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1.
采用金属螯合亲和层析法,纯化了小鼠腹水来源的抗乙肝核心抗原单克隆抗体,对上样缓冲液的pH和离子强度、洗脱液种类和洗脱方式进行优化。结果表明,采用降低pH分步洗脱时,最佳上样缓冲液为pH8.0,20mmol/LPB+0.5mol/LNaCl,抗体在pH5.0被洗脱下来,抗体回收率80%,纯度85%。采用咪唑浓度梯度洗脱时,最佳的上样缓冲液为pH8.0,20mmol/LPB+5mmol/L咪唑,抗体纯度大于95%,回收率65%;在上样缓冲液中不添加NaCl而添加少量的咪唑,更有利于抗体分离。以上洗脱方式都能较好地保持mAb的生物学活性,为该抗体的应用提供了必要的实验基础。 相似文献
2.
Ye X Yoshida S Ng TB 《The international journal of biochemistry & cell biology》2000,32(11-12):1143-1150
A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. beta-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, alpha-lactalbumin could not be adsorbed onto the resin. alpha-Lactalbumin and beta-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. alpha-Lactalbumin and beta-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. alpha-Lactalbumin was eluted using a linear (0-0.15 M) concentration gradient of NaCl in 0.05 M Tris-HCl buffer (pH 8.5). Subsequently, beta-lactoglobulin B and beta-lactoglobulin A were eluted from the column with 0.05 M Tris-HCl (pH 6.8), using a linear (0.1-0.25 M) concentration gradient of NaCl. The yields were 1260 mg alpha-lactalbumin, 1290 mg beta-lactoglobulin B and 2280 mg beta-lactoglobulin A from 1 l rennet whey. 相似文献
3.
Santos MJ Teixeira JA Rodrigues LR 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2011,879(7-8):475-479
A method for the recovery and fractionation of whey proteins from a whey protein concentrate (80%, w/w) by hydrophobic interaction chromatography is proposed. Standard proteins and WPC 80 dissolved in phosphate buffer with ammonium sulfate 1 M were loaded in a HiPrep Octyl Sepharose FF column coupled to a fast protein liquid chromatography (FPLC) system and eluted by decreasing the ionic strength of the buffer using a salt gradient. The results showed that the most hydrophobic protein from whey is α-lactalbumin and the less hydrophobic is lactoferrin. It was possible to recover 45.2% of β-lactoglobulin using the HiPrep Octyl Sepharose FF column from the whey protein concentrate mixture with 99.6% purity on total protein basis. 相似文献
4.
Xiuyun Ye Shigeru Yoshida T. B. Ng 《The international journal of biochemistry & cell biology》2000,32(11-12)
A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. β-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, α-lactalbumin could not be adsorbed onto the resin. α-Lactalbumin and β-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. α-Lactalbumin and β-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. α-Lactalbumin was eluted using a linear (0–0.15 M) concentration gradient of NaCl in 0.05 M Tris–HCl buffer (pH 8.5). Subsequently, β-lactoglobulin B and β-lactoglobulin A were eluted from the column with 0.05 M Tris–HCl (pH 6.8), using a linear (0.1–0.25 M) concentration gradient of NaCl. The yields were 1260 mg α-lactalbumin, 1290 mg β-lactoglobulin B and 2280 mg β-lactoglobulin A from 1 l rennet whey. 相似文献
5.
Makoto Shimizu Takamoto Kamiya Kunio Yamauchi 《Bioscience, biotechnology, and biochemistry》2013,77(11):2491-2496
Whey proteins adsorbed on fat globule surfaces during emulsification with coconut oil at pH 3 ~ 9 were examined. The amount of proteins adsorbed on the fat surface was dependent on the pH during emulsification. At any pH examined here, however, tightly-adsorbed proteins which were not extracted from the fat surface with urea or guanidine-HCl were 2 ~ 3 mg/m2. Marked selectivities in the adsorption of individual whey proteins were observed at any pH. No correlation between the adsorbabilities and the surface hydrophobicities of individual whey proteins was observed. Whey proteins adsorbed on the emulsified fat were much more easily digested with proteases compared to the native whey proteins, indicating that conformational changes of whey proteins occurred at the fat surface. The results suggested that conformational properties, such as flexibility of the structure, of whey proteins are important in the adsorption and possibly affect their emulsifying ability. 相似文献
6.
Bovine lactoferrin was enriched in various whey samples by affinity chromatography using immobilized gangliosides. Bovine gangliosides were isolated from fresh buttermilk using a combination of ultrafiltration and organic extraction. Isolated gangliosides were covalently immobilized onto controlled-pore glass beads. The immobilized matrix contained 66 micrograms of gangliosides per gram of beads. After loading the matrix with reconstituted whey protein isolate (WPI) or whey protein concentrate (WPC), the matrix was washed with sodium phosphate buffer (pH 7) followed by sodium acetate buffer (pH 4) before elution of lactoferrin with 1 M NaCl in sodium acetate buffer. From the intensities of the protein bands in SDS-PAGE, lactoferrin constituted a minimum of 40% of the total protein in the salt eluted sample. WPI, pretrated by heating and ultrafiltration, showed the highest lactoferrin purity among protein sources, while WPI (10% wt/vol) showed the highest recovery. These results show that immobilized gangliosides can be used to enrich the lactoferrin content of whey. 相似文献
7.
Glenda M. Roberson Larry D. Barnes 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,544(1):20-28
Cyclic AMP and cyclic GMP can be separated from thymidine and its possible metabolites, electrolytes, and polyvalent nucleotides using columns of acidic alumina. Electrolytes and thymidine are not adsorbed on acidic alumina at pH 4.4 while cyclic nucleotides and polyvalent nucleotides are adsorbed at this pH. Cyclic AMP and cyclic GMP are eluted together from acidic alumina with 0.2 M ammonium formate (pH 6.0) and the polyvalent nucleotides remain adsorbed. The cyclic nucleotides are separated by chromatography on Dowex AG 1 X 8 resin. Recovery is 60–64% for cyclic AMP and cyclic GMP isolated from renal tissue samples. This methodology permits the separation of tritiated thymidine from cyclic nucleotides which are present in tissue preparations used in studies on the role of cyclic nucleotides in cellular growth. 相似文献
8.
Cyclic AMP and cyclic GMP can be separated from thymidine and its possible metabolites, electrolytes, and polyvalent nucleotides using columns of acidic alumina. Electrolytes and thymidine are not adsorbed on acidic alumina at pH 4.4 while cyclic nucleotides and polyvalent nucleotides are adsorbed at this pH. Cyclic AMP and cyclic GMP are eluted together from acidic alumina with 0.2 M ammonium formate (pH 6.0) and the polyvalent nucleotides remain adsorbed. The cyclic nucleotides are separated by chromatography on Dowex AG 1 X 8 resin. Recovery is 60--64% for cyclic AMP and cyclic GMP isolated from renal tissue samples. This methodology permits the separation of tritiated thymidine from cyclic nucleotides which are present in tissue preparations used in studies on the role of cyclic nucleotides in cellular growth. 相似文献
9.
《The International journal of biochemistry》1984,16(6):587-591
- 1.1. A relatively rapid, high-resolution Chromatographic procedure, using mini-columns of DEAE cellulose equilibrated with 10mM sodium phosphate, pH 6.0, is described in sufficient detail to permit ready replication.
- 2.2. This initial paper demonstrates the quantitative separation, using suction, of the acidic nucleosides, 3-deazauridine and 6-azauridine, from their phosphorylated derivatives.
- 3.3. The chemically stable, tritium-labeled nucleosides are eluted from the mini-columns (capacity ≈ 1.8 ml) with 10mM sodium phosphate, pH 6.0; subsequently, the nucleotides are eluted completely with 0.5 M HCl/0.5 M NaCl.
- 4.4. Quantitation is based on liquid scintillation counting of aliquots of the eluates.
10.
Chromatography of blood-clotting factors and serum proteins on columns of diatomaceous earth
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MILSTONE JH 《The Journal of general physiology》1955,38(6):743-755
1. In batch adsorptions with prothrombin solutions, hyflo was the weakest adsorbent, standard super-cel intermediate, and filter-cel strongest. Of these three grades of diatomaceous earth, hyflo has the smallest surface area per gram and filter-cel the largest. In parallel breakthrough experiments, a column of standard super-cel had a capacity almost six times that of a hyflo column. 2. After partial removal of impurities by diatomaceous earth, prothrombin preparations contained less thrombokinase, were more stable, and displayed less tendency to form thrombin "spontaneously." Thrombokinase (or its precursor) was removed from a preparation of prothrombin by passage through a filter cake of standard super-cel. The specific activity of the prothrombin was increased; and 62 per cent of the activity was recovered. 3. Prothrombin was adsorbed from an ammonium sulfate solution at pH 5.26 by columns of hyflo or standard super-cel. When eluted by phosphate solutions, the protein moved down the columns more readily at higher pH and higher concentration of phosphate salts, within the pH range 5.0 to 6.6, and within the phosphate range 0.1 to 1.0 M. 4. Thrombin was adsorbed on a column of standard super-cel at pH 5.11. As successive eluents passed through the column, the thrombin emerged between two bands of impurities. The specific activity of the thrombin was raised; and 83 per cent of the activity was recovered. 5. With a column of standard super-cel, and with a series of eluents within the pH range 5.1 to 6.3, total serum proteins were separated into four major bands. About 94 per cent of the protein was recovered. 相似文献
11.
A complex of histones H2A, H2B, H3 and H4 has been isolated from purified rat liver nuclei by a method which is both gentle
and rapid. Nuclei were homogenised in 0.25 M sucrose and the residual nuclear material obtained after centrifligation was
adsorbed on calcium phosphate gel. After removing histone H1 from the adsorbed material by washing with 1M NaCl in 25 mM sodium
phosphate buffer, pH 6.0, histones H2A, H2B, H3 and H4 were eluted together, with 2 M NaCl in 25 mM sodium phosphate buffer,
pH 7.0. The core histones so obtained migrated as a single sharp band on polyacrylamide gel electrophoresis under non-denaturing
conditions. Fractionation of the freshly prepared core histones on a Sephadex G-100 column yielded two major protein peaks.
The peak having the larger elution volume contained histones H2A and H2B in equal amounts while the peak with the smaller
elution volume contained all the four histones. Histones H3 and H4 were present in larger proportions in the second peak. 相似文献
12.
ABSTRACT A previous study1 of the selective solubility of myelin basic protein (MBP) of tissue extracts at pH 9.0 has raised issues of its quantitative recovery, and the differential solubility of its charge isomers. The pH-dependent solubility of proteins of acid extracts of delipidated tissue of bovine spinal cord was therefore reexamined. MBP of whole extracts was completely soluble up to pH 8.0 only, and less so by 25 % at pH 9.0, and 43 % at pH 10.0. The proteins other than MBP were virtually insoluble between pH 5.0 to 6.0, and 9.0 to 10.0. The solubility of the main charge isomers I to III of MBP of 18.5 kDa was found not to be affected by pH. Either pH 5.0 or 9.0 is therefore suitable for the selective isolation of MBP from whole tissue extracts, only pH 5.0 providing for the complete recovery of MBP. The pH-dependent solution behaviour was also examined following the separation of proteins of whole extracts by anion exchange chromatogra phy at pH 10.4. Purified MBP and several related minor cationic components of lower molecular weight were soluble throughout. In contrast, the anionic proteins were only partly soluble between pH 4.0 to 10.0, i.e. by 4 to 20%. The results are consistent with specific protein-protein interactions of the proteins of whole extracts, either enhancing the solubility of non-MBP proteins, e.g. at pH 7.0, or impairing that of MBP between pH 8.0 to 10.0. 相似文献
13.
Non-dialyzable fraction of sweet whey was chromatographed on a column of phenyl-agarose equilibrated with 0.01 M sodium phosphate buffer, pH 6.8 containing 5 M NaCl. Most whey proteins were adsorbed on the column, while the glycomacropeptide (GMP) was not. Amino acid analysis of the GMP fraction showed presence of traces (each < 1 residue/peptide) of arginine, histidine and phenylalanine which are not found in GMP. The estimated yield of GMP fraction was approximately 1.6 g l-1 of sweet whey. 相似文献
14.
In this study we describe the design, preparation and testing of superparamagnetic anion-exchangers, and their use together with cation-exchangers in the fractionation of bovine whey proteins as a model study for high-gradient magnetic fishing. Adsorbents prepared by attachment of trimethyl amine to particles activated in sequential reactions with allyl bromide and N-bromosuccinimide yielded a maximum bovine serum albumin binding capacity of 156 mg g(-1) combined with a dissociation constant of 0.60 microM, whereas ion-exchangers created by linking polyethylene imine through superficial aldehydes bound up to 337 mg g(-1) with a dissociation constant of 0.042 microM. The latter anion-exchanger was selected for studies of whey protein fractionation. In these, crude bovine whey was treated with a superparamagnetic cation-exchanger to adsorb basic protein species, and the supernatant arising from this treatment was then contacted with the anion-exchanger. For both adsorbent classes of ion-exchanger, desorption selectivity was subsequently studied by sequentially increasing the concentration of NaCl in the elution buffer. In the initial cation-exchange step quantitative removal of lactoferrin (LF) and lactoperoxidase (LPO) was achieved with some simultaneous binding of immunoglobulins (Ig). The immunoglobulins were separated from the other two proteins by desorbing with a low concentration of NaCl (< or = 0.4 M), whereas lactoferrin and lactoperoxidase were co-eluted in significantly purer form, e.g. lactoperoxidase was purified 28-fold over the starting material, when the NaCl concentration was increased to 0.4-1 M. The anion-exchanger adsorbed beta-lactoglobulin (beta-LG) selectively allowing separation from the remaining protein. 相似文献
15.
Chromatofocussing was used for the separation of brush-border membrane proteins from calf kidney into 4 or 5 fractions over the pH range 4.0 to 7.4. These fractions were reconstituted into proteoliposomes by gradient centrifugation. Determination of the sodium-dependent solute uptake by proteoliposomes reconstituted from different chromatofocussed fractions showed that the sodium-D-glucose cotransport system was present in the fraction eluted between pH 5.3 and 5.8, and that the sodium-phosphate cotransport was present in fractions eluted between pH 4.6 and 5.3 and between pH 5.8 and 6.6, sodium-alanine cotransport could be detected in almost all fractions. Marker enzymes of the brush-border membrane, such as alkaline phosphatase, gamma-glutamyltransferase and aminopeptidase M etc. were also found to be eluted at pH 7.0-7.4, 4.0-4.1 and 5.6-5.8, respectively. These results suggest that chromatofocussing is a promising tool for the separation of membrane proteins and for pre-purification of the sodium-D-glucose cotransport system. It can be further concluded that the sodium-dependent phosphate transport across the brush-border membrane is not dependent upon alkaline phosphatase activity. 相似文献
16.
J. Szczodrak 《Engineering in Life Science》1999,19(3):235-250
A new low-cost β-galactosidase (lactase) preparation for whey permeate saccharification was developed and characterized. A biocatalyst with a lactase activity of 10 U/mg, a low transgalactosylase activity and a protein content of 0.22 mg protein/mg was obtained from a fermenter culture of the fungus Penicillium notatum. Factors influencing the enzymatic hydrolysis of lactose, such as reaction time, pH, temperature and enzyme and substrate concentration were standardized to maximize sugar yield from whey permeate. Thus, a 98.1% conversion of 5% lactose in whey permeate to sweet (glucose-galactose) syrup was reached in 48 h using 650 β-galactosidase units/g hydrolyzed substrate. After the immobilization of the acid β-galactosidase from Penicillium notatum on silanized porous glass modified by glutaraldehyde binding, more than 90% of the activity was retained. The marked shifts in the pH value (from 4.0 to 5.0) and optimum temperatures (from 50°C to 60°C) of the solid-phase enzyme were observed and discussed. The immobilized preparation showed high catalytic activity and stability at wider pH and temperature ranges than those of the free enzyme, and under the best operating conditions (lactose, 5%; β-galactosidase, 610–650 U/g lactose; pH 5.0; temperature 55°C), a high efficiency of lactose saccharification (84–88%) in whey permeate was achieved when lactolysis was performed both in a batch process and in a recycling packed-bed bioreactor. It seems that the promising results obtained during the assays performed on a laboratory scale make this immobilizate a new and very viable preparation of β-galactosidase for application in the processing of whey and whey permeates. 相似文献
17.
A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms 总被引:4,自引:0,他引:4
As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine. 相似文献
18.
Arakawa T Philo JS Tsumoto K Yumioka R Ejima D 《Protein expression and purification》2004,36(2):244-248
Acidic pH is commonly used to elute antibodies from Protein-A affinity column, although low pH may result in aggregation of the proteins. As an alternative, here arginine was tested as an eluent and compared with a more conventional eluent of citrate. Using purified monoclonal antibodies, recovery of antibodies with 0.1M citrate, pH 3.8, was less than 50% and decreased further as the pH was increased to 4.3. At the same pH, the recovery of antibodies was greatly increased with 0.5M arginine and more so with 2M arginine. Even at pH 5.0, 2M arginine resulted in 31% recovery, although the elution under such condition showed extensive tailing. Such tailing was observed at pH 3.8 when 0.1M citrate was used. Size exclusion analysis indicated that the eluted antibodies were mostly monomeric whether eluted with citrate or arginine. This demonstrates the usefulness of arginine as an efficient eluent for Protein-A chromatography. 相似文献
19.
Bovine transferrin (BTF) was fractionated from bovine whey using ganglioside affinity chromatography. After loading the immobilized matrix with a 2% whey solution, the matrix was washed with sodium acetate buffer at pH 4 containing 1 M NaCl before elution of BTF with sodium phosphate buffers at pH 7. Concanavalin-A affinity and ion exchange chromatography were used for further purification. The ganglioside column showed a 74.2% BTF recovery from whey and BTF was enriched to 61% purity with ion exchange chromatography. Bovine transferrin was identified by SDS-PAGE and western analysis. The Concanavalin-A affinity and ion exchange chromatography steps enriched BTF in the samples and removed other whey proteins from ganglioside purified fractions. These results indicate that immobilized ganglioside can be used to fractionate BTF from bovine whey. Our novel ganglioside affinity chromatography is rapid and efficient for the fractionation of BTF from whey. 相似文献
20.
The selective separation of whey proteins was studied using colloidal gas aphrons generated from the cationic surfactant cetyl trimethyl ammonium bromide (CTAB). From the titration curves obtained by zeta potential measurements of individual whey proteins, it was expected to selectively adsorb the major whey proteins, i.e., bovine serum albumin, alpha-lactalbumin, and beta-lactoglobulin to the aphrons and elute the remaining proteins (lactoferrin and lactoperoxidase) in the liquid phase. A number of process parameters including pH, ionic strength, and mass ratio of surfactant to protein (M(CTAB)/M(TP)) were varied in order to evaluate their effect on protein separation. Under optimum conditions (2 mmol/l CTAB, M(CTAB)/M(TP) = 0.26-0.35, pH 8, and ionic strength = 0.018 mol/l), 80-90% beta-lactoglobulin was removed from the liquid phase as a precipitate, while about 75% lactoferrin and lactoperoxidase, 80% bovine serum albumin, 95% immunoglobulin, and 65% alpha-lactalbumin were recovered in the liquid fraction. Mechanistic studies using zeta potential measurements and fluorescence spectroscopy proved that electrostatic interactions modulate only partially the selectivity of protein separation, as proteins with similar surface charges do not separate to the same extent between the two phases. The selectivity of recovery of beta-lactoglobulin probably occurs in two steps: the first being the selective interaction of the protein with opposite-charged surfactant molecules by means of electrostatic interactions, which leads to denaturation of the protein and subsequent formation and precipitation of the CTAB-beta-lactoglobulin complex. This is followed by the separation of CTAB-beta-lactoglobulin aggregates from the bulk liquid by flotation in the aphron phase. In this way, CGAs act as carriers which facilitate the removal of protein precipitate. 相似文献