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1.
Arakawa T Tsumoto K Ejima D Kita Y Yonezawa Y Tokunaga M 《Journal of biochemical and biophysical methods》2007,70(3):493-498
In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column. 相似文献
2.
Under basic conditions β-ecdysone can be covalently linked to the oxirane residue of epoxy-activated Sepharose 6B. Such an ecdysone-modified column matrix retards antibodies to β-ecdysone while permitting, however, the free passage of other antibodies in rabbit antiserum. The bound anti-ecdysone antibodies can subsequently be eluted by a low pH (3.8), high salt (0.5 m) buffer. The utility of such an affinity column for the isolation of ecdysone receptors is discussed. 相似文献
3.
Levison PR 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,790(1-2):17-33
This review describes the performance of various column designs available to process-scale users of low-pressure chromatography for protein purification. By carrying out a range of ion-exchange separations using Whatman microgranular ion-exchange celluloses we are able to compare and contrast the practical performance issues associated with several designs of axial and radial flow columns. 相似文献
4.
Zatloukalová E Kucerová Z 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,809(1):99-103
BAY 43-9006 is a selective Raf-1 kinase inhibitor with antitumor activity against a variety of human cancers. A highly sensitive HPLC method for determination of BAY 43-9006 in small volumes of serum (30 microl) was developed. Sample preparation involved a liquid-liquid extraction procedure with tolnaftate as internal standard followed by linear gradient elution at a reversed phase C18 column and UV detection. The method was selective and the calibration curves were linear over the concentration range of 80-2000 ng/ml. The intra-day accuracy ranged from 99.9 to 107.6% and the inter-day accuracy from 94.6 to 115%. The lower limit of quantitation (LOQ) was 80 ng/ml with an accuracy of 105.8%. Thus, this method has been validated and can be applied for the drug monitoring or pharmacokinetic studies of BAY 43-9006 in small volumes of serum samples. 相似文献
5.
R Gopalakrishna S H Barsky W B Anderson 《Biochemical and biophysical research communications》1985,128(3):1118-1124
S-100-binding proteins, and calmodulin-binding proteins were isolated from S-100- and calmodulin-depleted bovine brain extract by Ca2+-dependent affinity chromatography using S-100- and calmodulin-coupled Sepharose columns respectively. The majority of the protein (80 to 90%) including calcineurin that bound to S-100 also bound to calmodulin and vice versa, suggesting both proteins may regulate common targets. However these two regulatory proteins also bind few other proteins specific for each. These include cyclic nucleotide phosphodiesterase, 55k, and 220k proteins for calmodulin and 24k, 42k, and 90k proteins for S-100. Certain proteins also specifically bound to S-100 both in Ca2+-dependent and independent ways. In glial cells S-100 protein may replace calmodulin in regulating Ca2+-influenced functions. 相似文献
6.
Dexamethasone (9-fluoro-16α-methyl-116,17,21-trihydroxy-1,4-pregnadiene-3, 20-dione) binding proteins from rat liver cytosol were purified approximately 6470 fold by the use of an affinity column in which deoxycorticosterone was linked to CH-Sepharose 4B through a disulfide linkage. The receptor proteins were eluted from the column by washing with β-mercaptoethanol. A preliminary Sephadex G-200 filtration step of the cytosol was necessary in order to separate the dexamethasone binding proteins from other glucocorticoid receptors. 相似文献
7.
Tsutom Takegami Kazuo Yoshida 《Biochemical and biophysical research communications》1975,67(2):782-789
Cytokinin binding protein from tobacco leaves was isolated and purified to a single protein by means of affinity chromatography on benzyladenine-linked Sepharose column combined with polyacrylamide gel electrophoresis. In vitro binding of this protein to [14C] benzyladenine was inhibited remarkably by cold benzyladenine and kinetin and slightly by adenine, but not adenosine. The molecular weight of the protein was determined to be about 4,000 daltons by gel filtration and SDS polyacrylamide gel electrophoresis. 相似文献
8.
The binding of the E. coli ribosomal proteins L4, L5 and L21 to an erythromycin affinity column has been found to be co-operative. Binding does not occur in the absence of other ribosomal proteins. 相似文献
9.
Poly(beta-L-malic acid) is a cell type-specific polymer of myxomycetes (true slime molds) with the physiological role to organize mobility of certain proteins over the giant multinucleated plasmodia. We have developed an affinity chromatography employing 1,6-diamino-n-hexane-Sepharose-coupled poly(malic acid) to identify such proteins in cellular extracts of Physarum polycephalum. Molecular masses were measured by SDS-PAGE and non-denaturing PAGE after silver staining and/or Western blotting. Protein complexes/subunits were detected by 2-dimensional non-denaturing PAGE/SDS-PAGE. A simplified gel shift experiment displayed binding to fragmented calf thymus DNA. Nuclei were richest in poly(malate) binding proteins followed by cytoplasm and membranes. A protein of 370 kDa dissociated into 11 subunits of 11-29 kDa, indicative of a highly complex protein. This and other proteins displayed binding to nucleic acid in gel shift experiments. Poly(malate) is considered a structural and functional equivalent of long contiguous aspartate repeats in proteins of eukaryotes. 相似文献
10.
The use of affinity chromatography as means of isolating/purifying proteins which have an affinity for benzodiazepine is described. Three such drugs are employed: chlorazepate, clonazepam and delorazepam. The results presented in this paper indicate that the proposed technique only works for chlorazepate and delorazepam. In fact these benzodiazepine-Sepharose derivatives are able to retain specifically proteins from human serum and rat kidney, lung, skeletal muscle and brain. 相似文献
11.
Immobilized-biomembrane affinity chromatography for binding studies of membrane proteins 总被引:1,自引:0,他引:1
Gottschalk I Lagerquist C Zuo SS Lundqvist A Lundahl P 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,768(1):31-40
Analyses of specific interactions between solutes and a membrane protein can serve to characterize the protein. Frontal affinity chromatography of an interactant on a column containing the membrane protein immobilized in a lipid environment is a simple and robust approach for series of experiments with particular protein molecules. Regression analysis of the retention volumes at a series of interactant concentrations shows the affinity of the protein for the interactant and the amount of active binding sites. The higher the affinity, the fewer sites are required to give sufficient retention. Competition experiments provide the affinities of even weakly binding solutes and the non-specific retention of the primary interactant. Hummel and Dreyer size-exclusion chromatography allows complementary analyses of non-immobilized membrane materials. Analyses of the human facilitative glucose transporter GLUT1 by use of the inhibitor cytochalasin B (radioactively labeled) and the competitive substrate D-glucose (non-labeled) showed that GLUT1 interconverted between two states, exhibiting one or two cytochalasin B-binding sites per two GLUTI monomers, dependent on the membrane composition and environment. Similar analyses of a nucleoside transporter, a photosynthetic reaction center, nicotinic acetylcholine receptors and a P-glycoprotein, alternative techniques, and immobilized-liposome chromatographic approaches are presented briefly. 相似文献
12.
13.
T P King 《Biochemistry》1972,11(3):367-371
14.
Zhuang R Zhang Y Zhang R Song C Yang K Yang A Jin B 《Protein expression and purification》2008,59(1):138-143
GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein. 相似文献
15.
16.
Prof. Dr. Eng. S. Furusaki E. Haruguchi Dr. Agr. T. Nozawa 《Bioprocess and biosystems engineering》1987,2(2):49-53
Chromatographic separation of proteins by the gradient elution method using DEAE Toyopearl 650® was carried through. The concentration gradient was effected by changing the ionic strength of NaCl in the carrier buffer solution. Bovine serum albumin and hemoglobin were used as model proteins for separation. The experimental chromatogram was compared with theoretical results of Yamamoto et al. [1, 2]. Adsorption equilibria of the proteins onto the carrier were measured and expressed by a function of the ionic strength. The retention volume and peak width of the resulting chromatogram can be calculated from the equilibrium data using the Yamamoto theory. The calculated results agreed well with the experimental data.The method presented in this paper will be useful to predict the viability of ion-exchange chromatography in protein separation.List of Symbols c kg m–3
concentration in the liquid phase
-
c
s kg m–3
concentration in the solid phase
-
D
s m2 s–1
intraparticle diffusivity
-
d
p m
particle diameter
-
E
z m2 s–1
longitudinal diffusivity of the protein
-
E
z I m2 s–1
longitudinal diffusivity of ionic strength
-
H
/(1 – )
-
I kmol m–3
ionic strength
-
I
O kmol m–3
initial ionic strength
-
I
p kmol m–3
ionic strength at the peak
-
I
s kmol m–3
ionic strength in the solid phase
-
I/V mol (dm3)–2
slope of the ionic gradient elution
-
m
distribution coefficient
-
m
distribution coefficient at I
-
m
I
distribution coefficient for ionic strength
-
Q cm3s–1
flow rate
-
R m
particle radius
-
R
s
degree of separation
-
r m
radial position inside particles
-
t s
time
-
u m s–1
linear velocity
-
V cm3
eluted volume of liquid
-
V
p cm3
eluted volume of liquid at the peak
-
V
T cm3
volume of the packed bed
-
W cm3
peak width
-
Z m
bed height
-
z m
vertical position in the bed
-
z
p m
peak position from the inlet of the bed
-
(t)
delta input at time
-
void fraction
-
1 s
first moment
-
2 s2
second central moment
-
s
superficial space time 相似文献
17.
In proteomic analysis, one of the major limitations is the detection of low-abundance proteins. To detect low-abundance RNA-binding
proteins in mature dry seeds of rice, fractionation by single stranded DNA (ssDNA) affinity column chromatography was carried
out before analysis by two-dimensional gel electrophoresis (2-DE). Proteomic analysis of the ssDNA-binding fraction revealed
the existence of three types of RNA-binding proteins, including a K homology (KH) domain containing protein, a putative RNA-binding
protein and a glycine-rich RNA-binding protein, in mature seeds. In addition, decreases in the putative RNA-binding protein
and glycine-rich RNA-binding protein after absorbing water in seeds appear to be associated with seed germination. 相似文献
18.
R K Scopes 《Analytical biochemistry》1981,114(1):8-18
Quantitative studies on the binding of a variety of enzymes to CM-cellulose have been carried out, and the magnitude of the affinity elution effect in the presence of substrates of the enzymes has been determined. In most cases the weakening of binding in the presence of substrate corresponded closely to the amount expected as a result of the overall charge change, but in a few examples the effect was greater. Some calculations have been made demonstrating the range of strengths of interactions between enzyme and adsorbent, and the energy involved per charge on the protein molecule. 相似文献
19.
Open-air hydrophobic interaction chromatography with alkyl carbon functional groups coupled to agarose beads has been used to desalt large volumes of ammonium sulfate-fractionated bacterial cell lysates. The protein of interest can be simultaneously desalted and concentrated in less than 4 h, and the yield is significantly better than that obtained by the standard technique of precipitation, centrifugation, and dialysis. 相似文献