ISSN:0173-0835    

DOI:10.1002/elps.1150090102

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

年代:1988

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150090103

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

年代:1988

数据来源: WILEY

摘要:

AbstractPhastSystem, an integrated system for horizontal electrophoresis and isoelectric focusing in small gels, including automated staining and destaining, is described. Buffers for electrophoresis are supplied to the gel from buffer strips made of agarose. The separation bed is cooled by Peltier elements. All conditions of significance to the results, both during separation and development, are controlled by a microprocessor. For separation, nine programs are available, each with 9 steps; for developement, there are nine programs with 20 steps each. In this paper, we also report results using PhastSystem for purity checking and characterization of monoclonal antibodies after affinity chromatography and also for determining their digestion rate with papain.

ISSN:0173-0835    

DOI:10.1002/elps.1150090104

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

年代:1988

数据来源: WILEY

摘要:

AbstractThe development of equipment for fast automated staining is described. It is possible to handle staining procedures with up to 20 steps and nine different solutions. To increase the reaction rate in the reaction chamber, the gels are rotated and high temperatures are used. The temperature in the reaction chamber is controlled between room temperature and 50 °C. Increased temperature, above 20 °C, generally results in faster staining and destaining. However, some reactions proceed better at a low temperature, including fixation of proteins with TCA, and the development step in silver staining, where increased temperatures cause a high background stain. Silver in silver staining, where increased temperatures cause a high background stain. Silver staining using acidic silver nitrate solution is preferred, due to easy preparation and good storage stability of the reagents. This method also causes little precipitation of silver on the walls of the reaction chamber. Silver staining is accomplished within one hour. Staining with PhastGel Blue is accomplished within 30 mi

ISSN:0173-0835    

DOI:10.1002/elps.1150090105

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

年代:1988

数据来源: WILEY

摘要:

AbstractA new modification of silver staining of proteins in sodium dodecyl sulfate polyacrylamide gels is adapted to automated staining in PhastSystem Development Unit. The use of a reduction step, after fixation, with thiosulfate in alcoholic sodium acetate buffer results in a considerable increase in sensitivity without the need for a recycling step. The detection limit is tenfold lower than in the silver staining procedure recommended so far for PhastSystem and corresponds to 0.05–0.1 ng protein per band. Total staining time with the new procedure is 75 mi

ISSN:0173-0835    

DOI:10.1002/elps.1150090106

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

年代:1988

数据来源: WILEY

摘要:

AbstractA device for the measurement of voltage across tube gels was designed and constructed which allows one to measure voltage during electrophoresis without any manipulation of the gel electrophoresis apparatus or gel tube and with the elimination of a source of inaccuracy in previous such devices.

ISSN:0173-0835    

DOI:10.1002/elps.1150090107

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

年代:1988

数据来源: WILEY

摘要:

AbstractHorizontal two‐dimensional (2‐D) electrophoresis with immobilized pH gradients (IPG) in the first dimension for buffer soluble proteins and for complex proteins solubilized in the presence of Nonidet P‐40 (Görget al., Electrophoresis1987,8, 45–51), has been extended to analyze basic proteins of yeast cells focused under non‐equilibrium and equilibrium conditions. Transient state isoelectric focusing (IEF) in IPG gels revealed sample smearing and background staining, displaying horizontal streaks in the resultant 2‐D patterns. Inclusion of 0.5 % carrier ampholytes (CA) to the IPG gel (IPG‐CA), resulted in the formation of many sharp protein bands after transient state IEF with resultant distinct spots in the 2‐D patterns; however, resolution was poor and the gel contained heavy background staining. With prolonged focusing time, background staining disappeared and there was less difference in the final steady state IEF patterns obtained with IPG and IPG‐CA. Reduction of the Immobiline concentration to one third the manufacturer's recommended amount did not improve IEF resolution with respect to streaking and background staining under either transient state or equilibrium conditions. In general, spot intensities were less on 2‐D gels using diluted IPG gels than with “standard” IPG gels. Optimization of 2‐D electrophoresis with IPGs in the first dimension was strongly related to IEF conditions. The use of IPG gels focused to equilibrium should not only improve inter‐gel reproducibility and resolution but also the quality of the final 2‐D patterns with respect to background sta

ISSN:0173-0835    

DOI:10.1002/elps.1150090108

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

年代:1988

数据来源: WILEY

摘要:

AbstractRats were fed liquid food containing ethanol in concentrations ranging from 1–5 % for 13 weeks. Livers were removed for histopathology and the liver cytosolic protein fraction was prepared and used for two‐dimensional gel electrophoresis (2D‐PAGE). Polypeptides were visualized by silver staining. Scanning was made for estimation of the relative abundance of protein in each polypeptide spot in the gels and for comparison between rats. Visual inspection and scanning of gels with the stained polypeptide spots obtained after equilibrium isoelectric focusing and non‐equilibrium pH gradient electrophoresis revealed that: (1) within the control rat and ethanol‐treated rat livers the numbers of polypeptide spots detected using isoelectric focusing in the first dimension were approximately 500 and for non‐equilibrium pH gradient electrophoresis 400; (2) in the control group the variation in the estimated amount of protein in each spot was remarkably small; (3) pronounced differences in the relative abundance of protein in several of the spots was observed in the ethanol‐exposed rats as compared to controls. Dose‐response relations and possible causes for the effects of ethano

ISSN:0173-0835    

DOI:10.1002/elps.1150090109

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

年代:1988

数据来源: WILEY

摘要:

AbstractA rapid, inexpensive method for the salt‐free concentration of small quantities of proteins for analysis by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) has been developed. Proteins adsorbed to diatomaceous earth are subsequently removed using either sodium dodecyl sulfate or urea solubilization reagents for 2D‐PAGE analysis. This procedure has been found to concentrate proteins having wide ranges of molecular weight and charge. It is also valuable for the concentration of large numbers of small samples from cells culturedi

ISSN:0173-0835    

DOI:10.1002/elps.1150090110

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

年代:1988

数据来源: WILEY

摘要:

AbstractProtocols for horizontal two‐dimensional electrophoresis with immobilized pH gradients in the first dimension were modified for horizontal micro two‐dimensional electrophoresis using PhastSystem. Different equilibration conditions of the first‐dimensional immobilized pH gradient gel strip prior to second‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis were evaluated. Silver stained two‐dimensional patterns were obtained w

ISSN:0173-0835    

DOI:10.1002/elps.1150090111

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

年代:1988

数据来源: WILEY