origin(s) of replication; pUC19 also has a major apparent bend center near the promoter of the ampicillin resistance gene. These apparent bend centers are observed under a variety of experimental conditions, suggesting that they correspond to sites of stable curvature in the parent plasmids. Both plasmids also contain minor bend centers that are observed under a sub‐set of electrophoretic conditions and disappear when divalent cations are added to the solution, suggesting that these apparent bend centers may correspond to localized regions of variable flexibility.
Dynamic symmetry breaking in a model of polymer reptation
Authors: Aalberts Daniel P. , Van Leeuwen J. M. J.
Published: May 2005

A numerical study of the transition between oriented and nonoriented polymers in a model for reptation is presented. An electric field biases a charged polymer to drift in the direction of its leading end. At low fields this bias is not enough to orient the polymer, but at larger fields it succeeds in orienting the polymer with a definite head and tail. The resulting motion depends on the relative magnitudes of the field and the chain length. For a given chain length, the field plays the role of inverse temperature in this transition. The critical field between these “phases” is found to be a non‐trivial power of the length of the chain. This scaling extends over two full decades in the coupling parameter and the simulations performed involved 2 × 109 Monte Carlo steps per data point.
Theory of gel electrophoresis of DNA: Orientational effects
Authors: Subbotin Andrei V. , Semenov Alexander N.
Published: May 2005

Some aspects of the reptation model of gel electrophoresis of DNA, with emphasis on the orientational effects, are considered. A master equation for the reptational dynamics with the end boundary conditions is proposed. Based on this equation, the orientation and dynamics of the persistent‐chain are investigated. In particular, the role of trap conformations for this chain, which essentially reduce the chain mobility, was analyzed. A transient orientation of the chain during pulsed field gel electrophoresis is also discussed.
Cyclic migration of DNA in gels: DNA stretching and electrophoretic mobility
Authors: Åkerman Björn
Published: May 2005

Available data from spectroscopic and microscopy studies of electrophoretic orientation of long DNA (above 40 kbp) in agarose gels is analyzed on the basis of the fact that the migration in constant fields is cyclic in nature. Defining a cycle period as the time between two consecutive compact states, a simple model is used to obtain data on the average time period (〈T〉) and the step length (〈L〉) of the migration cycle from spectroscopic measurements of the dynamics of helix orientation and center‐of‐mass velocity. Furthermore, the degree of orientation is used to analyze tube‐orientation and DNA stretching contributions to 〈L〉 and 〈T〉. Finally, the average electrophoretic velocity v = 〈L〉/〈T〉 is analyzed in terms of 〈L〉 and 〈T〉 for different DNA sizes (Lc), field strengths (E), and gel concentrations (A). The main results of the analysis are: (i) the increase and saturation of the electrophoretic mobility with increasing E is mainly governed by 〈L〉 via the degree of DNA stretching, (ii) DNA molecules of different sizes migrate with the same velocity because 〈L〉 and 〈T〉 both increase approximately linearly with Lc, and (iii) migration in a denser gel is slower mainly because 〈T〉 increases, while the step length is approximately constant. Assuming the charge Q of DNA is the same as in free solution, these results suggest that the reason the fundamental reptation equation for the electrophoretic mobility μ = (Q/ζ) < (hx/Lt)2 > also applies in the presence of strong fluctuations in the tube length Lt and end‐to‐end distance hx, is that the friction coefficient ζ for motion along the tube is lower the more stretched the DNA is.
Pulsed‐field electrophoresis in microlithographic arrays
Authors: Duke Thomas A. J. , Austin Robert H. , Cox Edward C. , Chan Shirley S.
Published: May 2005

Transverse pulsed‐field electrophoresis of DNA has been conducted in a silicon array engineered by optical lithography and the motion of individual molecules observed by fluorescence microscopy. In strong fields, the molecules can be maintained in highly stretched, linear conformations. When the field is switched through an obtuse angle, they head off in the new direction led by what was formerly their tail end. This backtracking gives rise to fractionation that is linear with molecular weight. A simple prescription exists for choosing the field parameters to obtain a particular range of separation. Since the molecular motions are much more uniform than those that occur in a gel, it is anticipated that the arrays will permit more efficient fractionation than traditional pulsed‐field gel electrophoresis. Arrays suitably scaled down in size may be useful for pulsed‐field sequencing.
Theory of solitary waves in electrophoresis
Authors: Kist Tarso B. L.
Published: May 2005

A theoretical study about the generation of solitary waves (SW) in electrophoresis and capillary electrophoresis is performed. Two models that use velocity terms in Fick's first law and absorption and decaying terms in Fick's second law are presented. These models give the time evolution of the band or zone shape in electrophoresis and capillary electrophoresis in the presence of electromagnetic radiation. In particular, the effect of electrophoretic mobility changes of the analytes due to radiation excitation is included in the present models. The analytes are represented either by three‐level or four‐level systems. It is shown that the resulting system of coupled nonlinear partial differential equations governing the spatial motion of these bands over time exhibits SW solutions for some values of the equation parameters. We analyze the conditions in which these SWs, which propagate with constant velocity, constant area, constant standard deviation, and without change of form, are generated. The results of the present models are compared with those from a previous two‐level model (Phys. Rev. Lett. 1995, 75, 1210–1213). The velocities of these SWs are calculated analytically. The time evolution of their standard deviations is shown. The numerical integration of a two‐component electrophoresis run shows higher resolutions under some conditions. The expected practical difficulties which may be encountered when observing this phenomenon are discussed. Some practical difficulties that are likely to limit its useful application in electrophoresis and capillary electrophoresis are mentioned.
Effect of temperature and viscosity of sieving medium on electrophoretic behavior of sodium dodecyl sulfate‐proteins on capillary electrophoresis in presence of pullulan
Authors: Nakatani Manabu , Shibukawa Akimasa , Nakagawa Terumichi
Published: May 2005

Pullulan was used as a sieving medium in high‐performance capillary gel electrophoresis to study the effect of temperature and viscosity on electrophoretic behavior of sodium dodecyl sulfate (SDS)‐proteins with molecular masses in the range of 14 400–116 000 Da. The migration time decreased with increasing capillary temperature. Equations were derived relating mobility of SDS‐proteins to capillary temperature and viscosity of pullulan solution. Linear relationships were obtained from experiments between logarithm of mobility and reciprocal temperature and between double logarithmic values of mobility and viscosity of pullulan solution. The experimental results agree well with the equations.
Vancomycin as a chiral selector in capillary electrophoresis: An appraisal of advantages and limitations
Authors: Vespalec Radim , Billiet Hugo A. H. , Frank Johannes , Boček Petr
Published: May 2005

The properties of the macrocyclic antibiotic vancomycin, used as a chiral selector, were studied with aminoquinolycarbamate derivatives of amino acids, containing sulfur and selenium, as well as with other organic ions. Vancomycin combines the ability to resolve fully ionized anionic enantiomers, typical of proteins, with excellent separation efficiency, exceeding that of cyclodextrins. It allows better than baseline chiral separations of several anionic analytes within 3–5 min. The resolving power of vancomycin results from its great skill in discriminating enantiomers rather than from high affinities to the separated enantiomers. The association constants of vancomycin are of the same order of magnitude, 102 L/mol, as that found for β‐cyclodextrin (β‐CD). The difference in association constants of separated cystine enantiomers with vancomycin, 2 × 102 L/mol, is one order of magnitude higher than that of enantiomers separated with β‐CD. Analytically convenient mobility differences up to 1–2 × 109 m2V−1S−1, with only one of the enantiomers appreciably decelerated, are obtained at submillimolar vancomycin concentrations. Typical separation efficiencies are close to 250 000 theoretical plates per meter of capillary. Deceleration of various organic ions by millimolar vancomycin implies that chiral separations with vancomycin need not be restricted to carboxylic acids. The vancomycin‐analyte interactions are strongly affected by the chemical composition and concentration of the buffer. An additional experimental variable, highly effective in manipulating the separation selectivity of analytes, is the buffer pH.
Purification of glycopeptide antibiotics by isoelectric focusing in multicompartment electrolyzers with Immobiline membranes
Authors: Bossi Alessandra , Righetti Pier Giorgio , Riva Ernesto , Zerilli Luigi
Published: May 2005

The purification of small glycopeptides (a Hepta‐Tyr of the teicoplanin family, exhibiting broad activity against highly glycopeptide‐resistant enterococci) by isoelectric focusing in multicompartment electrolyzers with buffering, isoelectric membranes, is described. The main obstacle to such a preparative technique, in common with all focusing methodologies, is the poor solubility of the analyte at the pI value with resultant precipitation and coprecipitation of all impurities with the main fraction. A good solubilizing power was obtained in hydro‐organic solvents, particularly a mixture of 6 M urea and 20–25% trifluoroethanol. Best results, however, were obtained with mixtures of 8 M urea and zwitterionic detergents, notably the 3‐[(3‐cholamidopropyl)dimethylammonia]‐1‐propanesulfonate (CHAPS) family. A unique behavior of the peptide was found in concentration gradients of CHAPS: solubility increases up to 3.5% CHAPS, but the curve shows a maximum and then solubility decreases again at 5% CHAPS. In mixtures of 8 M urea and 3.5% CHAPS, sample loads of 500 up to 1000 mg Hepta‐Tyr could be purified in a single run, with recoveries > 90% and purity in excess of 99%. The main glycopeptide fraction (pI 8.56) was collected into an isoelectric trap delimited by pI 8.46 and pI 8.65 membranes. Attempts at purifying the glycopeptide by most known reversed phase‐high performance liquid chromatography (RP‐HPLC) techniques failed completely.
Successful deoxyribonuclease I (DNase I) phenotyping from a small piece of used sock
Authors: Yasuda Toshihiro , Takeshita Haruo , Sawazaki Kazumi , Iida Reiko , Kishi Koichiro
Published: May 2005

The forensic application of the DNase I polymorphism for individualization of used socks is described. We devised a technique that combines a simple extraction, method, including a partial purification step, using a Phenyl‐Sepharose CL‐4B column, isoelectric focusing and activity staining, for DNase I typing from socks. As a result, DNase I types were determined from socks kept at room temperature for a month or more in all of the samples tested. The DNase I types determined from socks exactly matched those determined from corresponding sweat and urine samples. By contrast, AB0 groupings could not be determined from these samples, using the conventional serological method. Therefore, we have shown that the DNase I polymorphism is the first genetic marker found to be well suited for individualization of used socks.
Protein alterations associated with gene amplification in cultured human and rodent cells
Authors: Conner Elizabeth A. , Wirth Peter J.
Published: May 2005

Genomic stability was investigated in Chinese hamster ovary (CHO) and human hepatocellular carcinoma HepG2 cells selected for growth in the presence of cytotoxic concentrations of N‐(phosphonacetyl‐L‐asparate) (PALA). In CHO cells selected with 9 × LD50 PALA the carbamyl p‐synthetaase, asparate transcarbamylase and dihydroorotase (CAD) gene complex was amplified two‐fold while in HepG2 cells selected at comparable PALA concentrations a 7‐ to 10‐fold increase in the CAD gene was observed. Concomitant with amplification of the CAD gene were increases in CAD mRNA and protein expression in both CHO and HepG2 cells. In long‐term cultures of HepG2 cells the CAD gene underwent spontaneous amplification (5‐fold) in the absence of PALA treatment with increasing passage number. In an attempt to define proteins and/or family of proteins that may either directly or indirectly influence DNA amplification potential through a mechanism of enhanced genomic instability, immobilized pH gradient‐two‐dimensional polyacrylamide gel electrophoresis (IPG 2‐D PAGE) analysis of silver‐stained nuclear cytoplasmic polypeptides comcomitant with PALA resistance and CAD amplification was performed. Analysis of silver‐stained polypeptides from 3 × LD50 PALA‐selected CHO and HepG2 cells revealed no significant alterations in polypeptide expression. In CHO cells selected at 5 × and 7 × PALA LD50, and HepG2 cells selected at 5 × and 9 × PALA LD50, one subset of 4–8 polypeptides (pl: pI 7.2–7.6/36–38 kDa) were increased 2‐ to 3‐fold in both 5 × and 7 ×‐ and 5 × and 9 × LD50 PALA‐selected CHO and HepG2, respectively, while five relatively neutral‐to‐basic, low Mr polypeptides (p2: 18/7.30; p3: 16/7.00; p4: 14/7.00; p5: 14/7.40; and p6: 13.5/7.00) were markedly increased in CHO cells selected at 7 × LD50 PALA. In addition to these PALA‐associated increases, four polypeptides (p7a: pI 6.50/40 kDa; p7b: 6.55/40; p7c: 6.60/40: and p7d: 6.65/40) were significantly increased in high‐passage (p159) HepG2 cells undergoing spontaneous CAD gene amplification in the absence of PALA exposure. In CHO cells, polypeptides p7 a, b, d were increased while the expression of p7c (pI 6.60/40 kDa) was unaltered in 7 × LD50‐treated CHO cells. Although neither the identity nor biological function of polypeptides 1–7 is known, a proposed mechanism involving interaction with certain growth regulatory proteins such as p53 for mediating genomic instability is given.
Immune response to different doses of a hapten of fluorescein isothiocyanate analyzed by two‐dimensional affinity electrophoresis
Authors: Wang Pei , Nakamura Kazuyuki , Mimura Yusuke , Takeo Kazusuke , Tanaka Tatehiko , Fujimoto Masanori
Published: May 2005

The immune response to different doses of a hapten of fluorescein isothiocyanate (FITC) in BALB/c mice was analyzed by two‐dimensional affinity electrophoresis (2‐D AEP). The mice were immunized with different doses (0.5 μg, 5 μg, 50 μg, 500 μg and 2.5 mg) of FITC‐conjugated bovine serum albumin (BSA). The antibodies to FITC bovine serum albumin (BSA) were separated into a large number of IgG spots due to differences in their isoelectric points (pI) and binding affinity to FITC ligand immobilized in the gel. The IgG spots. showing identical affinity to the ligand but different pI, have been considered as an IgG family. The affinity and quantity of IgG families changed with the increase in FITC‐BSA dosage. With a low dose (5 μg) most of the families showed high affinity (Kd < 1 μM). When the dose was increased, not only high affinity antibodies but also intermediate (1 μM < Kd < 50 μM) and low affinity (Kd > 50 μM) antibodies were produced. The increase of FITC‐BSA up to 500 μg markedly increased the quantity of IgG spots showing a variety of affinity to FITC. However, 2.5 mg FITC‐BSA did not increase the quantity and heterogeneity of IgG spots significantly. The changes in the heterogeneity and quantity of anti‐FITC antibodies and the subclass switch were observed over the course of immunization. The heterogeneity and the quantity of IgGl, IgG2b and IgG3 antibodies increased markedly during the first and the second immunization, whereas an increase in the heterogeneity and the quantity of IgG2a antibody was observed in the third immunization. This suggests that the subclass switch to IgGl, IgG2b and IgG3 and the somatic mutation of IgG1, IgG2b and IgG3 occur during the first and the second immunization, but the subclass switch to IgG2 and the somatic mutation of IgG2a seem to occur later than that of the other IgG subclasses.
Rapid detection of sequence polymorphisms in the human mitochondrial DNA control region by polymerase chain reaction and single‐strand conformation analysis in mutation detection enhancement gels
Authors: Alonso Antonio , Martin Pablo , Albarran Cristina , Garcia Oscar , Sancho Manuel
Published: May 2005

The article describes a rapid approach for the detection of sequence polymorphisms in the mitochondrial (mt)DNA control region that involves enzymatic amplification of each entire mtDNA control region (HV1 and HV2) and the subsequent analysis of the PCR products by single‐strand conformation analysis (SSCA) in mutation detection enhancement (MDE) gels, followed by silver stain detection. HV1 and HV2 SSC reference ladders were developed to standardize the classification of the different mtDNA types. Twenty‐five mtDNA types were observed among the 45 Spanish individuals analyzed: 11 types were observed in the HV1 region as compared with 10 types in the HV2 region. This mutation scanning strategy could be a promising method of potential use not only in forensic genetics but also in population and evolutionary studies.
Dynamics of protein isoelectric focusing in immobilized pH gradient gels
Authors: Stoyanov Alexander V. , Righetti Pier Giorgio
Published: May 2005

The protein migration velocity profile through the pH gradient in immobilized pH gradient gels (IPG) was investigated. Strong nonuniformities of migration velocity distribution, which could not be remedied by adding carrier ampholytes, were observed at the initial stages of a run (up to several thousand volthour products for wide pH gradients). The final (after a prerun) velocity distribution in IPG gels differs from the one containing carrier ampholytes, with maximum velocity being achieved not at the pH extremes but closer to neutrality. In some uniform pH media, created by either a single or by several Immobilines, essentially a nonconstant migration profile was also obtained immediately after a prerun. After a prerun, followed by a pause in the absence of current, the phenomenon of fast evolution of the final velocity profile was found.
Characterization of competitive binding, fluorescent drug immunoassays based on micellar electrokinetic capillary chromatography
Authors: Steinmann Lone , Thormann Wolfgang
Published: May 2005

This paper characterizes competitive binding, electrokinetic capillary‐based immunoassays for various drugs in human serum using reagents which were commercialized for fluorescence polarization immunoassays. After incubation of serum with the reactants, a small aliquot of the mixture is applied onto a fused‐silica capillary and tracers (fluorescein‐labeled drugs) and the antibodytracer complexes are separated and analyzed by micellar electrokinetic capillary chromatography with on‐column laser‐induced fluorescence detection. Examples studied include serum assays for theophylline, ethosuximide, paracetamol, salicylate and quinidine. With these assays, concentration‐dependent peaks produced by the free tracers or the antibody‐tracer complexes serve as the basis for quantitation. The sizes of the peaks produced are shown to be dependent on the applied power and the proportions of the reactants and serum employed. The separation medium permits effective characterization of tracers and antibody selectivities. Based on the high selectivity of the antibodies employed, the feasibility of the simultaneous performance of different immunoassays is demonstrated. For capillaries of 50 μm internal diameter (ID), separations are best performed at electric fields < 500 V/cm, this resulting in electrokinetic analyses within 4 to 10 min (capillaries of 20 to 50 cm effective length).
Some examples of the analysis of complex samples by micellar electrokinetic capillary chromatography with photodiode array detection
Authors: Vandepeer Jasmine , Trenerry V. Craige , Keogh Geraldine
Published: May 2005

The use of micellar electrokinetic capillary chromatography with photodiode array detection to assist in identifying the minor components in crude morphine, poppy straw and opium preparations, for the analysis of synthetic colours and other food additives in cordials and confectionery and for the identification of the ring substituted amphetamine derivative methylenedioxyethylamphetamine in illicit drug samples is demonstrated.
Dispersion coefficients of λ DNA in agarose gels during electrophoresis by fringe recovery after photobleaching
Authors: Tinland Bernard
Published: May 2005

By combining an electrophoretic cell with a fringe recovery after photobleaching setup, we have measured both the electrophoretic mobility μ and the dispersion coefficient D of double‐stranded λ‐DNA in agarose gel as a function of the applied electric field E. Besides the determination of the mobilities, experimental values of the exponential decay of the signal gives, for the first time, experimental values of the longitudinal Dx and transverse Dy dispersion coefficients (field E perpendicular and parallel to the light fringes, respectively). Time dispersion measurements are complicated by the existence of a bleached area drift time. We present an analysis of their different contributions. Both dispersion coefficients increase linearly with increasing field E and, in the range of field we used, Dx is larger than Dy. Our results are consistent with the recent throretical predictions of Slater (Electrophoresis 1993, 14, 1–7) and Duke et al. (Biopolymers 1994, 94, 239–247).
The effect of ethidium bromide on mobility of DNA fragments in agarose gel electrophoresis
Authors: Sigmon Janie , Larcom Lyndon L.
Published: May 2005

Ethidium bromide (EtBr) is sometimes added to running buffer during the separation of DNA fragments by agarose gel electrophoresis. It is used because upon binding of the molecule to the DNA and illumination with a UV light source, the DNA banding pattern can be visualized. The mode of binding of EtBr is intercalation between base pairs. This binding changes the charge, weight, conformation, and flexibility of the DNA molecule. Since DNA molecules are sized by their relative movement through a gel compared to a molecular weight standard, mobility measurements can be critical to size determinations. After running two identical gels, one without EtBr and one with 0.25, 0.5, 0.75 or 1.0 μg/mL EtBr in the running buffer, the mobilities of λ HindIII DNA fragments were compared. The mobility of DNA was always less in the gels with EtBr. Using the reptation theory equation, which describes the mobility of DNA molecules through a gel, changes in frictional coefficients were calculated. It was determined that the change in frictional coefficients brought about by the addition of EtBr is directly proportional to the fraction of base pairs of a fragment bound to EtBr. This change in friction is greatest in the largest fragments, which suggests that the stiffening of the molecule by the EtBr binding is the cause for the decreased mobility.
Evaluation of the ALF DNA sequencer for high‐speed sizing of short tandem repeat alleles
Authors: Decorte Ronny , Cassiman Jean‐Jacques
Published: May 2005

DNA typing of short tandem repeaat (STR) loci with automated real‐time analysis of the fluorescent polymerase chain reaction (PCR) products has given forensic DNA analysis a new dimension. In the present work, the ALF DNA sequencer was evaluated for automated size determination of tetra‐nucleotide STRs at high speed. Short gel plates, with a well‐to‐laser distance of 10 cm, allowed for the analysis of four STR loci (HUMvWF, HUMTH01, D21S11 and HPRT) in one gel lane in less than 75 min. Allele size determination was done with two external allelic ladders for each locus. Lane‐to‐lane variations were overcome by the inclusion in each lane of two fluorescent PCR products of constant size (123 and 375 bp) that migrated below and above the multiplex of the four STR loci. The accuracy of sizing and allele detection within and between different gels was high (99.89%) for all four STR systems investigated and the gels could be reloaded without a decrease in accuracy of the allele size estimation. This way, the throughput of the system was increased, which is of interest for linkage studies, gene mapping and population diversity studies.
Validation of the applied biosystems Prism™ 377 automated sequencer for forensic short tandem repeat analysis
Authors: Frazier Rachael R. E. , Millican Emma S. , Watson Stephanie K. , Oldroyd Nicola J. , Sparkes Rebecca L. , Taylor Karen M. , Panchal Sasikala , Bark Linda , Kimpton Colin P. , Gill Peter D.
Published: May 2005

The Applied Biosystems (ABI) PrismTM 377 DNA sequencer has been evaluated in an attempt to increase the throughput of samples for short tandem repeat (STR) analysis, in both forensic casework and the UK National Criminal Intelligence DNA Database. The gel system assessed consisted of 0.2 mm, 4% acrylamide 6 M urea gels, with a well‐to‐read distance of 36 cm. Gels were run at a constant voltage of 3 kV and constant temperature of 51°C. The run time of our second generation multiplex (SGM) STR system was achieved in less than 2 h. Rigorous validation has been performed on the instrument hardware and software Complete resolution of 1 base difference was obtained, up to and beyond 350 bases; sizing precision across gels was more than 2‐fold higher than the 373A and the sensitivity was increased by one third.
Multilocus fingerprinting and single locus analyses in the great tit for paternity determination
Authors: Lubjuhn Thomas , Epplen Cornelia , Epplen Jörg T.
Published: May 2005

In order to arrive at detailed information about the occurence of alternative reproductive tactics in the great tit (Parus major) we examined parental lineages by multilocus fingerprinting in 51 great tit broods containing 429 nest‐lings. In 45% of the broods at least one nestling was found to stem from copulations with males other than the social mate of the female. Single broods contained up to 62.5% extra‐pair young (EPY). Single locus profiling revealed how many fathers were involved in extra‐pair paternity (EPP) within each brood: In most cases a single male is the genetic father of all EPY in a given brood.
Quick and easy molecular weight determination with Macintosh computers and public domain image analysis software
Authors: Seebacher Thomas , Bade Ernesto G.
Published: May 2005

The program “molecular weights” allows a fast and easy estimation of molecular weights (Mr), isoelectric point (pI) values and band intensities directly from scanned, polyacrylamide gels, two‐dimensional protein patterns and DNA gel images. The image coordinates of Mr and pI reference standards enable the program to calculate Mr and pI values in a real time manner for any cursor position. The program requires NIH‐Image for Macintosh computers and includes automatic band detection coupled with a densitometric evaluation.
Channel thickness variations could degrade resolution in electrophoresis
Authors: Holzwarth George
Published: May 2005

Variations in the lateral dimensions of capillaries and ultrathin gels along their length could lead to a distortion of the sample zone because charged particles traveling near the wall must travel a longer distance under a smaller field than particles traveling along the centerline. The size of the effect is computed by solving Laplace's equation for the potential everywhere in a conducting slab with a discontinuity in thelateral dimensions and integrating the travel time along different streamlines.
Capillary electrophoresis of closely related intrinsic thylakoid membrane proteins of the photosystem II light‐harvesting complex (LHC II)
Authors: Zolla Lello , Bianchetti Maria , Timperio Anna Maria , Mugnozza Giuseppe Scarascia , Corradini Danilo
Published: May 2005

The electrophoretic migration behavior of three closely related hydrophobic intrinsic membrane proteins of the photosystem II light‐harvesting complex (LHC II) was investigated in free solution capillary electrophoresis at pH 8.0–10 with running electrolyte solutions containing either anionic, zwitterionic or nonionic detergents. The complete and repeatable separation of these proteins was accomplished with a running electrolyte solution of 25 mM Tris/192 mM glycine, pH 8.8, containing either sodium dodecyl sulfate or n‐octyl β‐D‐glucopyranoside at concentration up to 5.0 and 7.0 mM, respectively. Migration times and resoltuion of the individual LHC II intrinsic membrane proteins were sensitive to the type of detergent. The effect of detergent concentration on the electrophoretic behavior of the LHC II proteins was also investigated. Electroelution of the LHC II components separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was used to isolate these intrinsic membrane proteins, which were then injected onto the capillary electrophoresis system for peak identification.
The determination of choline in vitamin preparations, infant formula and selected foods by capillary zone electrophoresis with indirect ultraviolet detection
Authors: Carter Nicole , Trenerry V. Craige
Published: May 2005

A method for the determination of choline in vitamin preparations, infant formula and selected foods by capillary zone electrophoresis with indirect ultraviolet detection at 214 nm is described. The “free” choline in the vitamin preparations was simply extracted with water, the solutions filtered and analysed. The “free” and “bound” choline in the other products was extracted with methanol. The near dry extract was then treated with hot saturated aqueous calcium hydroxide to hydrolyse the “bound” choline before analysis. A 75 cm × 75 μm uncoated fused silica capillary column with an electrolyte consisting of 5 mM 1‐methylimidazole, pH 4.5, and an operating voltage of 30 kV was used for all of the determinations. Using these conditions, choline migrates in less than 3 min and is well separated from the other components in the electropherograms. The tetramethylammonium ion was used as the internal standard. The instrument repeatability data for area calculation and migration time variation were good (coefficient of variation, CV, area calculation 1.4%, n = 10; CV for migration time variation 1.0%, n = 20). The level of detection for choline using this procedure is 5 mg/100 g.
Analysis of the biotin‐binding protein actinavidin using affinity capillary electrophoresis
Authors: Okun Vadim M. , Bilitewski Ursula
Published: May 2005

Affinity capillary electrophoresis (ACE) was applied to study the bioaffinity of ligand‐receptor interaction between the microbial biotin‐binding protein actinavidin and biotin. The ACE method is based on short time incubation of a mixture of actinavidin and increasing concentrations of biotinylated oligonucleotide (bio‐ON), which was found to be an effective affinity ligand. Separation of intermediate loading forms of actinavidin from unbound ligand in the presence of micellar phase and by capillary zone electrophoresis enabled the quantitation of free bio‐ON, permitting the evaluation of the biotin‐binding capacity of actinavidin in absence and presence of sodium dodecyl sulfate (SDS). Although in the latter case actinavidin lost a part of its binding capacity (not more than 12%), it was still possible to develop an indirect, noncompetitive assay for the determination of actinavidin in culture liquid, utilizing the combination of micellar electrokinetic capillary chromatography (MEKC) and ACE. Due to the affinity inteaction, actinavidin in the sample decreases the amount of bio‐ON added, enabling quantitation of the protein. SDS, which is required in this assay to prevent protein adsorption to the capillary wall, greatly enhances the reproducibility and peak shape. Actinavidin levels determined are in agreement with those obtained by commonly used solid‐phase analysis. The limit of detection was about 500 ng/mL. Thus the proposed method was found to be well suited for the evaluation of actinavidin affinity and monitoring of its levels in cultivation process.
The interface of capillary electrophoresis with high performance fourier transform ion cyclotron resonance mass spectrometry for biomolecule characterization
Authors: Severs Joanne C. , Hofstadler Steven A. , Zhao Zack , Senh Robert T. , Smith Richard D.
Published: May 2005

The interfacing of capillary electrophoresis (CE) with Fourier transform ion cyclotron resonance‐mass spectrometry (FTICR‐MS) and the factors which dictate obtainable performance (i.e., sensitivity, mass resolution, scan rate and duty cycle) are described. We demonstrate the current status of the technique with examples of capillary zone electrophoresis (CZE) and capillary isotachophoresis (CITP) with FTICR analyses of proteins and oligonucleotides, and describe current limitations on sensitivity and scan speed. The first on‐line interfacing of capillary isoelectric focusing (CIEF) with FTICR is also demonstrated and shown to be effective for separating minor components of protein mixtures for on‐line mass spectral analysis. Finally, the potential for greatly improved performance based upon recent advances in FTICR instrumentation and methods is briefly described.