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排序方式: 共有264条查询结果,搜索用时 19 毫秒
1.
Fractionation techniques in a hydro-organic environment. I. Sulfolane as a solvent for hydrophobic proteins 总被引:1,自引:0,他引:1
Sulfolane (thiophene, tethrahydro-1,1-dioxide), at concentrations of 4 M or above, is an efficient solubilizing agent for water-insoluble proteins (e.g., zein or globin chains). In comparison with urea, it appears indefinitely stable in aqueous solutions and does not chemically modify proteins upon storage. Moreover, it favors protein structure, i.e., it increases their alpha-helix content, while urea decreases it. Sulfolane is compatible with electrophoretic techniques (it only slightly reduces polyacrylamide polymerization efficiency and it does not interfere with protein and peptide detection methods) and with chromatographic methods (it has negligible A280 nm). With hydrophilic proteins, sulfolane behaves as a mild denaturant and precipitates them at concentrations between 5 and 7 M. 相似文献
2.
Benito Casu Giangiacomo Torri Margherita Legramandi Pier Giorgio Righetti 《Carbohydrate research》1982,104(2):299-308
The binding of heparin (Hep) and Hep fractions with oligo- and poly-(alkylenimines) having the general formula H2N(CH2-CHR-NH)nH, where R = H or Me, has been investigated by spectroscopy, by evaluating the competition of the amines and Methylene Blue for the anionic sites of Hep. The strongest-binding was observed at pH 3.5, with the essentially linear triethylenetetramine and the slightly branched tetraethylenepentamine giving the most stable complexes. For N (number of nitrogen atoms per molecule) >5, a decrease of the binding ability of the amines was observed. The apparent stoichiometry of the complexes was a function of the relative concentration of Hep and the amine, indicating an equilibrium between different types of complexes. Beef-lung Hep and a Hep fraction consisting mainly of trisulphated disaccharide blocks gave stronger complexes than the more heterogeneous, pigmucosal Hep and a Hep fraction of lower sulphate content. The results are interpreted in terms of polyelectrolyte-type associations involving sulphate groups on adjacent residues of the Hep chain and sequences of charged nitrogen atoms on the polyamine. 相似文献
3.
Isoelectric focusing in polyacrylamide gels 总被引:45,自引:0,他引:45
4.
Combinatorial peptide ligand libraries have been extensively used for the enrichment of very low-abundance proteins, while concomitantly reducing the concentration of major species. A number of biological extracts have been reported with great success. Nevertheless, there are examples where the enrichment was not as good as expected. It has been demonstrated that the protocol itself may be responsible for the final results and is not necessarily applied as it should. In this paper, technical details along with important suggestions are given for an optimized enrichment of gene products present at trace levels among very many other proteins. 相似文献
5.
6.
P Wenger M de Zuanni P Javet C Gelfi P G Righetti 《Journal of biochemical and biophysical methods》1987,14(1):29-43
Amphoteric, isoelectric agarose membranes, as devised by Martin and Hampson [Martin, A.J.P. and Hampson, F. (1978) J. Chromatogr. 159, 101-110], are found unsuitable for blocking electroendosmosis in multi-compartment electrolysers during preparative isoelectric focusing, due to the poor and highly unpredictable incorporation of carboxyls and amino groups on the polysaccharide moiety. New, polyacrylamide-based membranes are described, containing as buffers and titrants the Immobiline chemicals used to produce immobilized pH gradients. These new membranes are supported on both faces by a non-woven polypropylene cloth, a material exhibiting minimal adsorption properties for proteins. Due to the extensively developed Immobiline technology, membranes with highly predictable isoelectric points, well-defined buffering capacity and conductivity can be synthesized at any pH value along the pH 3-10 scale. They are effective in blocking electroendosmosis even when the delta pH on either side of the membrane is as high as 1.5 pH unit. 相似文献
7.
P G Righetti B Barzaghi M Luzzana G Manfredi M Faupel 《Journal of biochemical and biophysical methods》1987,15(3-4):189-198
A modification of the previously described apparatus (Faupel et al. (1987) J. Biochem. Biophys. Methods 15, 147-162), for recycling isoelectric focusing in a segmented immobilized pH gradient, is here reported. The most important improvements are: (1) a horizontal, vs. the previously vertical assembly; (2) a reduction of the thickness of the central flow chamber to 6 mm, vs. the previous 3 cm length and (3) the introduction, at both gel extremities of each Immobiline segment, of polypropylene filters, thus efficiently blocking the gel in situ. The advantages are: (i) the spontaneous removal of air bubbles, which in the vertical apparatus tend to accumulate in the ceiling of the flow chamber and to obstruct the flow of electric current; (ii) a more efficient hydraulic flow with a reduced chance of heating the liquid stream in the flow chamber, due to its reduced length along the separation path and (iii) a reduced risk of gel detachment from the tube walls, due to osmotic swelling caused by focused protein zones in the gel phase and by the fixed Immobiline charges in the polyacrylamide matrix. 相似文献
8.
B Barzaghi P G Righetti M Faupel 《Journal of biochemical and biophysical methods》1987,15(3-4):177-187
A method is described for keeping a constant salt background during protein purification in a segmented immobilized pH gradient. It is based on an external hydraulic flow replenishing the salt loss due to combined electric and diffusional mass transport (similar to the concept of Ribes' steady-state rheoelectrolysis). Such a minimum of ionic strength might be needed for proteins which tend to precipitate and aggregate at or in vicinity of the isoelectric point. However, it is found that any salt level in the sample feed (already at 1 mM concentration) deteriorates transport of non-isoelectric proteins, because of the much larger current fraction carried by the ions themselves as opposed to proteins. In addition, high salt levels in the sample reservoir might form cathodic and anodic ion boundaries, alkaline and acidic, respectively, which might hamper protein migration and even induce denaturation. Thus, when high salt backgrounds are needed in the sample feed, external pH control should be exerted, e.g. with a pH-stat. Three parameters influence protein transport in the segmented IPG chamber: (a) cross-sectional area of the Immobiline membranes; (b) delta pI between the isoelectric protein and the contaminants and (c) salt molarity in the sample reservoir. The first 2 show a positive, the last a negative correlation. 相似文献
9.
Elisabetta Gianazza Francesco Chillemi Marcello Duranti Pier Giorgio Righetti 《Journal of biochemical and biophysical methods》1983,8(4):339-351
A new method for peptide analysis and purification is described, based on isoelectric focusing in immobilized pH gradients. On the analytical scale, the peptide zones can now be revealed by an stain for primary and secondary amino group (e.g. ninydrin, fluorescamine, dansyl chloride) since the buffering species, unlike conventional carrier ampholytes, contain only carboxyl and tertiary amino groups. For preparative purposes, conditions have been described to remove most contaminants (e.g. unreacted monomers, non-cross-linked, short polyacrylamide chains) from the gel matrix before the electrophoretic run. However, ca. 2% of the gel dry mass is still present as extractable material. The focused peptides can be recovered in higly yields (ca. 90%) with a fairly high degree of purity (75%), the contaminants being mostly components eluted from the polyacrylamide gel. 相似文献
10.
Elisa Fasoli Mara Colzani Giancarlo Aldini Attilio Citterio Pier Giorgio Righetti 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(8):1484-1491
Combinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteomes of lemon peels and pulp, of a home-made alcoholic infusion of peels and of a very popular Italian liqueur called “Limoncello”, stated to be an infusion of the flavedo (the outer, yellow skin of lemons). The aim of this study was not only to perform the deepest investigation so far of the lemon peel proteome but also to assess the genuineness of the commercial liqueur via a three-pronged attack. First, different extraction techniques have been used for the characterization of the peel (and additionally of the pulp) proteome, secondly a home-made infusion has been analysed and finally the proteome of the commercial drink was checked. The peel (the flavedo, not the underlying layer called albedo) proteome has been evaluated via prior capture with CPLLs at different pH values (2.2 and 7.2). Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could identify a total of 1011 unique gene products in the peel extracts and 674 in the pulp, 264 proteins in the home-made infusion and just 8 proteins (and protein fragments), together with 12 peptides, in one Italian Limoncello produced in the Sorrento Region, thus proving the genuineness of this product. On the contrary, cheaper Limoncellos were devoid of any protein/peptide, casting doubts on their production from vegetable extracts. This could be the starting point for investigating the genuineness and natural origin of commercial drinks in order to protect consumers from adulterated products. 相似文献