Search results for "Polyacrylamide gel electrophoresis"
showing 10 items of 89 documents
Improving Fractionation of Human Milk Proteins through Calcium Phosphate Coprecipitation and Their Rapid Characterization by Capillary Electrophoresis
2018
This work describes a simple sample pretreatment method for the fractionation of human milk proteins (into their two main groups, whey and caseins) prior to their analysis. The protein-extraction protocol is based on the addition of calcium phosphate to nonadjusted pH human milk. The combination of calcium ions with phosphate results in an effective coprecipitation of caseins. To assess the suitability of this fractionation protocol, the protein extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), LC-MS/MS, and capillary electrophoresis (CE) analysis. The results evidence a significant decrease in contamination of casein fraction with whey proteins…
Identification of a cell surface-associated protein involved in mouse neural cell aggregation by means of antibodies against the sponge aggregation f…
1989
Polyclonal antibodies were raised against the purified aggregation factor (AF) from the sponge Geodia cydonium to elucidate possible immunological relationships between adhesion molecules of lower multicellular eukaryotic systems (sponges) and those of vertebrates. This anti-AF recognized a series of polypeptides associated with the AF, among them also a polypeptide with a Mr of 47,000 (p47). The formation of the antibody-p47 immunocomplexes could be prevented by adsorbing the anti-AF with a brain extract from DBA/2J mice. Moreover, this brain polypeptide inhibited the AF-mediated aggregation of sponge cells. Interestingly, the anti-AF recognized a p37 molecule in the brains of 2- to 3-day-…
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 …
Methodical Studies in the Separation of Chlorophyll-Protein Complexes by Polyacrylamide Gel Electrophoresis
1980
Summary Chlorophyll-protein complexes from thylakoids of Sinapis alba plants were separated, using Tris-HCl and Tris-borate buffer in the sodium dodecyl sulphate acrylamide gel electrophoresis. In the electrophoretogram with Tris-borate buffer, 6 bands could be characterized in the gel pattern. In addition to the three major green zones (P-700-chlorophyll a-protein, light-harvesting chlorophyll a/b-protein, free pigment zone), which were also observed in experiments with the Tris-HC1 buffer, three further bands (LHCP 1 , LHCP 2 , CPa) were electrophoretically separated. An elongation of the electrophoretical period resulted especially in a decrease of the proportion of the light-harvesting …
Chlorophyll-Protein Complexes of Chlorella fusca
1980
Chlorophyll-protein complexes from thylakoids of the normal type and two mutants of Chlorella fusca were separated using sodium dodecyl sulfate acrylamide gel electrophoresis (PAGE). The properties of the chlorophyll-protein complexes of the three strains of Chlorella were compared. Standard curves were set up for the characterization of the chlorophyll-proteins. In every electrophoretic separation of chlorophyll-protein complexes, a certain amount of pigment is separated from the protein. We tried to keep that amount as low as possible by mild solubilization and by working in low temperature. Under these conditions, we obtained several new chlorophyll-proteins in addition to the P-700-chl…
A novel cholesterol-based detergent
2005
Design, synthesis and characterization of CHAPSTEROL, a novel cholesterol-based detergent developed for functional solubilization of cholesterol-dependent membrane proteins are described. To validate CHAPSTEROL, we employed the oxytocin receptor, a G protein-coupled receptor requiring cholesterol for its high-affinity binding state. Using the photoactivatable cholesterol analogue [3H]6,6-azocholestan-3beta-ol[3alphaH], we demonstrate that solubilization by CHAPSTEROL leads to an enrichment of cholesterol-binding proteins whereas the widely used bile acid derivative CHAPSO leads to a significant depletion of cholesterol-binding proteins. Similar to Triton X-100 and CHAPS, CHAPSTEROL maintain…
Membrane Protein Subunit Fractionation by Means of Inverse Pore Gradient Elution Polyacrylamide Gel Electrophoresis
1996
We report here the preparative scale isolation of the four subunits of the nicotinic acetylcholine receptor (nAChR) applying short inverse pore gradient SDS gels on an elution-PAGE apparatus. The nAChR subunits are of similar molecular weights (alpha, 50.2 kDa; beta, 53.7 kDa; gamma, 56.3 kDa; delta, 57.6 kDa) and isoelectric point (approx 5.5) and share the typical properties of amphiphatic membrane proteins that are difficult to separate by chromatographic procedures. Preparative PAGE, which has proved to be the method of choice for nAChR-subunit fractionation, however, is time-consuming and achieves only moderate resolutions yielding dilute fractions. We present here the fractionation of…
Purification and Characterization of <I>Bacillus cereus</I> Protease Suitable for Detergent Industry
2005
An extracellular alkaline protease from an alkalophilic bacterium, Bacillus cereus, was produced in a large amount by the method of extractive fermentation. The protease is thermostable, pH tolerant, and compatible with commercial laundry detergents. The protease purified and characterized in this study was found to be superior to endogenous protease already present in commercial laundry detergents. The enzyme was purified to homogeneity by ammonium sulfate precipitation, concentration by ultrafiltration, anion-exchange chromatography, and gel filtration. The purified enzyme had a specific activity of 3256.05 U/mg and was found to be a monomeric protein with a molecular mass of 28 and 31 kD…
Use of an enzyme-assisted method to improve protein extraction from olive leaves.
2013
The improvement of protein extraction from olive leaves using an enzyme-assisted protocol has been investigated. Using a cellulase enzyme (Celluclast® 1.5L), different parameters that affect the extraction process, such as the influence and amount of organic solvent, enzyme amount, pH and extraction temperature and time, were optimised. The influence of these factors was examined using the standard Bradford assay and the extracted proteins were characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum extraction parameters were: 30% acetonitrile, 5% (v/v) Celluclast® 1.5L at pH 5.0 and 55°C for 15min. Under these conditions, several protein extracts…
Identification and isolation of the primary aggregation factor from the cell membrane of the sponge Geodia cydonium
1985
The primary aggregation factor (pAF) of sponge cells is a glycoprotein that is firmly associated with the cell membrane. Polyspecific antibodies (anti-GM) prepared from sera raised against membranes of cells from the siliceous sponge Geodia cydonium were found to inhibit initial aggregation of homologous cells. The inhibition of aggregation, caused by anti-GM was neutralized by pAF. The pAF had been successfully solubilized and enriched by affinity chromatography, gel filtration and density gradient centrifugation, if checked by polyacrylamide gel electrophoresis in the presence of urea. The Mr of the native pAF was approximately 40 000 as estimated by gel filtration; under denaturing condi…