摘要:

AbstractThe electric field variation across moving boundaries in three component systems is analyzed with a model based on electromigration only (moving boundary equation and regulating function ω). A decrease in the electric field strength across a boundary in the direction of migration is the most general requirement for the establishment of steady state migrating boundaries. The formation of that electric field gradient is shown to be based on two fundamental constraints. These are (i) the higher net mobility of the leading component compared to that of the terminating constituent and (ii) the higher concentration ratio of the leading component to the terminating component ahead of the moving boundary compared to that in the terminating zone. The relations are discussed with respect to strong and weak electrolytes where all three components are present on either side of the boundary. The predictions are verified experimentally. Limitations of the model based on migration only are discussed

ISSN:0173-0835    

DOI:10.1002/elps.1150061002

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

年代:1985

数据来源: WILEY

摘要:

AbstractIn the design of continuous electrophoresis devices, the main variables are the gap width, the flow rate and inlet temperature of the buffer, the electric field strength, the point of injection of the proteins, and the orientation of the flow to the gravitational field. Best values for many of these can be derived from first principles by requiring that the flow be stable, and that streak spreading be minimized. The result is a downflow device, with larger gap width than is usual, warm inlet buffer solution, and protein injection slightly downstream of the start of the electrodes. If cooling is improved by increasing the thermal conductivity of the cooling walls, the gap width must be reduced and the buffer flow increased. The performance possible with perfect cooling walls is similar to that obtained in space because, in both cases, the limiting factor is not thermal convection but the high temperature generated at the mid‐point between the cooling wall

ISSN:0173-0835    

DOI:10.1002/elps.1150061003

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

年代:1985

数据来源: WILEY

摘要:

AbstractProtein concentration using step pH and conductivity gradients in a free‐flow electrophoretic apparatus is described. The experiments were carried out in a vertical planar 36.5 × 5.0 × 0.05 cm separation chamber with 48 channels both at the inlet and outlet. The gradients were generated by injecting a 2.5 mM borax solution (pH 9.2, conductivity = 0.5 mS) into the chamberviachannels No. 1–12 counting from the cathode, and 0.45 M boric acid (pH 4.3, conductivity = 0.025 mS) together with the protein to be concentratedviathe remaining channels. The gradients were suitable for the concentration of the proteins with isoelectric points ≥ pH 7.0 (human immunoglobulin G, bovine hemoglobin). From mixtures of hemoglobin and albumin, the latter was removed during concentration. The protein concentration of the starting solution does not affect the results of concentration. At constant current of 30 mA and a residence time of 45 s, the proteins were concentrated at a rate of 400 ml of starting solution per hour with a maximum concentration facto

ISSN:0173-0835    

DOI:10.1002/elps.1150061004

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

年代:1985

数据来源: WILEY

摘要:

AbstractA sensitive technique of antibody‐affinity blotting was developed for the detection of small amounts of human α‐fetoprotein (AFP) in the presence of high background lectins and other serum proteins in agarose‐gel affinity electrophoresis. AFP was specifically transferred by blotting to nitrocellulose membranes which were precoated with affinity‐purified horse or goat polyclonal antibodies to human AFP. The AFP transfers were treated with rabbit immunoglobulins to human AFP, followed by affinity‐purified goat anti‐rabbit IgG(H+L)‐horseradish peroxidase conjugate for color development with 3,3′‐diaminobenzidine. The method allowed us to detect as low as 4 pg/mm2AFP (or 4 ng/ml AFP with a 3 μl sample volume applied to a 0.5 × 6 × 1 mm trough). In lectin affinity electrophoresis, AFP bands constituting more than 10% of the total AFP were quantitatively detected with 5 μl of 125–500 ng/ml AFP applied to a 1 × 5 × 1 mm trough. When horse antiserum to AFP or its immunoglobulin fraction was used to precoat nitrocellulose membranes, the background stain due to concanavalin A(Con‐A) in agarose gel became marked. This was eliminated by washing the transfers with 0.2 M α‐methyl‐D‐mannoside before subsequent antibody treatments. The washing with α‐methyl‐D‐mannoside also counteracted the uneven staining of separated AFP bands by Con‐A. The antibody‐affinity blotting suits best for the quantitative transfer of a specific protein to nitrocellulose membrane in the presence o

ISSN:0173-0835    

DOI:10.1002/elps.1150061005

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

年代:1985

数据来源: WILEY

摘要:

AbstractThe sensitivity of group specific component (Gc) detection by immunofixation after isoelectric focusing (IEF) has been examined by using two IEF methods: ultrathinlayer IEF and immobilized pH gradient (IPG) focusing followed by two staining methods: Ponceau S and a combination of Ponceau S with silver staining (SS). Both diluted plasma and bloodstain extracts were examined by these methods at two dilutions of immunofixation antibody ‐ 1:6 and 1:24. The cellulose acetate membranes containing the Gc precipitates were washed with sodium chloride after the SS process. This greatly improved the visibility of the Gc bands. The method was found to be more sensitive than Ponceau S and its use reduced the cost of immunofixation by 75%. Ultrathin‐layer IEF was carried out in gels containing the pH 4.5–5.4 Pharmalytes. The separator N‐2‐hydroxyethylpiperazine‐N′ 2‐ethanesulfonic acid (HEPES) was added to some gels and the effect on the resolution of the Gc 1 subtypes was examined and compared with the separation achieved with IPG gels. It was found that ultrathin‐layer IEF using narrow range Pharmalytes and the separator HEPES was the more sensitive method; however, IPG gels were ea

ISSN:0173-0835    

DOI:10.1002/elps.1150061006

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

年代:1985

数据来源: WILEY

摘要:

AbstractThe effects of concentration on immunoglobulin G (IgG) oligoclonal bands after isoelectric focusing in cerebrospinal fluids (CSF) were examined using six concentration procedures, and the percent recoveries of IgG varied. The procedures, % IgG recoveries, and corresponding degrees of concentration are as follows: (1) Amicon CF25 membrane cone, 75 %, 45 ×; (2) Amicon CF50A membrane cone, 65 %, 3 ×; (3) Amicon CS15 Minicon, 47 %, 80 ×; (4) Microprodicon, 55 %, 35 ×; (5) collodion bag negative pressure dialysis, 50 %, 16 ×; and (6) microcollodion bag in Permasorb, 40–80 %, 10–60 ×. Ultracentrifugation of CSF at 100 000gfor 1 h prior to concentration using CF 25 membrane cones introduced not more than 5 % variation in the IgG concentrations. Isoelectric focusing was performed on 20 CSF samples concentrated by CF25 membrane cone, CS15 Minicon, and collodion bag, and no effects on the oligoclonal IgG banding patterns w

ISSN:0173-0835    

DOI:10.1002/elps.1150061007

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

年代:1985

数据来源: WILEY

摘要:

AbstractIn electropherograms of whole‐cell protein extracts a background absorbance due to many partially overlapping bands is usually observed. Since this background is in general not relevant for the identification of a given pattern, its removal will increase the discrimination between patterns, especially when the distance between patterns is measured on a correlation basis. Here we present an algorithm for the estimation of background envelope. The algorithm selects a set of concave kernels of the total profile and subsequently performs a Fourier smoothing of the trace linking these kernels. A fraction of this smoothed trace is then the background estimate. The method is illustrated for the analysis of total protein extracts of bacterial strain

ISSN:0173-0835    

DOI:10.1002/elps.1150061008

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

年代:1985

数据来源: WILEY

摘要:

AbstractSplenocytes from inbred mice strains were stimulated with concanavalin A (Con A) or lipopolysaccharide (LPS) to form T‐ or B‐blasts. Following three days of mitogenic stimulation, immunofluorescence of the splenocytes with a rabbit antimouse‐immunoglobulin antiserum conjugated with fluorescein isothiocyanate revealed an extensive enrichment of T‐blasts by Con A and of B‐blasts by LPS. The cell preparations, however, represented enriched but not pure populations of T‐ or B‐blasts. Two‐dimensional electrophoretic resolution of cellular proteins from Con A or LPS‐stimulated murine splenocytes and subsequent silver staining of the polyacrylamide gels showed approximately 450 spots (polypeptides). Comparative analysis of the resulting patterns revealed 13 differences specific to the T‐cells and 8 specific to the B‐cells. In order to ascertain the reproducibility of the differences of two‐dimensional protein patterns from Con A and LPS stimulated splenocytes, the experiment was performed several times using two different inbred mice strains (C57B1/6 and BALB/c). Interstrain specific differences were also found at a molecular mass of about 68 kDa and at a pIran

ISSN:0173-0835    

DOI:10.1002/elps.1150061009

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

年代:1985

数据来源: WILEY

摘要:

AbstractTwo‐dimensional electrophoresis and Western blotting with lectins were used to provide information about a glycoprotein without requiring its previous purification. The oestrus‐associated glycoprotein from ovine oviductal fluid has an isoelectric point of 4.7 and a subunit molecular weight (Mr) range of 70 000–90 000. Under non‐denaturing conditions, it is present as a high molecular weight aggregate or polymer although at low pH some monomer is also detected. The surface carbohydrate on this protein includes fucose, galactose and N‐acetyl galactosamine residues but not glucose, mannose or si

ISSN:0173-0835    

DOI:10.1002/elps.1150061010

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

年代:1985

数据来源: WILEY

摘要:

AbstractPlasma specimens were phenotyped for FXIIIA in local Whites and Blacks using agarose isoelectric focusing in the range pH 4–7, followed by immunoblotting. Gene frequencies for the alleles FXIIIA*1, FXIIIA*2 and FXIIIA*4 in Whites were 0.774, 0.225 and 0.001, respectively. In Blacks FXIIIA*1 was observed at a frequency of 0.771 and FXIIIA*2 at 0.229. The rare phenotype FXIIIA 3‐1, which by conventional electrophoresis demonstrates two bands, was found to clearly show three bands using isoelectric focusing and immunoblotting. The technique used in this study is inexpensive, easy to perform and can differentiate the gene products of all the currently known variants of FXI

ISSN:0173-0835    

DOI:10.1002/elps.1150061011

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

年代:1985

数据来源: WILEY