Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society最新文献

{"title":"Automated DNA sequencing: an image processing approach.","authors":"Y Wu,&nbsp;D Mislan","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>An automatic DNA sequencing analysis system has been developed by Scanalytics/CSPI. The strategies used to solve some of the common problems in the automation of DNA sequencing systems will be discussed. Algorithms developed include fast image background correction, automatic base detection, resolution enhancement by deconvolution, desmiling and artificial intelligence algorithms for sequence interpretation.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"3 5","pages":"223-8"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19207911","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The trajectories of spheres during agarose gel electrophoresis.","authors":"G A Griess,&nbsp;R A Harris,&nbsp;P Serwer","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>To develop a physical description of the gel-induced retardation of spheres during gel electrophoresis, the microscopic motion of single electrically charged latex spheres is statistically quantified here, by digital image analysis. To obtain adequate resolution in space, comparatively large spheres, 240 nm in radius, are used. The following observations are made during electrophoresis in a 0.2% agarose gel at 22 degrees C: (a) When a comparatively high field (3.0 V cm-1) is used, inelastic collisions result in field-induced trapping of spheres; no elastic collisions are observed. (b) Reduction of the field from 3.0 to 0.0 V cm-1 results in reverse migration of previously trapped spheres. (c) In the absence of trapping, the electrical field does not cause an alteration in the tortuosity of motion (i.e. motion in a field-perpendicular direction). (d) When results are obtained for a constant time between images (0.2 s), gel-dependent deviations from a true random walk are not observed in the absence of trapping. (e) When results are obtained as a function of time between images, significant gel-dependent deviation from a random walk is observed. In the absence of trapping, the data presented here indicate that retardation is derived primarily from dissipative processes that are concentrated near gel fibers. However, steric effects have not yet been distinguished from hydrodynamic effects.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"3 6","pages":"305-15"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19189490","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Increased apolipoprotein-E concentrations in individuals suffering chronic low back syndrome identified by two-dimensional gel electrophoresis.","authors":"D M VanderPutten,&nbsp;B M Cameron,&nbsp;C R Merril","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Non-biased screening of plasma proteins by two-dimensional gel electrophoresis from individuals suffering low back syndrome revealed a polypeptide spot that was increased 2-5-fold over the concentration found in normal control individuals. The apparent molecular weight (34-36 kDa) and pI (5.7) of this spot suggested that it might be apolipoprotein-E. Immunoblot analysis showed that the polypeptide was reactive with anti-apolipoprotein-E antibodies. N-terminal amino acid microsequence confirmed the identify of this polypeptide as apolipoprotein-E. We have determined that elevated plasma levels of apolipoprotein-E is associated with inflammation and nerve damage.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"3 5","pages":"247-52"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19207914","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Purification of recombinant adenomatous polyposis coli polypeptide chains from E. coli extracts by continuous-elution electrophoresis.","authors":"C Kraus,&nbsp;E Klein,&nbsp;W G Ballhausen","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>A polypeptide chain encoded by the exons 1-3 of the human adenomatous polyposis coli (APC) tumor suppressor gene was expressed as a maltose binding fusion protein (MBP) in E. coli to be used for immunization purposes. It turned out, that the APC-MBP fusion product of 60 kDa was deposited in bacterial inclusion bodies and, in addition, it was not retarded by an amylose affinity column, most probably due to an altered conformation of the chimeric molecule. For this reason, we established an alternative purification scheme, which took advantage of SDS-extraction followed by a high-resolution two-step continuous-elution electrophoresis (CEE) procedure. This purification method allowed us to obtain high yields of pure human APC exon 1-3-encoded proteins. The final yield of the pure APC polypeptide chains was estimated to represent 5-8% of the amount of SDS-extracted E. coli lysate subjected to the first cycle of CEE. The purified APC molecules were successfully used for the development of specific antibodies. The CEE procedure described here represents a general purification method which is valuable in cases where fusion proteins are deposited as inclusion bodies in bacteria, or if affinity chromatography is precluded due to a conformation-induced lack of ligand binding of the chimeric molecule.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"3 6","pages":"271-5"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19188911","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exact behaviour of single-stranded DNA electrophoretic mobilities in polyacrylamide gels.","authors":"P Mayer,&nbsp;G W Slater,&nbsp;G Drouin","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>We demonstrate the existence of a gel edge effect where the velocity of the samples varies in the first and last centimetres of the gel. In spite of this effect, a differential method of velocity determination leads to exact mobility data. Further analysis against DNA length N and electric field E shows that the molecules always migrate according to an A/N+B(E) law, except in a limited range of E and N, whereas a 1/N1.6 entropic driven regime is found. Below a threshold electric field intensity B(E) varies as E2, in good agreement with the biased reptation mechanism, while at stronger electric fields intensities, B(E) stays constant. This second mechanism is not described by any actual theory, but might be attributed to a geometration-like mechanism. Implications of our findings in sequencing electrophoresis are discussed.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"3 3-4","pages":"147-55"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19494978","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification and use of constitutive proteins for the normalization of high resolution electrophoretograms.","authors":"C R Merril,&nbsp;G J Creed,&nbsp;J Joy,&nbsp;A D Olson","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Quantitative inter-gel comparisons of proteins separated by high resolution two-dimensional protein electrophoresis present a number of problems. These problems may arise from: variations in pipetting and other mechanical manipulations of samples, protein loss during transfer from the first to the second gel dimension, variations in staining, and/or variations in film development during autoradiography, in the case of radioactively labeled proteins. This study presents a discussion of these issues and a normalization algorithm to deal with variations, which relies on a class of proteins present in most biological samples which by their nature may be considered internal standards. This class consists of proteins which are controlled by constitutive genes. Constitutive genes are genes that are expressed constantly. We have developed an algorithm which is currently available as a subroutine, 'FINDCONS', in the computerized densitometry and protein comparison analysis program, developed by Olson & Miller (1988). This algorithm identifies potentially 'constitutive' proteins. A normalization method employing these potentially 'constitutive' proteins was compared to several others by examining 2D-electrophoretograms of proteins from developing gypsy moths (Lymantria dispar L.) insect tissue. Following normalization, inter-gel comparisons of spots, which were 'identified' as 'constitutive', were observed to vary less in density than when no normalization method was used, or when normalization based on total integrated spot density was used. In addition to its use as a normalization tool, this algorithm and the subroutine FINDCONS may be useful as an aid in biological studies to identify 'constitutive' proteins.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"3 6","pages":"329-33"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19189493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparison of the Bio Image Visage 2000 and the GELLAB-II two-dimensional electrophoresis image analysis systems.","authors":"J E Myrick,&nbsp;P F Lemkin,&nbsp;M K Robinson,&nbsp;K M Upton","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>To compare the Visage 2000 analysis system (Bio Image, Ann Arbor, MI, USA) with the GELLAB-II analysis system (National Cancer Institute, Frederick, MD, USA), we used each to perform image analysis of the same 29 silver-stained two-dimensional electrophoresis (2DE) gel image files from a study of urinary proteins in metal recovery plant workers who had confirmed body burdens of cadmium. Visage, aided by interactive analysis, detected an average of 890 +/- 177.6 spots per gel, or a total of 25,800 spots, whereas GELLAB-II detected 1971 +/- 198.5 spots per gel, or a total of 57,160 (a 222% increase over the Visage system), without operator intervention. Visage automatically quantified 52.5% (13,556) of the spots; 47.2% (12,173), consisting mostly of larger spots, had to be quantified interactively with an image editor, and 0.3% (71) were not quantified. GELLAB-II automatically quantified all detected spots. After we interactively assigned the maximum allowed number of landmarks (30 for Visage and 52 for GELLAB-II), we found that Visage matched 657 +/- 211.2 spots per gel, and GELLAB-II matched all detected spots and also extrapolated an average of 1269 virtual spots per gel. Plots of densities from the two systems on selected spots showed excellent agreement, and both systems showed high correlation between their measurements of the beta-2-microglobulin spot densities and an independent radioimmunoassay quantification of the original urine samples. By comparing the regression of the densities of all spots with urinary cadmium (UCD) levels, we found that several of the same detected spots from each system were highly correlated. The densities of four acidic proteins with relative molecular weights of approximately 112,000 Da (as quantified by GELLAB-II but not by Visage) were highly correlated with UCD concentrations. These proteins are new candidate biomarkers of cadmium toxicity. We compared the estimated labor costs of using each system to analyse a hypothetical 20-sample (60 gels) 2DE study and found that GELLAB-II was six times less expensive to use than Visage, primarily because of the operator time required to do interactive error correction with the Visage system.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"3 6","pages":"335-46"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19189494","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of a brain-specific human cerebrospinal fluid glycoprotein, beta-trace protein.","authors":"M G Harrington,&nbsp;R Aebersold,&nbsp;B M Martin,&nbsp;C R Merril,&nbsp;L Hood","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>A prominent human cerebrospinal fluid (CSF) protein, P5, identified at mass 19-24 kDa and charge 5.5, by two-dimensional electrophoresis (2DE) and silver staining, has been previously demonstrated to be reduced in quantity in the CSF of patients with multiple sclerosis and schizophrenia. We report the purification and partial amino acid sequences from five tryptic fragments of P5. These sequences are not those of any known sequence in the Protein Identification Resource (PIR release 31) database. Synthetic peptides from two of the sequences were used to raise rabbit polyclonal antibodies. These antibodies detected P5 on 2DE blots of normal CSF proteins and other proteins of the same mass with a charge distribution between 5.17-8.5. These proteins comprise 5-10% of the total CSF protein and their mass, charge, abundance and predominance in CSF over plasma are consistent with a protein that had been initially characterized with antibodies, beta-trace protein. Glycosidase studies confirm that most of these proteins are due to sialic acid modifications that are N-linked to an 18 kDa protein, but other charge and mass variations also exist. 2DE blots of 26 types of human tissue and body fluid were immunostained. Of these, anti-P5 serum detected proteins of the same mass and charge as beta-trace protein only in brain samples. Proteins of different mass and charge from beta-trace protein were clearly immunostained in samples of eight tissues.(ABSTRACT TRUNCATED AT 250 WORDS)</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"3 5","pages":"229-34"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18695760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DAPI, a simple sensitive alternative to ethidium bromide staining of DNA in agarose gels.","authors":"E Buel,&nbsp;M Schwartz","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>We describe a simple procedure to stain DNA in horizontal agarose gels using 4',6-diamidino-2-phenylindole (DAPI). The method has the speed and simplicity of ethidium bromide while localizing the dye in the sample, limiting incidental dye contamination.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"3 5","pages":"253-5"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18695761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Plasma/serum protein patterns in human fetuses and infants: a study by high-resolution two-dimensional polyacrylamide gel electrophoresis.","authors":"J D Tissot,&nbsp;P Hohlfeld,&nbsp;F Forestier,&nbsp;J F Tolsa,&nbsp;D F Hochstrasser,&nbsp;A Calame,&nbsp;E Plouvier,&nbsp;H Bossart,&nbsp;P Schneider","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Plasma/serum proteins of fetal blood samples (N = 88) obtained under ultrasound guidance between the 18th and the 39th week of pregnancy, of blood samples collected from premature infants (N = 19), newborns at term (N = 20) and children of less than 5 years of age (N = 55) were analysed by high-resolution two-dimensional polyacrylamide gel electrophoresis. By comparison with adult 'reference' protein maps, tens of different proteins (and some of their genetic variants) were identified on the electrophoretograms. After the 18th week of gestation, albumin, transferrin, Factor B, glu- and lys-plasminogen, antithrombin III, Gc-globulin, alpha 1-antitrypsin, alpha 2-HS-glycoprotein, several apolipoproteins (apo A-I, A-II, A-IV, C-II, C-III, D, E, J), retinol-binding protein, transthyretin and alpha-fetoprotein could be observed. During intrauterine life, the size of the spots corresponding to alpha-fetoprotein progressively decreased, whereas the protein pattern globally showed an increase in the number and in the size of the spots. These modifications were particularly apparent in the regions of the electrophoretograms restricted to the heavy and light chains of IgG and to alpha 1-antichymotrypsin. In addition, we observed an unidentified fetal polypeptide characterized by an apparent molecular weight (M(r)) of 46 kDa (P46) and a pI of 5.0. P46 was present in all fetuses and all infants of less than 2 years of age.(ABSTRACT TRUNCATED AT 250 WORDS)</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"3 3-4","pages":"183-90"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19494982","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}