摘要:

AbstractA method is described for electroblotting of proteins, separated by gradient polyacrylamide gel electrophoresis, onto an agarose gel matrix containing specific antibodies. Three proteins of different molecular weight, including human albumin, isolated and in plasma, human plasma transferrin and C3complement were tested. Immunoblotting on agarose, compared with nitrocellulose, was quantitative and highly sensitive, with small amounts of protein (i.e., 100 pg) being detected. Moreover, albumin aggregates (i.e., dimer, trimer and tetramer) were blotted quantitatively in addition to the monomer, and their percentages were calculated. This method is sensitive, quantitative, reproducible, and includes fewer manipulations; furthermore, it is less expensive and does not require the use of toxic or carcinogenic agents.

ISSN:0173-0835    

DOI:10.1002/elps.1150140112

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe PhastTransfer system is a semi‐dry electrophoretic unit designed to optimize the transfer of small amounts of protein. Because of its efficiency, we adapted the PhastTransfer system for the detection of labeled membrane molecules. Biotin was used as the membrane molecule label because it permitted the long‐term storage of labeled lysates as well as the flexibility of derivatizing several different functional groups. After immunoaffinity separation using magnetic microspheres, the protein was electrophoretically separated with the PhastSystem and transferred with the PhastTransfer unit. Using an avidin‐linked enzyme amplification system, less than 10 ng of loaded protein could be detected on the transfer membrane. Based on these findings, the PhastTransfer system is a fast, reproducible, and convenient method for the transfer of small quantities of labeled cell surface pr

ISSN:0173-0835    

DOI:10.1002/elps.1150140113

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

年代:1993

数据来源: WILEY

摘要:

AbstractA silver‐staining procedure for enhancing the sensitivity of protein detection on nitrocellulose membranes in immunoblotting is described. After completing any peroxidase‐Ni‐diaminobenzidine immunostaining, nitrocellulose sheets are placed in a physical developer, containing sodium tungstate and ascorbic acid, until the desired grade of silver‐intensification has been reached. In this way a 16‐ to 64‐fold amplification of intensity of the initial immunostaining can be achieved. False positive silver staining of protein bands and of background are suppressed by replacing bovine serum albumin, the blocking agent most frequently used in immunoblotting, with s

ISSN:0173-0835    

DOI:10.1002/elps.1150140114

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

年代:1993

数据来源: WILEY

摘要:

AbstractImmune response to a hapten of fluorescein isothiocyanate (FITC) in a single BALB/c strain mouse was analyzed by two‐dimensional affinity electrophoresis (2D‐AEP). Anti‐FITC antibodies were induced by immunization with FITC‐conjugated bovine serum albumin. The antibodies were separated into a large number of spots of IgG due to differences in their isoelectric points(pI) and binding affinities to the FITC ligand. These spots consisted of IgG families which were composed of several spots having an identical affinity to the ligand but a different pI. The spots were not clearly detected in the antiserum taken on day 7 after the primary immunization, but on day 21 the spots of IgG were clearly detected, with a high diversity and specificity for the ligand. The size and number of IgG spots were markedly increased by the secondary immunization; however, the third immunization did not increase the size and number of IgG spots. The IgG spots of each family were specifically stained with an antimouse IgG subclass antibody. Furthermore, a monoclonal antibody (FL‐D6) was separated by 2D‐AEP into a single family which consisted of seven IgG1spots having an identical affinity to FITC but different pIs. Therefore, each of the IgG families of anti‐FITC antibodies in the antiserum can be generated by a single clone of anti‐FITC antibody‐producing cells. The substitution of dextran T 2000 or lipopolysaccharide for bovine serum albumin as a carrier for FITC induced much smaller amounts of anti‐FITC antibodies with a low diversity but high

ISSN:0173-0835    

DOI:10.1002/elps.1150140115

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

年代:1993

数据来源: WILEY

摘要:

AbstractA new procedure for the analysis and detection of phosphate‐activated glutaminase (EC 3.5.1.2) by native electrophoresis has been developed. The method is based on thein situdetection of glutaminase activity in two different systems of native polyacrylamide gradient gels, containing 3‐(3‐cholamidopropyl)‐dimethyl‐ammonio‐1‐propane sulfonate (CHAPS) or Triton X‐100 as nondenaturant detergents. Crude Triton X‐100 extracts of mitochondria were resolved by electrophoresis. The enzyme was specifically revealed by incubation of the gel with glutamine and coupling the oxidation of the glutamate formed to the reduction of a tetrazolium dye, in the presence of glutamate dehydrogenase trapped in a 1% agar solid overlay. Both Ehrlich ascitic cell and mouse kidney glutaminases were resolved by native electrophoresis and specifically detected with the activity staining. Moreover, the redox‐cycling staining was tested in solution, showing linearity with the amount of glutamate or glutaminase activity present. The method described could be a useful tool for native polyacrylamide gel electrophoresis o

ISSN:0173-0835    

DOI:10.1002/elps.1150140116

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

年代:1993

数据来源: WILEY

摘要:

AbstractInclusion of gelatin in polyacrylamide gels provides a sensitive way of detecting multiple proteolytic activities in crude extracts from any source. The present study describes a method allowing discrimination between cysteine and serine proteinases in plant extracts, using gelatin‐containing gels in combination with class‐specific proteinase inhibitors. Preincubation of extracts with 4 mMphenylmethylsulfonyl fluoride, a serine proteinase inhibitor, or with 25 μML‐trans‐epoxysuccinyl‐L‐leucylamido(4‐guanidino) butane, a cysteine proteinase inhibitor, allowed the identification of enzymes from both classes in extracts of tomato fruit and papaya latex. The efficiency of the two low molecular weight inhibitors used was very high, and the irreversibility of the inhibiting effect was maintained during electrophoresis conducted in the presence of sodium dodecyl sulfate. The analytic procedure described here, with a detection threshold of less than 100 pg enzyme, is the first that allows quick and accurate discrimination of plant cysteine and serine proteinases separated in electrophoretic gels. This simple and rapid technique could be of interest for studying the evolution of class‐specific proteinases in plant extracts during various developmental, physiological, and pathogenic processes. It is also potentially applicable to the majority of eucaryotic and pro

ISSN:0173-0835    

DOI:10.1002/elps.1150140117

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe influence of the ion permeable diaphragms, separating the electrode compartments of continuous flow electrophoresis chambers from the separation space, are described in detail. A set of equations is derived from a simplified phenomenological model of the ion transport across media boundaries. These equations are useful to predict the magnitude of concentration changes near the membranes due to the electrophoretic ion transport. Adaption to any CFE geometry, membrane material and electrolyte system is possible with only a few measurements, which yield the transference number difference as a characteristic transport property of the membrane material for a special electrolyte. The validity of the equations is checked by experiments. Local electric field strength and local conductivity are measured with a potential gradient‐conductivity scanner developed in our laboratory. This device yields data on the dynamics of the concentration change

ISSN:0173-0835    

DOI:10.1002/elps.1150140118

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

年代:1993

数据来源: WILEY

摘要:

AbstractByin vivolabeling with [75Se]selenite and separation of the proteins in the tissue homogenates by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), several selenium‐containing proteins or protein subunits were detected in rat tissues (liver, lung, spleen and prostate). Their distribution among the cell components was investigated after fractionation by means of differential centrifugation. The selenium‐containing proteins in the 16 kDa range were found to be mainly membrane‐bound. By two‐dimensional electrophoresis they were resolved into three labeled spots, two with the same relative molecular mass and pIvalues of about 4.8 and 5.0 and the third with a slightly lower molecular mass and a pIof 4.8. For further investigation they were concentrated and separeted from the other labeled compounds by SDS‐PAGE using preparative flow‐through

ISSN:0173-0835    

DOI:10.1002/elps.1150140119

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe technique of two‐dimensional gel electrophoresis was used for analysis of tyrosine phosphorylated polypeptide substrates after epidermal growth factor (EGF)‐induced stimulation of receptor tyrosine kinase activity in a brush border fraction of human placental syncytiotrofoblast cells. After incubation with [γ32P]ATP, followed by autoradiography of the gels, 35 phosphorylated components were detected, of which 8 were strongly tyrosine phosphorylated by EGF. Using a more sensitive assay with phosphotyrosine‐specific antibody, an additional 12 polypeptide components were found to be strongly tyrosine phosphorylated by EGF. A number of the phosphorylated substrates could be aligned with components in a protein catalog of the human brush border membrane fraction that was characterized by glycoprotein staining, Triton X‐114 fractionation, immunoreaction with specific antibodies, and comigration with35S‐labeled AMA (transformed human amnion) cells. Identified components, stimulated by EGF, in addition to well‐recognized substrates (calpactin II, ezrin, EGF receptor) included β‐tubulin and serum albumin, while other cytoskeletal proteins and alkaline phosphatase were excluded as substrates. A notable feature of the catalog was that a number of glycoproteins were present in both the membrane and cytoskeletal fraction, suggesting involvement in membrane/cytoskeletal interactions. The data demonstrate the feasibility of using two‐dimensional gel electrophoresis in a global way to identify target substrates for tyrosine kinase activity. In addition they suggest that many of these are located in the vicinity of tyrosine kinase at the membrane/cytoskeletal border at a location which is probably involved, at the molecular level, in morphological changes of the plasma membrane associated with c

ISSN:0173-0835    

DOI:10.1002/elps.1150140120

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe methodology for the simultaneous analysis of protein synthesis concomitant with protein phosphorylation/dephosphorylation is described. The technique consists of metabolic labeling of rat liver epithelial (RLE) cells with [32P]orthophosphate and [35S]methionine, performing two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) of the mixed samples, followed by silver staining and subsequent autoradiography of the dried silver stained 2‐D PAGE electrophoretograms using two films placed back‐to‐back. The first film, which is positioned in direct contact with the dried silver‐stained gel, visualized both exposure to35S and32P while the second film recorded exposure to only32P due to the differential energy levels of the two isotopes. The juxtapositioning of the silver‐stained images with the two autoradiographic film images permits the unambiguous mapping of the phosphorylated polypeptides back to their corresponding silver‐stained and methionine‐labeled counterparts. This strategy provides quantitative information utilizing both silver staining (measure of constitutive levels of protein expression) and metabolic labeling to measure rates of protein synthesis and/or degradation and phosphorylation and/or dephosphorylation using [35S]methionine and [32P]orthophosphate, respectively. We have utilized this methodology for thein vitroanalysis of transforming growth factor type β1(TGF‐β1)‐mediated signal transduction in RLE cells and have identified three nuclear polypeptides, 1 (pI4.95/Mr97 kDa), 2 (5.00/85 kDa) and 3 (4.90/84 kDa) whose phosphorylation status is rapidly and transiently modulated by TGF‐β1. The methodology described should have wide applications in studies where it is desirous to measure protein synthesis and/or degradation concomitant with signal transduction pathways involvi

ISSN:0173-0835    

DOI:10.1002/elps.1150140121

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

年代:1993

数据来源: WILEY