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1.
Column chromatography was used to investigate the interaction of human alpha-fetoprotein and albumin with different immobilized dyes. Binding of alpha-fetoprotein to the dye conjugates was studied at pH 7.0. Between 56 and 93% of the total alpha-fetoprotein applied to the column was recovered in the break-through fractions of the respective runs. Of all the dyes, Cibacron Blue F3G-A adsorbs alpha-fetoprotein most strongly. This interaction clearly depends on the degree of dye substitution of the gel. A relatively weak interaction exists between alpha-fetoprotein and immobilized Procion Red HE-3B. This is used in the purification of alpha-fetoprotein by negative chromatography resulting in a 16.6-fold enrichment of this protein. Human albumin binds tightly to immobilized Cibacron Blue F3G-A as well as to Cibacron Brilliant Blue FBR-P and shows a lower affinity to Procion Blue MX-R. Procion Red dyes, which are structurally different from Cibacron Blue F3G-A are also capable of interacting with serum albumin. The results obtained are discussed in terms of the present theoretical conceptions about dye-protein interactions. 相似文献
2.
Cibacron Blue F3-GA, Basilen Blue E3-G and Procion Red HE-3B are dyes currently used in affinity purification, and are commonly determined by spectrophotometry with limited sensitivity. An assay method is described based on a specific immunochemical recognition of the dyes amplified by a final enzymatic reaction. The sensitivity is close to 1 ng/ml of dye and the method is applicable any time that sensitive and accurate results are necessary. This method has actually been applied with success to the determination of trace amounts of dyes in the presence of affinant protein. The method was also applied to the demonstration of dye leaching from affinity sorbents when treated under acidic and/or alkaline conditions. 相似文献
3.
An extract from porcine muscle containing soluble enzymes has been partitioned between the two liquid phases of an aqueous,
biphasic system consisting of dextran, polyethylene glycol, and water. The influence of polymer-bound triazine dyes (Cibacron
blue F3G-A and Procion yellow HE-3G) on the partition of lactate dehydrogenase and total protein was studied. The effects
of pH and concentrations of polymers and buffer on this so-called affinity partitioning were also determined. The two-phase
systems were used in extraction procedures for purification of lactate dehydrogenase to a specific activity of 456–494 U (7.6–8.4
μkat) per mg protein. The use of these systems for extraction of enzymes in technical scale is discussed. 相似文献
4.
《Journal of membrane science》1998,142(1):13-26
Cibacron Blue F3GA, Procion Red HE-3B and Procion Blue MX-R were immobilized on macroporous chitosan and chitin membranes with concentrations as high as 10–200 μmol/ml membrane. These dyed membranes were chemically and mechanically stable, could be reproducibly prepared, and operated at high flow rates. Human serum albumin (HSA) and bovine serum albumin (BSA) were selected as model proteins, and their adsorption on and desorption from the dyed chitosan membranes investigated. The Cibacron Blue F3GA membranes had a higher protein adsorption capacity, much greater for HSA than BSA, than the other dyed membranes. About 8.4 mg HSA/ml membrane were adsorbed at saturation by Cibacron Blue F3GA–chitosan membranes from a 0.05 M Tris–HCl/0.05 M NaCl, pH 8 solution. The chitin membranes had a lower dye content and hence a lower protein adsorption capacity than the chitosan membranes. The effects of important operation parameters (flow rate, protein concentration and loading) were also investigated. Cibacron Blue F3GA–chitosan membranes were employed for the separation of HSA from human plasma and high purity HSA thus obtained. This suggests that these membranes could be used for large-scale plasma fractionation. 相似文献
5.
Immobilized triazine dye affinity chromatography has been widely used for protein purification. In this paper, cibacron Blue F3G-A was immobilized,through a spacer arm, onto a rigid hydrophilic porous polymer by reacting an epoxy-group-containing poly(vinyl alcohol) with 6-aminohexyl-N'-Cibacron Blue F3G-A,which was obtained by reacting Cibacron Blue F3G-A with excess of 1,6-diaminohexane, in a pH 8.6 buffer. The epoxy-group-containing poly(vinyl alcohol) was prepared by hydrolysis of macroporous crosslinded poly(vinyl acetate),which was synthesized by suspension copolymerization of vinyl acetate and triallyl isocyanurate in the presence of butyl acetate and n-heptane as diluents. The cibacron Blue F3G-A-immobilized poly(vinyl alcohol)was packed in a stainless steel column (250×5 mm I. D.) and the chromatographic behaviors of several proteins (cytochrome c, lysozyme, bovine serum albumin, insulin, and lactate dehydrogenase) were determined. 相似文献
6.
Yan Xu Michele Vitolo Cristina Northfleet de Albuquerque Adalberto Pessoa Jr. 《Applied biochemistry and biotechnology》2003,108(1-3):853-865
To improve the selectivity of glucose-6-phosphate dehydrogenase (G6PDH) extraction by an aqueous two-phase system, a simple
and inexpensive affinity aqueous two-phase system using unbound reactive triazine dyes as ligands was introduced. In a polyethylene
glycol (PEG)/hydroxypropyl starch (PES) system, the unbound free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, partitioned
unevenly in the top PEG-rich phase and thus showed an affinity effect on G6PDH, but no influence on hexokinase. The various
parameters investigated were pH of the system, buffers, molecular weight of PEG, and ligand type and concentration. A two-step
affinity extraction process was established for the purification of G6PDH from baker’s yeast. The total yield of G6PDH was
66.9% and purification factor was 2.35. 相似文献
7.
Thyroxine-binding globulin was isolated from pooled human serum by a two-step method involving affinity chromatography on thyroxine-Sepharose and preparative disc-electrophoresis. The final product was found to be homogeneous by polyacrylamide gel electrophoresis and has a molecular weight of 59,000. Isoelectric focusing resolved the protein into seven bands with isoelectric points ranging from 3.9 to 4.3. The isolated protein showed affinity to a number of different dyes as recognized by affinity phase partitioning. The interaction of the protein with the dye Cibacron Blue F3G-A was found to be strongly competitive with the natural ligand thyroxine. 相似文献
8.
Unsal E Durdu A Elmas B Tuncel M Tuncel A 《Analytical and bioanalytical chemistry》2005,383(6):930-937
In this study, a new affinity high-performance liquid chromatography (HPLC) stationary phase suitable for protein separation
was synthesized. In the first stage of the synthesis, uniform porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(HEMA-co-EDM), beads 6.2 μm in size were obtained. Homogeneous distribution of hydroxyl groups in the bead interior was confirmed
by confocal laser scanning microscopy. The plain poly(HEMA-co-EDM) particles gave very low non-specific protein adsorption with albumin. The selected dye ligand Cibacron blue F3G-A (CB
F3G-A) was covalently linked onto the beads via hydroxyl groups. In the batch experiments, albumin adsorption up to 60 mg
BSA/g particles was obtained with the CB F3G-A carrying poly(HEMA-co-EDM) beads. The affinity-HPLC of selected proteins (albumin and lysozyme) was investigated in a 25 mm×4.0-mm inner diameter
column packed with CB F3G-A carrying beads and both proteins were successfully resolved. By a single injection, 200 μg of
protein was loaded and quantitatively eluted from the column. The protein recovery increased with increasing flow rate and
salt concentration of the elution buffer and decreased with the increasing protein feed concentration. During the albumin
elution, theoretical plate numbers up to 30,000 plates/m were achieved by increasing the salt concentration. 相似文献
9.
Bead cellulose derivatives as supports for immobilization and chromatographic purification of proteins 总被引:3,自引:0,他引:3
H F Boeden K Pommerening M Becker C Rupprich M Holtzhauer F Loth R Müller D Bertram 《Journal of chromatography. A》1991,552(1-2):389-414
Characteristic data are presented for Divicell, a macroporous bead cellulose with excellent flow parameters. The preparation of Divicell derivatives and their properties are described with respect to their application as chromatographic supports. The ion exchangers Divicell DEAE and Divicell CM were manufactured in two types with different exclusion limits and an available capacity for proteins of up to 100 mg/ml gel. Divicell Blue is a bead cellulose with covalently bound Cibacron Blue F3G-A and was found to be a very suitable adsorbent for the selective separation and purification of human serum albumin. Activation of Divicell with sodium periodate, epichlorohydrin and 5-norbornene-2,3-dicarboximido carbonochloridate provided activated supports used for immobilization of ligands in organic solvents and in aqueous solutions. Coupling of amines, diamines, amino acids, carbohydrates and proteins is described. The immobilized ligands retained their biological activity as determined by their specific adsorption of proteins. Divicell alkyl derivatives were tested in hydrophobic interaction chromatography with bovine serum albumin as a model. Examples are presented of the application of Divicell derivatives to the purification of biomacromolecules such as immunoglobulins and lectins by affinity chromatography. The results were comparable to those obtained using the corresponding Sepharose-derived absorbents. 相似文献
10.
A simple and rapid analytical method for the determination of trace levels of five sulphonated and azo sulphonated reactive dyes: Cibacron Reactive Blue 2 (C-Blue, trisulphonated dye), Cibacron Reactive Red 4 (C-Red, tetrasulphonated azo dye), Cibacron Reactive Yellow 2 (C-Yellow, trisulphonated azo dye), Levafix Brilliant Red E-4BA (L-Red, trisulphonated dye), and Levafix Brilliant Blue E-4BA (L-Blue, disulphonated dye) in water is presented. Initially, the dyes were preconcentrated from 250 ml of water samples with solid-phase extraction using natural zeolite sample previously modified with a microemulsion. The modified zeolite exhibited an excellent extraction for the dyes from solution. The parameters that influence quantitative recovery of reactive dyes like amount of extractant, volume of dye solution, pH, ionic strength, and extraction-elution flow rate were varied and optimized. After elution of the adsorbed dyes, the concentration of dyes was determined spectrophotometrically with the aid of principle component regression (PCR) method without separation of dyes. The results obtained from PCR method were comparable to those obtained from HPLC method confirming the effectiveness of the proposed method. With the aid of SPE by M-zeolite, the concentration of dyes could be reproducibly detected over the range 25-200 ppb for C-Yellow and L-Blue and from 50 to 250 ppb for C-Blue, C-Red, and L-Red. The multivariate detection limits of dyes were found to be 15 ppb for C-Yellow and L-Blue and 25 ppb for C-Blue, C-Red, and L-Red dyes. The proposed chemometric method gave recoveries from 85.4 to 115.3% and R.S.D. from 1.0 to 14.5% for determination of the five dyes without any prior separation for solutes. 相似文献
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染料壳聚糖微球的制备及其对牛血清白蛋白(BSA)吸附性能的研究 总被引:11,自引:0,他引:11
采用反相悬浮交联法制备壳聚糖微球,对微球进行羟丙基氯化及氨基化,并偶联色素配体Cibacron Blue F3GA,得到一种新型染料亲和吸附剂.以牛血清白蛋白(BSA)为目标蛋白,考察了该染料亲和吸附剂的吸附性能,发现其对BSA有较高的吸附量(95.2mg/g),吸附行为满足Langmuir吸附等温式.负载牛血清白蛋白的微球容易洗脱,洗脱率高达99%. 相似文献
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Serap
enel Ahmed Kassab Yakup Arca Ridvan Say Adil Denizli 《Colloids and surfaces. B, Biointerfaces》2002,24(3-4):265-275
Commercially available microporous polyamide hollow fibres are modified by acid hydrolysis to activate the reactive groups and subsequently binding of the ligand, i.e. Cibacron Blue F3GA. Then the Cibacron Blue F3GA-derived hollow fibres were loaded with different metal ions (i.e. Zn(II), Cu(II), Ni(II)) to form the metal chelate. The internal polymer matrix was characterised by scanning electron microscopy. The effects of pH, initial concentration of lysozyme, metal type and temperature on the adsorption of lysozyme to the metal–chelated hollow fibres were examined in a batch reactor. The non-specific adsorption of lysozyme onto the polyamide hollow fibres was 1.8 mg/g. Cibacron Blue F3GA immobilisation increased the lysozyme adsorption up to 62.3 mg/g. Metal–chelated hollow fibres showed a significant increase of the adsorption efficiency. Lysozyme adsorption capacities of Zn(II), Cu(II) and Ni(II)-chelated hollow fibres were different. The maximum capacities of Zn(II), Cu(II) or Ni(II)-chelated hollow fibres were 144.2, 75.2 and 68.6 mg/g, respectively. Significant amount of the adsorbed lysozyme (up to 97%) was eluted in 1 h in the elution medium containing 1.0 M NaSCN at pH 8.0 and 25 mM EDTA at pH 4.9. Repeated adsorption–desorption process showed that this novel metal–chelated polyamide hollow fibres are suitable for lysozyme adsorption. 相似文献
15.
High-performance liquid chromatography was utilized for the purification of bovine alpha-fetoprotein (BAFP) from fetal calf serum (FCS). An initial step in the purification involved absorption of charcoal delipidated FCS on Cibacron Blue F3GA gel. The Cibacron Blue pre-purified FCS was then chromatographed on a Polyanion SI weak anion-exchange column. The BAFP isolated had a purity of greater than 93% with an overall yield of 48% from FCS. The procedure was applicable for semi-preparative scale purification of BAFP. 相似文献
16.
López-Mas JA Streitenberger SA García-Carmona F Sánchez-Ferrer A 《Journal of chromatography. A》2001,911(1):47-53
Interactions between Cibacron Blue F3GA (CB F3GA), as a model of triazine dye, and 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD), as a model of cyclodextrin, were investigated by monitoring the spectral shift that accompanies the binding phenomena. Matrix analysis of the difference spectral titration of CB F3GA with HP-beta-CD revealed only two absorbing species, indicating a host-guest ratio of 1:1. The dissociation constant for this HP-beta-CD-CB F3GA complex, Kd, was found to be 0.43 mM. The data for HP-beta-CD forming inclusion complexes with CB F3GA were used to develop the concept of competitive elution by inclusion complexes in dye-affinity chromatography. When this concept was applied to the elution of L-lactate dehydrogenase from a CB F3GA affinity matrix, it was shown to be an effective elution strategy. It provided a 15-fold purification factor with 89% recovery and sharp elution profile (0.8 column volumes for 80% recovery), which is as good as that obtained by specific elution with NADH (16-fold, 78% recovery and 1.8 column volumes). In addition, the new elution strategy showed a better purification factor and sharper elution profile than traditional non-specific elution with KCl (4.5-fold, and 1.4 column volumes). Hence, competitive elution by inclusion complexes may be a promising strategy for eluting proteins with high recoveries and purification factors in dye-affinity chromatography. 相似文献
17.
The interaction of Cibacron blue F3G-A with two therapeutic proteins, recombinant human growth hormone and recombinant human interferon-alpha2b, has been examined by applying gel-permeation chromatography in combination with the absorption difference spectroscopy. The complexes of these proteins with Cibacron blue F3G-A have been isolated, and their absorbance spectra have been registered. The influence of Cibacron blue F3G-A on the oligomeric state of proteins has been investigated. It was found that Cibacron blue F3G-A promotes the generation of interferon-alpha2b dimers at pH 5.0. 相似文献
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Cibacron Blue F3GA was immobilized on poly(hydroxyethyl methacrylate) cryogel and it was used for selective and efficient depletion of albumin from human serum. The poly(hydroxyethyl methacrylate) was selected as the basic component because of its inertness, mechanical strength, chemical and biological stability, and biocompatibility. Cibacron Blue F3GA was covalently attached to the poly(hydroxyethyl methacrylate) cryogel to produce poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel affinity column. The poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel was characterized with respect to gelation yield, swelling degree, total volume of macropores, Fourier Transform Infrared spectroscopy, and scanning electron microscopy. It was found that the maximum amount of adsorption (343 mg/g of dry cryogel) obtained from experimental results is very close to the calculated Langmuir adsorption capacity (345 mg/g of dry cryogel). The maximum adsorption capacity for poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel column was obtained as 950 mg/g of dry cryogel for nondiluted serum. The adsorption capacity decreased with increasing dilution ratios while the depletion ratio of albumin remained as 77% in serum sample. Finally, the poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel was optimized for using in the fast protein liquid chromatography system for rapid removal of the high abundant proteins from the human serum. 相似文献