摘要:

AbstractThe separation of enantiomeric forms of dansylated amino acids by isoelectric focusing in immobilized pH gradients (IPG) is demonstrated for the first time. Separations occur in a pH 3.0–4.0 IPG interval, in presence of 7murea, 10% methanol and 60 mM β‐cyclodextrin (CD) as chiral discriminator. It is found that the inclusion complex formed between the D‐form and CD has a lower pIthan the uncomplexed form (ΔpI= 0.05 for DL‐Phe and ΔpI= 0.025 for DL‐Trp); from this, it is calculated that the pKof the tertiary amino group in the dansyl moiety is lowered by 0.1 pH unit in the former case (D‐Phe) and by 0.05 in the case of D‐Trp (both values referring to 60 mM CD gels). For some racemates (e.g., DL‐Phe) the separation mechanism is still operative with CD concentrations as low as 20 mM. In our system 60 mM CD appears to be the solubility limit of CD. As the complex is stable in the electric field for at least 15 h, this separation mechanism could be exploited for purifying large quantities of pure D and L forms racemates in multicompartment electrolyzers with isoelectric Im

ISSN:0173-0835    

DOI:10.1002/elps.1150110102

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments ≤ 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained ≤ 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris‐borate buffer (TBE) than in gels cast and run in Tris‐acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incroporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature‐corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments ≤ 12 kbp in size in agarose gels ≤ 1.4 % in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments ≤ 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments ≥ 1 kbp in size in agarose gels ≥ 2 % in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an “intrinsic” DNA mobility of 2.7 x 10−4cm2/Vs at 20°C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments. If the volume occupied by each DNA molecule is approximated by its root‐mean‐square (rms) radius of gyration, the median pore radius,rp, of an LE agarose gel cast and run in TAE buffer isrp= 98A−0.90, whereAis the agarose concentration in % andrpis given in nm. For gels cast and run in TBE buffer, the apparent median pore radius isrp= 66A−0.95. If the volume occupied by each DNA molecule is represented by its mean geometric radius instead of the rms radius of gyration, the median gel pore radius in both buffers depends onA−0.5, as predicted by the extended Ogston theory. From the corresponding retardation coefficients, the

ISSN:0173-0835    

DOI:10.1002/elps.1150110103

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe electrophoretic properties of purified calmodulin were investigated. High performance capillary electrophoresis of this Ca2+‐binding protein in free solution at pH 2.5 resulted in an elution of a single peak with a retention time of approximately 4.7 min. Addition of [ethylene‐bis(oxyethylenenitrilo)]N, N, N', N'‐tetraacetic acid (EGTA) to the protein prior to capillary electrophoresis completely abolished this electrophoretic profile. Polyacrylamide gel electrophoresis of calmodulin under denaturing and nondenaturing conditions also revealed a single polypeptide band. However, the relative electrophoretic mobilities of this protein could vary, depending on the presence or absence of Ca2+. The pIof calmodulin was estimated to be 3.7 by using isoelectric focusing techniques. Analysis of this acidic protein by high performance capillary electrophoresis at pH 8.0 revealed that it could be resolved into two major and one minor polypeptide peaks, regardless of the presence or absence of Ca2+. These findings suggest that capillary electrophoresis at near physiological pH may differentiate the microheterogeneity of calmo

ISSN:0173-0835    

DOI:10.1002/elps.1150110104

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe electrophoretic mobilities of monosaccharides, oligosaccharides, sugar alcoho and sugar acids were determined in 0.3 M borate buffer, pH 10, using thin‐layer electrophoresis on silanized silica gel, pretreated with octanol‐1. A rapid separation of number of sugars, occurring in foods, could be achieved. Using a 0.05–0.1 M neutra solution of barium acetate as electrolyte, thin‐layer electrophoresis allowed exceller and rapid separation as well as identification of all common uronic acids which are constituents of many acidic polysacc

ISSN:0173-0835    

DOI:10.1002/elps.1150110105

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

年代:1990

数据来源: WILEY

摘要:

AbstractAcidic oligosaccharides derived from glycosaminoglycan heparin were separated by polyacrylamide gradient gel electrophoresis (PAGE). The gel could be visualized using Alcian Blue dye to give a pattern of highly resolved, well defined bands. The particular banding pattern obtained was the result of a heparinase catalyzed depolymerization which afforded oligosaccharide products that differed in size by one disaccharide unit. The separated oligosaccharides could be recovered prior to staining by electroelution onto a positively charged nylon membrane by a semi‐dry transfer procedure. Subsequent elution and quantitative recovery of individual oligosaccharides from the membrane was achieved. By using multiple membrane layers a second separation dimension was obtained, resulting in increased oligosaccharide purity proportional to transfer depth. Preparative gradient polyacrylamide gel electrophoresis followed by semi‐dry electro‐transfer and recovery represents a novel method for the preparation of homogeneous acidic oligosaccha

ISSN:0173-0835    

DOI:10.1002/elps.1150110106

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

年代:1990

数据来源: WILEY

摘要:

AbstractNondenaturing polyacrylamide gel electrophoresis was used to separate protein kinases in crude extracts and subcellular fractions of murine erythroleukemic cells. The kinases were detected using anin situphosphorylation assay. The electrophoretic patterns obtained using gel bound to GelBond and prepared with AcrylAide differed from those seen without GelBond and withN, N'‐methylenebisacrylamide as cross‐linker. In an attempt to improve the resolution of the bands in the membrane fractions, detergent‐treated preparations were electrophoresed through gels which contained either 0.1 % Triton X‐100 or 0.1 % Nonidet P‐40. The resolution of the bands in this fraction was not, however, improved with the inclusion of the nonionic detergent in the gels. When cytosol was electrophoresed through gels containing detergent, a major band of cAMP‐dependent protein kinase activity showed a marked shift in mobility. This may have been the result of a structural change, altering the shape and possibly affecting the charge on the molecule, or the enzyme may have formed aggregates with th

ISSN:0173-0835    

DOI:10.1002/elps.1150110107

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

年代:1990

数据来源: WILEY

摘要:

AbstractMyoglobin CNBr peptides, constituting the commercially available molecular weight calibration kits for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, were analyzed by microsequencing after electroblotting on polyvinylidene difluoride (Immobilon) membranes. An obious disagreement was found between peptide identification and the data provided by the manufacturers. We observed 6 peptides fromMr2500 to 17 000 corresponding, in increasing size order, to the 3 peptides resulting from the total CNBr digestion, to 2 incompletely cleaved peptides and to the intact myoglobin. Using a corrected calibration curve, a linear relationship was established fromMr6000 to 43 000 and a second one for shorter peptides. This method of electrophoresis and electroblotting, easily adapted for peptides, is a powerful tool for peptide identification correlated with size determination. It is especially useful for CNBr‐cleaved pepti

ISSN:0173-0835    

DOI:10.1002/elps.1150110108

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

年代:1990

数据来源: WILEY

摘要:

AbstractMultiple successive pressure blottings of a single agarose isoelectric focusing gel were performed on normal and CNBr‐activated nitrocellulose (NC) filters. The results obtained by multiple successive 10s immunoprints were compared to those obtained by a single 10 min immunoprint. To quantify the transfer efficiency of these techniques, a defined amount of radioactive material was separated by isoelectric focusing on agarose gel. After separation and pressure blottings of the gel the NC filters were submitted to autoradiography. The amount of radioactive material bound to the NC filters was determined by scintillation technique. The single 10 min pressure blot was more efficient than each of the multiple successive 10s prints. However, the latter procedure allowed equal resolution and resulted in a higher recovery of total radioactivity than the single immunoprint technique. The aim of this paper is to show how to obtain highly reproducible prints of electrophoretic patterns in an agarose gel of heterogeneous samples when accurate multiple immunodetections are to be performed. This technique was tested to characterize the grass pollen specific immunoglobulin classes and subclasses from an allergic patien

ISSN:0173-0835    

DOI:10.1002/elps.1150110109

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

年代:1990

数据来源: WILEY

摘要:

AbstractEnzyme‐linked immunofiltration assay (ELIFA) for labeling transferred proteins is an interesting and powerful technique for the rapid specific detection (15 min) of proteins immobilized on nitrocellulose or nylon membranes (0.20 and 0.45 μm). ELIFA does not require fastidious handling of the membranes. Saturation, specific labeling and washing procedures are achieved by filtration, controlled by a monitoring unit which regulates the flow rate and ensures excellent specificity, repetition and reproducibility. The recycling by closed circuit or by repetetive inversion of the flow direction offers the advantage of reducing the volumes of expensive reagents while simultaneously increasing the sensitivity of the technique. The detection limit is at least as low as 1–5 ng using directly or indirectly enzymatically labelled probes. ELIFA may be extended to the identification of glycoproteins using specific ligands such as lectins or to the immunocapture of an antigen using specific antibodies immobilized on an activated membrane. ELIFA complements fast separation, bye.g., isoelectric focusing, polyacrylamide gel electrophoresis, or sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and accelerated electrotransfer to membranes with rapid detection reducing the total time for separation transfer and detection to less th

ISSN:0173-0835    

DOI:10.1002/elps.1150110110

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe efficiency of semi‐dry electrophoretic transfer after sodium dodecyl sulfate (SDS)‐electrophoresis using PhastGel®media was investigated in a model system using three isotope labelled proteins. To give a full picture of the blotting process the amount of protein present in the gel, membranes, and filter papers was determined after different transfer times. The influence of the transfer buffer, commonly used additives such as methanol and SDS, and several different immobilizing matrices was investigated. Soybean trypsin inhibitor, bovine serum albumin, and ferritin were used as model proteins to study the effect of size on transfer efficiency. Basically, all three stages of the blotting process decide the result; the elution of protein from the gel, the immobilization of protein to the membrane, and the loss of material from the membrane during transfer. A theoretical explanation for the observed poor binding to a second membrane is discussed. Our results show that the buffer composition has little influence on the efficiency of transfer from the gel, but can be significant to the binding capacity of the membrane. In all experiments performed, there was never one moment during the transfer when all protein was eluted from the gel and simultaneously still bound to the membrane. The highest recovery in the membrane was obtained at different time intervals for different proteins. This indicates that quantitative transfer procedures cannot be generalized. However, obtaining an optimal method for reliable quantification of a specific protein or group of proteins is possible. For general protein staining of nitrocellulose and polyvinylidene difluoride membranes, a highly sensitive silver staining method requiring only 15 min has been

ISSN:0173-0835    

DOI:10.1002/elps.1150110111

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

年代:1990

数据来源: WILEY