摘要:

AbstractThe applicability of multiphasic buffer systems to electroendosmosis‐free agarose (IsoGel) was tested in order to be able to combine the benefits of agarose gel electrophoresis with those of steady‐state stacking. It was found that buffer systems of a negative polarity function normally over the entire pH range, whereas proteins with positive net charge are retarded at the gel surface in buffer systems with positive polarity. This retardation is not due to a failure to establish a moving boundary in agarose. Rather, it appears due to a negatively charged weakly acidic functional group in agarose which can be removed from agarose by pre‐electrophoresis at 0.1 M ionic strength. After its removal positively charged proteins also stack normally in multiphasic buffer systems of the positive pol

ISSN:0173-0835    

DOI:10.1002/elps.1150030302

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

年代:1982

数据来源: WILEY

摘要:

AbstractA previous observation by Nochumsonet al.[7], showing that agarose after reduction or abolition of its supercoiled structure by hydroxyethylation (SeaPrep 15/45), exhibits a molecular sieving effect similar to polyacrylamide in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was confirmed and quantitated by Ferguson plot analysis in PAGE. SeaPrep 15/45 exhibits an effective fiber radius of 0.8 nm, as compared to 23.8 nm for native agarose and 0.4 nm for 2 % crosslinked polyacrylamide. Its effective pore size (estimated as\documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {{\rm K}_{{\rm R polyacrylamide}} } /\sqrt {{\rm K}_{{\rm R agarose}} } $\end{document}) is 0.88 times that of polyacrylamide 2% crosslinked with N,N'‐methylene bisacrylamide (Bis). Although this is not a practical result as yet, due to the low melting temperature and poor gel strength of SeaPrep 15/45, it does sound the death knell for the use of crosslinked polyacrylamide in macromolecular separations, by demonstrating that a linear polymer capable of forming a gel on mere cooling is equivalent to the product of a laborious and relatively irreproducible free radical polymerization. It remains to find a linear polymer with higher melting point and better mechanical strength and adherence to glass walls than SeaPrep 15/45 to make crosslinked polyacrylamide obs

ISSN:0173-0835    

DOI:10.1002/elps.1150030303

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

年代:1982

数据来源: WILEY

摘要:

AbstractHuman plasma proteins separated by high‐resolution two‐dimensional electrophoresis have been electrophoretically transferred to sheets of nitrocellulose using a modification of the method of Towbin, Staehelin, and Gordon [8]. Although the proteins have been denatured in sodium dodecyl sulfate and separated into subunits, the nitrocellulose‐bound molecules still react with appropriate specific antisera even after storage of the transfer in air at room temperature for 5 months. Of 25 proteins whose location in the pattern had been previously determined, 24 are specifically revealed on transfers of whole plasma patterns by appropriate antiserum. In addition, 6 previously unidentified proteins (prothrombin, C1s, C4γ, C1s inhibitor, Ig J‐chain, and a1AP glycoprotein) have been identified in the pattern for the first time using the transfer technique. It therefore seems likely that a large majority of proteins (>96 % in this study) retain sufficient conformation throughout the analytical procedure (or can regain it easily afterwards) to be recognized immunologically. The transfer technique thus constitutes a generally useful immunological “third‐dimension” in the high‐resolution separation of proteins. Of three monoclonal antibodies similarly tested, none could detect antigen transferred to nitrocellulose from a two‐dimensional gel, while each bound specifically to the appropriate antigen absorbed in native form to

ISSN:0173-0835    

DOI:10.1002/elps.1150030304

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

年代:1982

数据来源: WILEY

摘要:

AbstractElectrofocusing has been adapted in order to make screening experiments for the detection of enzyme variants more efficient. The principle of the new technique consists in making agar copies of the polyacrylamide gel by contact; the original gel and the agar copies are then developed for different enzyme systems. By doing so, the number of enzymes,i.e.loci, per test animal screened after a single electrofocusing run is increased. This method has proved to be feasible for the detection of mouse variants with altered enzyme banding patterns due to parental treatment with chemical mutagens.

ISSN:0173-0835    

DOI:10.1002/elps.1150030305

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

年代:1982

数据来源: WILEY

摘要:

AbstractWagtail samples (Motacilla flava, ssp; song bird) have been collected in different regios of central and southern Europe. Due to sampling extending over long periods of time it was necessary to investigate the effect of storing conditions (liquid nitrogen or ‐36 °C). Isozyme separation was done by polyacrylamide ultrathinlayer isoelectric focusing (100 μm). Liver homogenates were diluted in appropriate manner and 5 μl aliquots were used for separations. Gels for isoelectric focusing (T = 5.4, C = 2.8) containing different kinds of carrier ampholytec (Servalyt, Pharmalyte and Ampholines were cast on 12.5 × 20 cm pretreated glass plates according to the procedure of Radola [9]. Enzyme staining was performed in solution (ACP and esterases) or with an agarose overlay technique (LDH, ADH, MDH, PGM and GPI). Wagtail liver samples were chosen to optimize the electrophoretic conditions and to test the usefulness of different staining methods. This — and investigations presently under way on samples of pectoral muscle, heart, brain and blood — should allow us to elucidate genetic differences at the molecular level of the various subspecies of wagtails. The smaller total amount of protein available in some of these samples stresses the necessity of choosing a reproducible and sensitive, as well as rapid, electrophoretic

ISSN:0173-0835    

DOI:10.1002/elps.1150030306

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

年代:1982

数据来源: WILEY

摘要:

AbstractIntracellular deoxyribonucleases (DNases) in human peripheral blood leukocytes have been identified and characterized by polynucleotide‐polyacrylamide gel electrophoresis (PPAGE). This technique combines the protein separating powers of polyacrylamide gel electrophoresis with the sensitivity of detection characteristic of an enzyme assay. Visualization of nuclease activity is made possible by the inclusion of high molecular weight DNA substrate within the sieving portion of a narrow polyacrylamide gel. No sodium dodecyl sulfate, urea, or other protein denaturants are used in this procedure. However, electrophoresis of the contents of up to 106cells/gel is accomplished using conditions under which the enzymes are inactive. The positions of DNases are then revealed by incubation of the gel in an appropriate buffer followed by staining for intact DNA. Colorless regions correspond to the presence of enzyme. Used as a pattern recognition tool, PPAGE revealed that leukocytes from normal donors contained lower levels of certain activities than did cells from donors with chronic myelogenous leukemia. The rapid simultaneous characterization of multiple activities is made possible by simply using different buffer compositions in the incubation step. For example, ionic strength and pH optima may be found directly for activities present in unpurified human intracellular contents. The reproducibility of this method permits comparison of individual activities by using Ferguson plot analysis to detect differences or similarities among enzymes. These analyses revealed that DNase activities in human peripheral blood leukocytes fell into a minimum of two families. Modifications of overall effective charge probably account for microheterogeneit

ISSN:0173-0835    

DOI:10.1002/elps.1150030307

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

年代:1982

数据来源: WILEY

摘要:

AbstractAfter agarose gel isoelectric focusing (AGIEF) and immunofixation, human antithrombin III (AT‐III) in plasma showed a microheterogeneous pattern which consisted of 5 major and 5 minor components with isoelectric points (pI) between pH 4.8 and 5.2. Treatment with neuraminidase reduced this microheterogeneity of AT‐III to 1 major, 3 sub‐major and 2 or 3 minor components with a shift in pI to between pH 5.5 and 5.8. Thus, differential sialylation of the isoproteins partially contributed to the microheterogeneity observed. The addition of heparin to plasma produced broad acidic precipitates with pIs between pH 4.2 and 4.7 on the gel. The same acidic precipitates were also obtained from heparin‐purified AT‐III mixture. The appearance of these precipitates was related to the decreasing in number or in staining intensity of the native AT‐III bands. These precipitates may be complexes of heparin and AT‐III. The addition of human or bovine thrombin to plasma produced several basic minor components, which were similar to those in serum, at a thrombin concentration of 48 or more NIH U/ml‐plasma. The basic minor components were not obtained from a purified AT‐III‐thrombin mixture. These might be complexes between AT‐III, thrombin and some of the othe

ISSN:0173-0835    

DOI:10.1002/elps.1150030308

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

年代:1982

数据来源: WILEY

摘要:

AbstractUsing 0.5 mm agarose gels for Tf subtyping by isoelectric focusing we were able to identify ten variants at the Tf locus in US Amerindians, Whites and Blacks. The variant TfC4was found only in the Amerindian populations. This population specific variant had gene frequncies ranging from 0.079 in the Walapi to 0.179 in the Apache. The Black sample studied had a lower incidence of TfC3than in whites and was the only population with the marker TfD1. Thin agarose gels provided good separation of TfC3from TfC1. Thin agarose gels provided good separation of TfC3from TfC1enabling us to demonstrate a new variant, TfC8, migrating between TfC1and TfC3.

ISSN:0173-0835    

DOI:10.1002/elps.1150030309

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

年代:1982

数据来源: WILEY

摘要:

AbstractA routine technique for phenotyping the PGM1marker system was developed using thin‐layer isoelectric focusing on agarose. Resolution of PGM1variants by thin‐layer isoelectric focusing on 0.5 mm gels was found to be superior to isoelectric focusing on ⩾ 1 mm gels. Isoelectric points were determined for 14 different PGM1variants and should serve as a useful tool for comparing variants between laborat

ISSN:0173-0835    

DOI:10.1002/elps.1150030310

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

年代:1982

数据来源: WILEY

摘要:

AbstractA simple technique for polyacrylamide gel isoelectric focusing of α1‐antitrypsin (PI) using thin, highly cross‐linked gels, restricted pH‐range carrier ampholytes and a separator was developed. This method enables easy typing of all known PI phenotypes including the four PI*M su

ISSN:0173-0835    

DOI:10.1002/elps.1150030311

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

年代:1982

数据来源: WILEY