摘要:

AbstractA two‐dimensional polyacrylamide gel electrophoresis approach for the separation of urea/detergent solubilized basic proteins is presented. An immobilized pH gradient (IPG) gel, pH range 7–10, containing 8 M urea, 2% Nonidet P‐40 and 10 mM dithioerythritol was used for the first‐dimensional isoelectric focusing separation. Approximately 400 to 500 basic polypeptides from myeloblasts or neutrophils were visualized with silver staining. The IPG gel in some cases included carrier ampholytes in the same pH range. The carrier ampholyte‐modified (“hybrid”) IPG gel exhibited improved resolution of some polypeptides, but at the expense of reproducibility in the focusing of other polypeptides. Transfer of sample into the IPG gel was facilitated by a combination of low initial voltage, presence of carrier ampholytes in the sample and application of the sample to the surface of the gel within a containment collar. 6 M urea was required in the SDS equilibration solution for the efficient transfer and high resolution separation of polypeptides in the second‐dimensional sodium dodecyl sulfate‐gel. Absolute spot position was highly reproducible among gels prepared simultaneously. Standard deviations of ± 0.84 mm for the focusing and ± 1.25 mm for the molecular weight dimensions of 70 randomly selected spots were obtained. Integrated spot intensities ranged from 0.17 to 80 O.D. × mm2with a medi

ISSN:0173-0835    

DOI:10.1002/elps.1150080402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractA database structure is needed for easy handling of the large quantity of data obtained from a series of gel electrophoresis experiments and also for defining the first statements of a standard between experiments from different origins. This paper describes the database concept and the database interrogation strategies in the field of two‐dimensional electrophoresis and protein expression data. First, the main characteristics of our database structure and the main capabilities offered to the user to handle and interrogate the structure, as well as to retrieve his information are described. Gel standardization and protein identification are then examined. A relational database, associated with the description of each spot according to internal and external (physicochemical) descriptors, provides a suitable structure for a standardization procedure. A dynamic updating of the actual position of each spot, each time new positional information is entered in the system, allows the encoding of standardized positions. A database structure can also be used to establish a link between different kinds of information from different areas required for a correct interpretation of two‐dimensional gel data. Particularly, information from the gene sequence of a given protein may offer insights into the regulation of this protein. Thus a link between the two‐dimensional gel database and sequence databases is implemented in our system. The information available from sequence data may help to interpret electrophoresis data. Furthermore, a formal presentation of the database structure is achieved in order to propose a standardized description of the database structure and concepts used. This feature allows comparisons between different databases and frees the user from a specific configuration. Logical formulations are introduced using predicate calculus and then lead to a description of manipulation tools in terms of a declarative programming lan

ISSN:0173-0835    

DOI:10.1002/elps.1150080403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractData analysis programs are essential tools for processing the large quantity of data produced by two‐dimensional gel electrophoresis computerized analysis. Global handling of two‐dimensional gel information can be achieved using both analysis programs and artificial intelligence techniques. First, a statistical multivariate approach is presented, used to sort gels and to obtain a gel classification according to protein patterns. Both clustering and multivariate techniques are used and the results show that a classification of protein expression patterns according to effector action can be achieved. Spots can also be sorted and compared according to their expression under effector action. Each spot is thus described using a set of variables (its intensity for each gel pattern) and its description is essentially given in a multivariate way. Therefore, each facet of the regulation of a particular protein according to the action of several effectors can easily be taken into account. Spot sets containing spots having common regulation characteristics are obtained using principal component analysis. We show that each group corresponds to a functional aspect of the regulation in a given cell line. The map obtained describes the organization of cell functions and the interactions of these functions. Another approach employs artificial intelligence techniques. Since the rules available for describing some aspects of knowledge in the field of two‐dimensional electrophoresis remain unknown, learning techniques are suitable for defining them. Their use for the automatic recognition of spot groups belonging to the same entity on a gel is illustrated. Our learning program is applied to the determination of glycosylation groups and to the identification of relevant spot pat

ISSN:0173-0835    

DOI:10.1002/elps.1150080404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractQuantitative analysis of polypeptide differences among sets of identically prepared two‐dimensional polyacrylamide gels is facilitated by the use of mathematical transformations of the spot integrated intensity data for each gel in order to make comparisons. Such adjustments are needed because technical factors cause the stained polypeptide spots displayed on a particular gel to simultaneously appear darker or lighter than on another gel. We have compared three different adjustment schemes by applying each to the same set of data and testing to determine which gives smaller coefficients of variation for a large battery of spots. The adjustment that gave the best results is one based on regressing the spot integrated intensities of a standard gelversuseach “target” gel and using the fitted line to transform the spot integrated intensities on the target gel. This adjustment is intuitive, easy to compute, and performed well regardless of whether the spots with large variability were included in the data o

ISSN:0173-0835    

DOI:10.1002/elps.1150080405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractTumor cells, thymocytes and lymph node cells in normal and tumor‐bearing mice treated with recombinant human tumor necrosis factor (rH‐TNF) were analyzed using automated cell electrophoresis to measure and classify cells according to cell surface charge. In the benzopyrene‐induced tumor system the intratumoral administration of rH‐TNF caused marked tumor necrosis and an increased cell surface charge on the tumor cells as detected by increased electrophoretic mobility (EPM). The thymus and lymph node are involved during murine tumor development characterized by abnormal weight and EPM pattern of lymphocyte histograms. Thymocyte and lymph node histograms were restored to their normal pattern by treatment of tumor bearers with rH‐TNF. The analysis of electrophoretic patterns of murine tumor cells, thymocytes and lymph node cells may be a simple method to evaluate drug effects on the immune system when automated techniques ar

ISSN:0173-0835    

DOI:10.1002/elps.1150080406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractAn ultrathin‐layer polyacrylamide gel isoelectric focusing technique is described for obtaining highly resolved transferrin (Tf) subtypes. N‐2‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid (HEPES) buffer was used to flatten the pH gradient in gels containing pH 5–7 Ampholines. The result was increased separation distances for the TfC subtype bands. The method is effective even with the most recent lots of pH 5–7 Ampholines which were not capable of providing an appropriate environment for effect

ISSN:0173-0835    

DOI:10.1002/elps.1150080407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractSoybean leghemoglobin subcomponents were separated with good resolution in immobilized pH gradients, both at the analytical and preparative scales. In the latter case, the tedious step of gel washing could be omitted without affecting resolution. This procedure appeared as the only one suitable for preparing large amounts of highly purified leghemoglobin c1, c2and c3, allowing their further characterization and an investigation of their specific roles in nitrogen fixation.

ISSN:0173-0835    

DOI:10.1002/elps.1150080408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150080409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150080401

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY