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1.
Gordon S. Rule 《Analytical biochemistry》1984,138(1):99-106
A method for the quantitative assay of nuclease activity in crude cell lysates after isoelectric focusing (IEF) in polyacrylamide slab gels is described. After IEF, an agarose overlay gel containing DNA is placed on the IEF gel and the nuclease activity quantified by the loss of ethidium bromide fluorescence of the DNA. With this method a linear response was obtained for 1 to 10 ng of DNase I. Various methods of pH equilibration after IEF were also evaluated. The use of a high buffer concentration in the overlay gel is recommended to control the pH during the enzyme reaction. An analytical solution for the diffusion of enzymes from the IEF gel to the overlay gel is also presented and an equation that may be used to choose optimum times for transfer of the enzyme from the IEF gel to the overlay gel is given. 相似文献
2.
3.
Effects of novel leukotrienes on neutrophil migration 总被引:2,自引:0,他引:2
Fibronectin was chromatographed on immobilized alkanes of various chain lengths. No binding of the protein to the hydrophobic matrix was observed with alkanes containing from 3–7 methylene groups; the protein bound, however, to immobilized alkanes with 8 or 10 methylene groups. Fibronectin could be quantitatively eluted from commercial Octylsepharose with a non-ionic detergent. A gelatin-based plasma expander did not interfere with the binding. Many proteolytic fibronectin fragments also bound to a hydrophobic matix. The results show that fibronectin posesses at least one hydrophobic binding site. 相似文献
4.
Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing 总被引:3,自引:0,他引:3
P Pernin 《International journal for parasitology》1984,14(5):459-465
Pernin P. 1984. Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing. International Journal for Parasitology14: 459–465. The isoenzymatic patterns of different strains of Naegleria were studied by isoelectric focusing (I.E.F.) on polyacrylamide gels for seven enzymatic activities (leucine amino peptidase; lactate dehydrogenase; glucose 6 phosphate dehydrogenase; propionyl esterase; glucose phosphate isomerase; malate dehydrogenase; acid phosphatase), two of which (lactate dehydrogenase and glucose 6 phosphate dehydrogenase) were being investigated for the first time. The three pathogenic N. fowleri strains share a common pattern for most of the enzymes tested except for glucose 6 phosphate dehydrogenase, and thus form a very homogeneous species, while thermophilic nonpathogenic strains show more heterogeneity particularly for leucine amino peptidase and glucose 6 phosphate dehydrogenase.I.E.F. must be considered as a supplementary and rapid method for the identification of N. fowleri and as a powerful tool to demonstrate the complexity of different genera of free-living amoebas. 相似文献
5.
Shahjahan Kabir 《FEMS microbiology letters》1986,37(2):155-162
Abstract Analytical isoelectric focusing (IEF) in thin layers of polyacrylamide gels resolved cholera toxin into 3 isomeric forms differing in charge (isoelectric points 6.80, 6.65 and 6.55). All these forms had identical molecular weights, and were also antigenically similar, as demonstrated by their reactivity to antisera to cholera toxin. Both the B and A subunits possessed charge heterogeneity. The B subunit was detected in a free form when a solution of cholera toxin was aged for a few days. Antisera to cholera toxin, irrespective of mode of immunisation, contained antibodies to both the intact cholera toxin and the free B subunit as demonstrated by the immunoblotting technique based on IEF. 相似文献
6.
Data‐independent acquisition (DIA) approaches, such as SWATH®‐MS, are showing great potential to reliably quantify significant numbers of peptides and proteins in an unbiased manner. These developments have enhanced interest in developing a single DIA method that integrates qualitative and quantitative analysis, eliminating the need of a prebuilt library of peptide spectra, which are created through data‐dependent acquisition methods or from public repositories. Here, we introduce a new DIA approach, referred to as “SWATH‐ID,” which was developed to allow peptide identification as well as quantitation. The SWATH‐ID method is composed of small Q1 windows, achieving better selectivity and thus significantly improving high‐confidence peptide extractions from data files. Furthermore, the SWATH‐ID approach transmits precursor ions without fragmentation as well as their fragments within the same SWATH acquisition period. This provides a single scan that includes all precursor ions within the isolation window as well as a record of all of their fragment ions, substantially negating the need for a survey scan. In this way all precursors present in a small Q1 window are associated with their fragment ions, improving the identification specificity and providing a more comprehensive and in‐depth view of protein and peptide species in complex samples. 相似文献
7.
J. L. WILLIAMS R. A. OLIVER A. L. G. MORGAN E. J. GLASS R. L. SPOONER 《Animal genetics》1991,22(5):407-415
This paper describes the production of alloantisera directed against bovine major histocompatibility complex (MHC) (BoLA) class II antigens in animals whose MHC phenotypes had been defined by one dimensional isoelectric focusing. Animals of closely matched BoLA class I types were selected by serology and subsequently typed for class I and class II by 1D-IEF of immunoprecipitated antigens. Those with similar class I type by both methods, but differing at the class II locus, were chosen for reciprocal immunization. Cross-immunization was by two skin implantations 6 weeks apart. The resulting antisera showed low titre after the first immunization and elevated titre 3 weeks after the second immunization. The sera reacted strongly with cells expressing specific BoLA class II antigens. The pattern of reactivity correlated well with IEF class II typing on a panel of animals representing all of the class II IEF types present in the Friesian population. 相似文献
8.
The examination of human hair keratin to obtain genetic information, which may be useful also in forensic sciences, has been
carried out with the use of isoelectrophoretic procedure obtaining considerable evidence for the existence of specific-species
patterns.
In this paper the keratins extracted from hairs of 280 subjects belonging to Sardinian people (113 males, 167 females, aged
from 1 to 89, belonging to 52 families) were analyzed using IEF in thin-layer polyacrylamide gel (0.5 mm) in the pH range
2.5–7.0, followed by the silver staining method.
Number, position and colour of the bands were the same in all the analyzed samples but a large individual variability was
revealed for the relative intensity of some bands.
Differences for a long time storage were not revealed as well hair's sample as protein extract: Neither were differences in
the number and position of the bands analyzing samples of hair from several sites of the head of the same individual revealed.
The results obtained are a useful indication to continue this research considering the numerous fields of application of this
analysis system. 相似文献
9.
Elisabetta Gianazza Francesco Chillemi Pier Giorgio Righetti 《Journal of biochemical and biophysical methods》1980,3(3):135-141
Iodine stain is used for the detection of peptides after isoelectric focusing has been developed. Ultrathin gels (240–360 μm) are cast and, after focusing, dried at 110°C on a filter paper sheet. The paper-pasted gel is then exposed to iodine vapors for a few seconds to a few minutes, depending on the peptide load. White peptide zones are visivle on a brown, uniform background. The reaction is fully reversible and can be used also for small-scale preparative purification of peptides. Better than 80% recoveries of peptide from the gel can be obtained by elution in 80% acetic acid. 相似文献
10.
Genetic polymorphism is found among the PIF (parotid isoelectric focusing variant) salivary proteins after separation by prolonged isoelectric focusing in pH 3.5–5.2 urea polyacrylamide slab gels subsequently stained for protein. Two PIF proteins are either present (PIF +) or absent (PIF –) from all salivas. The phenotypes are determined by autosomal inheritance of two alleles, PIF
+ and PIF
–. Gene frequencies in randomly collected samples show marked racial differences: among 148 whites, PIF
+ is 0.66 and PIF
– is 0.34; among 90 blacks, PIF
+ is 0.35 and PIF
– is 0.65; among 78 Chinese, PIF
+ is 0.56 and PIF
– is 0.44. Studies in 41 families including 129 children support the interpretation of control of PIF by a single autosomal locus. In 8 PIF+ × PIF– matings, there were 8 PIF– (6.34 expected) children. In 33 PIF+ × PIF+ matings, there were 7 PIF– (6.70 expected) children. Linkage studies indicate that PIF is closely linked to the proline-rich protein (PPP) gene complex (e.g., for six families, lod score at =0.00 of PIF/G1 is 3.58). In 107 randomly collected samples from whites, PIF is strongly associated with Db (x
1
2
=20.02; P<0.0001) and Gl (x
1
2
=12.58; P=0.0005) but not with Pr, Ps, Pm, and Pa proteins. These data (probably reflecting genetic disequilibrium) suggest that PIF may be closer to Db and G1 than to other identified loci of the PPP gene complex. The PPP gene complex includes at least seven genes (and probably more) that produce many acidic and basic proline-rich proteins, constituting about two-thirds of parotid salivary proteins that are thought to have important functions at the tooth surfaces.This study was supported by a grant from the National Institutes of Dental Research (DEO 3658-15). Paper No. 2435 of the Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706. 相似文献
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12.
Pier Giorgio Righetti Stellan Hjertén 《Journal of biochemical and biophysical methods》1981,5(5):259-272
A method is described for the synthesis of high-molecular-weight carrier ampholytes for preparative isoelectric focusing of peptides. A giant polyethylene imine () is mixed with a linear gradient of acrylic acid in a flow-through system and let to react at 80°C for 70 h. Giant carrier ampholytes () are thus obtained. These compounds interact very strongly among themselves, probably not by hydrogen bonds or hydrophobic interactions but ionic bonds. In fact, the aggregates are split by high salt (NaCl) or by zwitterionic compounds (Gly, taurine) or at acidic or alkaline pHs. They appear to interact only weakly and reversibly with proteins and no interactions are apparent with model dipeptides (His-Ser, His-Met, His-Phe and His-Lys). 相似文献
13.
Five major isoelectric focusing (IEF) parameters--volt-hours; concentrations of acrylamide, NaOH, and H3PO4; and equilibration time--were systematically varied to determine the effect of each on two-dimensional IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel patterns and to optimize IEF conditions. Alterations in each parameter affected the gel pattern, frequently causing uncertainty in the identification of spots between conditions. The results emphasize the need for internal analytical consistency, and indicate that gel pattern comparisons between laboratories can be complicated if different IEF conditions are employed. The systematic evaluation indicated that optimized patterns were obtained when increased concentrations of NaOH and H3PO4 (to 50 and 25 mM, respectively) and run durations of 10,000 V-h or longer were used. 相似文献
14.
Richard C. Caswell Peter Gacesa
Andrew J. Weightman
《International journal of biological macromolecules》1986,8(6):337-341A sensitive method has been developed for the rapid analysis of mutliple forms of alginate lyases in crude bacterial extracts. The technique is based on isoelectric focusing with a substrate-overlay technique for the direct measurement of enzyme activity. Isoelectric point values have been determined for the alginate lyases present in five strains of bacteria using, typically, 5.7 × 10− units of activity. Multiple forms of these enzymes have been observed in three of the five bacterial strains studied. The method has also been used to compare the pI value of the
-guluronate lyase from Klebsiella pneumoniae with those for the cloned gene products in strains of Escherichia coli. 相似文献
15.
Identification and quantification of actin isoforms in vertebrate cells and tissues 总被引:14,自引:0,他引:14
The cytoskeletal protein actin exists in vertebrates as six different isoforms, which are difficult to identify conclusively because of a high degree (greater than 90%) of overall sequence homology. We have used IEF immunoblotting in combination with a panel of isoform-specific and -selective antibodies to analyze the actin isoform composition of nine tissues from adult rat. In three nonmuscle tissues (lung, spleen, and testis), we detected a previously unreported isoform that we identified as smooth muscle alpha. The IEF immunoblot technique was also used to quantify the proportions of the isoforms expressed in these nine rat tissues. 相似文献
16.
Pier Giorgio Righetti Cinzia Macelloni 《Journal of biochemical and biophysical methods》1982,6(1):1-15
The major cause for pH gradient decay and cathodic drift during isoelectric focusing in polyacrylamide gels has been found to be electroendo-osmotic flow generated by fixed charges in the gel matrix. These charges have the following causes: (a) trace impurities of acrylic acid in the co-monomers; (b) covalent incorporation of catalysts (persulphate and riboflavin 5'-phosphate) as terminal groups in polyacrylamide chains; (c) hydrolysis of amide groups to acrylic acid in the gel layer underneath the cathodic filter paper strip. The result of these fixed negative charges in the matrix is a movement of counter-ions with hydration water towards the cathode (i.e. electroendo-osmosis) with concomitant drift of pH gradient and focused protein zones in the same direction. It is impossible to cure the cathodic drift by increasing the pH of the anolyte, or decreasing the pH of the catholyte, or both, as previously suggested. One way to reduce the cathodic drift efficiently is to incorporate covalently in the matrix tertiary or quaternary groups (3-dimethylaminopropylmethacrylamide) in stoichiometric amounts as compared with the negative charges.This ‘balanced’ polyacrylamide displays zero drift for at least 5000V·h, which is considered to be an ample time for equilibrium separation of protein species in isoelectric focusing. 相似文献
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18.
制备式等电聚焦测得湖南产五步蛇蛇毒磷脂酶A2为单一的吸收峰(OD280),等电点为5.32,证明了作者前报道的纯化的磷脂酶A2为纯品 相似文献
19.
Polymorphism for two autosomal alleles of equine plasminogen, PLG 1 and PLG 2 , was demonstrated in plasma by isoelectric focusing and immunofixation, with a goat anti-human plasminogen antibody. The frequency of PLC 2 was 0.16 in 150 Standardbreds, 0.20 in 96 Thoroughbreds, and 0.39 in 32 Shetland ponies. No evidence for linkage of PLG with any of 13 marker loci was found. 相似文献
20.
G. de Jong C. C. A. Ammerlaan W. L. van Noort H. G. van Eijk G. L. van Landeghem P. C. D'Haese M. E. de Broe 《Biometals》1995,8(4):352-356
Transferrin saturated with Al3+ subjected to isoelectric focusing (IEF) in a pH gradient can be separated into four fractions, representing the apotransferrin, transferrin with aluminum at the metal binding site in the C- or N-terminal lobe, or both. The electrophoretic mobilities of these four fractions are identical to those of the iron-transferrin counterparts. Simultaneous binding of aluminum and iron to transferrin can also be demonstrated. The decreased saturation after IEF indicates that the affinity of transferrin for aluminum is low compared with its affinity for iron. This effect is particularly evident when bicarbonate is used as the synergistic anion in the loading procedure. In contrast, loading of transferrin with aluminum in the presence of oxalate produces a di-aluminum-transferrin complex that is stable during IEF. 相似文献