Search results for "Isoelectric Focusing"
showing 10 items of 39 documents
A simple method to estimate the isoelectric point of modified Tomato bushy stunt virus (TBSV) particles
2017
We present a simple method to estimate the isoelectric point (pI) of Tomato Bushy Stunt particles. We demonstrate that the combination of agarose gels with different pH buffers can be used to electrophorese the virus particles and their migration patterns can be compared. This method allows us to estimate the pI of the virus particles (wild type, wt, and genetically modified particles) and to monitor the effect of the pI of modified peptide side chains of the viral capsid subunit on the pI of the whole virus particle.
Analysis of tear proteins by one- and two-dimensional thin-layer iosoelectric focusing, sodium dodecyl sulfate electrophoresis and lectin blotting. D…
1998
· Background: Isoelectric focusing (IEF) of tear proteins has not yet been carried out in a satisfactory way. Two-dimensional (2D) electrophoresis, especially in the combination of IEF with SDS, is able to differentiate between proteins in detail. The purpose of this study was therefore to analyze tear proteins by 1D IEF alone and in combination with a 2D pattern, and by IEF followed by lectin staining. · Methods: Ampholines, covering a broad range from pH 3 to pH 10, were applied. After IEF, semi-dry blotting and incubation with a group II lectin and two group V lectins was performed. · Results: Tear proteins could be separated into 31 single bands. Tear-specific pre-albumin (TSPA), lactof…
Human liver cytosolic epoxide hydrolases.
1988
Human liver epoxide hydrolases were characterized by several criteria and a cytosolic cis-stilbene oxide hydrolase (cEHcso) was purified to apparent homogeneity. Styrene oxide and five phenylmethyloxiranes were tested as substrates for human liver epoxide hydrolases. With microsomes activity was highest with trans-2-methylstyrene oxide, followed by styrene 7, 8-oxide, cis-2-With methylstyrene oxide, cis-1,2-dimethylstyrene oxide, trans-1, 2-dimethylstyrene oxide and 2, 2-dimethylstyrene oxide. With cytosol the same order was obtained for the first three substrates, whereas activity with 2, 2-dimethylstyrene oxide was higher than with cis-1,2-dimethylstyrene oxide and no hydrolysis occurred …
Polymorphism of the Complement C8A and -B Genes in Two Families with C8β Deficiency and Neisserial Infections
1994
Serum samples from members of two Italian families with complement C8 beta deficiency were studied by SDS-PAGE under nonreducing conditions and by IEF. The proband of family I had suffered from two episodes of purulent meningitis and two of her uncles had suffered from only one episode, while the proband of family II had suffered from three different episodes. In contrast to previous findings, where C8 beta deficiency was cosegregating with C8A (alpha-gamma) allotype A, the proband of family II had the C8A allotype B. In addition, in one of her sons a novel variant of the C8 beta chain was detected. Studies at the DNA level in family I, using a recently described PCR system, demonstrate the…
Oligomerization of Vibrio cholerae cytolysin yields a pentameric pore and has a dual specificity for cholesterol and sphingolipids in the target memb…
1999
Vibrio cholerae cytolysin permeabilizes animal cell membranes. Upon binding to the target lipid bilayer, the protein assembles into homo-oligomeric pores of an as yet unknown stoichiometry. Pore formation has been observed with model liposomes consisting of phosphatidylcholine and cholesterol, but the latter were much less susceptible to the cytolysin than were erythrocytes or intestinal epithelial cells. We here show that liposome permeabilization is strongly promoted if cholesterol is combined with sphingolipids, whereby the most pronounced effects are observed with monohexosylceramides and free ceramide. These two lipid species are prevalent in mammalian intestinal brush border membranes…
Characterization of the trimeric, self-recognizing Geodia cydonium lectin I.
1983
A D-galactose-specific lectin I was extracted from the sponge Geodia cydonium and purified by affinity chromatography. The molecular weight of lectin I as determined by high-pressure liquid gel chromatography, was found to be 36500 +/- 1300. Disc gel electrophoresis in the presence and in the absence of sodium dodecyl sulfate showed that lectin I is a trimer composed of three different subunits (Mr: 13800, 13000 and 12200); two of the three subunits are linked by one disulfide bond. Isoelectric focusing gave a pI of 5.6 for the native molecule and a pI of 4.4 and of 7.4 for the subunits. The three subunits carry carbohydrate side chains, composed of D-galactose (94%) and of arabinose (5%). …
High-performance and ion-exchange chromatography and chromatofocusing of the human uterine progesterone receptor: its application to the identificati…
1984
Two independent lines of evidence were used to identify the human uterine progesterone receptor. First, three differently tritiated progestogens (Org 2058, R 5020, progesterone) were used for reversible labelling of the receptor. Secondly, the highly potent affinity label 21-[3H]dehydro Org 2058 was used to label covalently the steroid-specific binding site of the receptor. The labelled cytosols were chromatographed on a Mono Q high-performance anion-exchange column in the absence or presence of a high molar excess of the respective unlabelled competitor steroids. In the case of 21-[3H]dehydro Org 2058, Org 2058 was used as the unlabelled competitor. After elution with a NaCl gradient, the …
Sample streaks and smears in immobilized pH gradient gels
1996
In immobilized pH gradient (IPG) gel formulations as wide as pH 4-9, encompassing neutrality and containing the pK 7.0 acrylamido buffer as one of the buffering ions, smears are directly proportional to the total amount of the pK 7.0 species. At a total level of 10 mM pK 7.0 in these gel formulations, severe smears occur not only for mildly hydrophobic proteins (e.g., recombinant alcalase and termamylase) but also for the relatively hydrophilic pI marker proteins. Streaks and smears are essentially abolished in recipes devoid of the pK 7.0 compound or in formulations containing a maximum of 3 mM of this component. Although partitioning in water/n-octanol has shown the pK 7.0 acrylamido buff…
Comparison of DNase, DNA-polymerase and RNA-polymerase activities present in the DNA-bindung proteins of normal human dermis, epidermis, horny layer …
1978
DNA-binding proteins (DBP) of normal human dermis, epidermis, horny layer and psoriatic scales represent a tissue-specific group of mostly nuclear nonhistone proteins. To analyse their function, the different DBP fractions were examined concerning the presence of DNase, DNA-polymerase and RNA-polymerase activities. DBP of normal epidermis and horny layer contain four different DNases. One DNase of both DBP fractions is active only at pH 5.0. Three DNases of epidermal DBP are active at a pH-range from 5.0--8.5, while the corresponding DNases of horny layer-DBP are most active at pH 7.4. Probably these DNases have changed their pH-optimum during keratinisation. DBP of psoriatic scales include…
Microdisc-electrophoretic study of deoxyribonucleases in cow snout epidermis
1983
Acid and neutral deoxyribonucleases (DNases) of the cow snout epidermis were investigated by the microdisc-electrophoresis of polyacrylamide gels containing highly polymerized DNA and by isoelectric focusing techniques. The nucleases were characterized with respect to their pH optimum. An acid DNase at pH 5.0 was detected as a single distinct band after the electrophoretic separation. After isoelectric focussing also, only one acid DNase activity with an isoelectric point (IP) of 6.2 was detectable. Neutral DNases at pH 7.4 were demonstrated as major and minor bands by their different electrophoretic mobilities. In the isoelectric focusing system also, two neutral DNases, a major one (IP, 4…