共查询到20条相似文献,搜索用时 0 毫秒
1.
2.
3.
Purification of antibodies to protein hormones by affinity chromatography on divinylsulfonyl sepharose 总被引:1,自引:0,他引:1
M R Sairam W C Clarke D Chung J Porath C H Li 《Biochemical and biophysical research communications》1974,61(1):355-359
Antibodies to human growth hormone and ovine interstitial cell stimulating hormone have been purified from rabbit antisera by affinity chromatography using a newly developed divinylsulfonyl activated agarose. Elution of the antibodies was accomplished by neutral solutions containing chaotropic ions. 相似文献
4.
J.Martyn Bailey R.W. Bryant S.J. Feinmark A.N. Makheja 《Prostaglandins & other lipid mediators》1977,13(3):479-492
[14C]-labelled thromboxane B2 and hydroxy fatty acids were isolated using thin layer and gas chromatographic procedures from human platelets incubated with [1-14C]-arachidonic acid. A number of TLC solvent systems were evaluated for differential separation of thromboxanes and hydroxy fatty acids from prostaglandins E2, A2, D2 and F2α. Chromatographic properties in nine different solvent systems are tabulated. Two dimensional TLC procedures suitable for complete resolution of mixtures of these compounds on a single plate were developed. The systems were used to demonstrate conversion of [1-14C]-arachidonic acid to thromboxane B2 and prostaglandin E2 by human lung fibroblasts in tissue culture. 相似文献
5.
6.
Effects of operational and system parameters on process scale-up of murine immunoglobulin (IgG2a kappa) purification using Protein A affinity chromatography are investigated. Parameters studied are those related to sample application, elution, ligand concentration on support, and column size change. Between sample application velocities of 0.004 and 0.030 cm/s (16-108 cm/h), the product concentration profiles in eluate did not show significant differences. With given system parameters, the retention time and bandwidth of the peak could be predicted by moment theory. The mean equilibrium constant during elution showed a strong effect of pH between 2 and 4. Within the range of protein A concentrations studied, 0.15-1.22 mg/mL of gel, the column capacity shows a linear relationship with the concentration of protein A immobilized. Dimensional scale-up of the column in the radial direction increased the total purification capacity linearly as the cross-sectional area increased without increasing pressure drop; however, the product concentration was diluted. Scale-up of the column dimension in the axial direction enables higher concentrations of product in the eluate, although the retention time increases linearly as the gel height increases. 相似文献
7.
8.
The testosterone-binding globulin (TeBG) from bovine serum was purified by affinity chromatography and hydroxylapatite chromatography. The affinity column used was prepared by coupling 17 alpha-carboxyethynyl-17-hydroxy-4-androsten-3-one to aminoethyl-Sepharose. The compound was replaceable by 17alpha-carboxyethynyl-17-hydroxy-5alpha-androstan-3-one, but not by testosterone 17-hemisuccinate, estradiol 17-hemisuccinate, or testosterone 3-(O-carboxymethyl)oxime. The TeBG isolated was homogeneous on analytical polyacrylamide gel electrophoresis and equilibrium centrifugation. The protein was a glycoprotein having a molecular weight of 89,500 and a carbohydrate content of 17%. The association constant (M-1) at 4 degrees C was 1.1 X 10(8) and the number of binding sites per molecule was 0.8. Treatment with guanidine-HCl dissociated the protein into subunits having a molecular weight of 28,400 (about one-third of that of the original molecule). SDS-gel electrophoresis showed that two of the three subunits were slightly larger than the other. The dissociation into subunits could also be accomplished by GEDTA treatment with concomitant loss of testosterone-binding activity. The activity and molecular size were reversibly restored by incubation with excess Ca2+. 相似文献
9.
A N Smirnov T A Shchelkunova V V Egorova I G Reshetova E I Chernoburova 《Biulleten' eksperimental'no? biologii i meditsiny》1984,98(9):377-380
A procedure has been developed for purification of a special rat liver estrogen-binding protein. It includes protein precipitation by ammonium sulfate, gel filtration, ion exchange chromatography, and affinity chromatography on estradiol sepharose. The protein is purified 2260-fold with a 27% yield. Upon electrophoresis in 10% PAG in the presence of sodium dodecylsulfate the protein gives one polypeptide strip with a molecular weight of 31.200 +/- 400 dalton. 相似文献
10.
Hitoo Iwase Atsushi Tanaka Yoshiyuki Hiki Tohru Kokubo Takashi Sano Ikuko Ishii-Karakasa Kazunori Toma Yutaka Kobayashi Kyoko Hotta 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,728(2):115
Immunoglobulin A1 (IgA1) from normal human serum is known to have O-linked sugar chains, sialylated Galβ1,3GalNAc, in the hinge portion. In order to reduce the microheterogenity of the sugar chain, the hinge glycopeptide prepared from IgA1 was sequentially treated with neuraminidase and β-galactosidase. The asialo-, agalacto-hinge glycopeptide (HGP-SG) composed of a 33-mer peptide (HP33) and N-acetylgalactosamine (GalNAc) residues was obtained. The HGP-SG was separated into three major peaks, A, B and C, by high-performance liquid chromatography (HPLC). Each glycopeptide fraction was further separated by capillary electrophoresis (CE). Peaks A, B and C with HPLC abundantly contained HP33 bearing five and six N-acetylgalactosamine residues (HGP33-5,6GN), HGP33-4,5GN and HGP33-3,4GN, respectively. Among these glycopeptide peaks, only the HGP33-5GN peak was partly split into two peaks based on the CE analysis – HGP33-5GN-α and -β. The glycopeptide, HGP25-5GN shortened by the thermolysin digest of HGP33-SG was also well separated into the α and β forms by CE analysis. No differences in their mass and peptide portion were observed between HGP25-5GN-α and -β. Therefore, the obtained result might indicate that HGP25-5GN-α was an isomer of HGP25-5GN-β differing in its stereospecific structure of the peptide portion and/or the attachment site of the GalNAc residue. 相似文献
11.
L-histidine coupled to aminohexyl-sepharose (H-AH) has been used as an affinity sorbent to separate IgG from human plasma. Two subclasses IgG1 and IgG2 were specifically bound to histidyl-AH-sepharose at pH 7.4 and eluted using 0.2 M and 1M NaCl. The specificity of the two subclasses were determined by immunoelectrophoresis. Quantitative determination of IgG1, IgG2 was carried out using radial immunodiffusion technique. 相似文献
12.
13.
Malondialdehyde, acetaldehyde, acrolein, and 4-hydroxynonenal are all products of fatty acid oxidation found in the fatty streaks of atherosclerotic arteries due to a lack of antioxidants and an increase in glycation products. Previously identified cross-links derived from these molecules have nearly always required more than one molecule of each type, although this is physiologically less likely than a reaction involving a single molecule. Here we provide indirect but strong evidence for a malondialdehyde-derived cross-link requiring just one malondialdehyde molecule to link arginine and lysine, giving 2-ornithinyl-4-methyl(1epsilon-lysyl)1,3-imidazole following a 4-day incubation of albumin with 8 mm malondialdehyde. This cross-link was identified as its partial degradation product Nepsilon-(2-carboxyl,2-aminoethane)-Nepsilon-methanoyl-lysine by NMR and mass spectrometry. Analysis of plasma from treated diabetic patients revealed that one patient levels had as high as 0.46%, 0.67% of their lysine/arginine residues modified by this cross-link, although others had lower levels. Alkaline hydrolysis of serum albumin also revealed two acid-labile malondialdehyde adducts of histidine in significant quantities, the isomers 4- and 2-ethylidene-histidine. These constituted up to 0.93% of the histidines in treated diabetic patients. Although collagen is readily cross-linked by malondialdehyde, none of these particular products could be found in incubations of collagen with malondialdehyde. 相似文献
14.
Peter Dimroth 《FEBS letters》1982,141(1):59-62
Aspartic acid can be covalently linked to yeast aspartyl-tRNA synthetase and to other proteins, in the absence of tRNA, under conditions where the synthetase activates the amino acid into aspartyl-adenylate, i.e., in the presence of ATP and MgCl2. The linkage between aspartic acid and the protein is acid and alkali resistant; thus it is likely a peptide-like amide bond formed between the activated carboxylate group of aspartic acid and the primary amine function of the side chain of lysine residues. 相似文献
15.
16.
Catherine Grenot Claude-Yves Cuilleron 《Biochemical and biophysical research communications》1977,79(1):274-279
An electrophoretic elution procedure of antibodies retained on affinity columns is described. It afforded a 60% recovery of the binding activity of a high affinity (Ka ~ 1010 M?1) antiserum to 5α-dihydrotestosterone retained on antigen-linked Sepharose 4B affinity columns. These purified unbound antibodies, (Ka ~ 1010 M?1) when applied again on identical antigen-linked affinity columns, were all retained and totally recovered after a new electrophoretic elution. Comparable results were obtained by elution with 1M NH4OH.The residual 40% binding activity remaining on the antigen-linked Sepharose gel after electrophoretic elution was totally recovered by elution with an excess of 5α-dihydrotestosterone. It corresponded to antibodies of higher affinity (Ka ~ 1011 M?1). On the other hand the residual 40% fraction of antibodies resistant to NH4OH elution was denaturated. 相似文献
17.
18.
A beta-D-galactosidase from bovine liver was purified to apparent homogeneity. The major purification step was affinity chromatography on a column of D-galactose attached to a Sepharose support activated with divinyl sulfone. Affinity media prepared by binding ligands to Sepharose activated with cyanogen bromide were unsuitable for purification of the enzyme, even though such media have been used to purify beta-D-galactosidases from other sources. The molecular weight of the denatured enzyme was 67,000. The molecular weight of the native enzyme at pH 7.0 was 68,000, and at pH 4.5 or 5.0, was 141,000. These data suggest that the enzyme has a single, fundamental subunit with a molecular weight of 67,000, and that the enzyme exists as a monomer at pH 7.0, and a dimer at pH 4.5 or 5.0. The Vmax values of the enzyme with p-nitrophenyl beta-D-galactoside, p-nitrophenyl beta-D-fucoside, lactose, and beta-Gal-(1----4)-beta-GlcNAc-1---- OC6H4NO2 -p were 10,204, 11,550, 9,479, and 8,859 nmol/min/mg of protein, respectively, and the Km values for these substrates were 0.08, 14.9, 14.2, and 1.6mM, respectively. D-Galactose, beta-D- galactosylamine , p-aminophenyl 1-thio-beta-D-galactoside, and D- galactono -1,4-lactone were competitive inhibitors of the enzyme, with Ki values of 0.9, 0.6, 0.6, and 0.8mM, respectively. The enzyme catalyzed the transfer of the D-galactosyl group from p-nitrophenyl beta-D-galactoside to D-glucose. The pH optimum of the enzyme was 4.5, and the pI was 4.7. 相似文献
19.
Some immunological studies on prothrombin fragment 1 from bovine prothrombin and its warfarin-induced precursor acarboxyprothrombin are reported. Based on the results, a rapid and simple immunoadsorption method for the isolation of prothrombin fragment 1 in good yield has been established. The method exploits the conformational change induced in the fragment by removal of Ca2+. The principle may be applicable to other gamma-carboxyglutamyl-containing proteins or fragments therof. 相似文献
20.
Yosef I Bloushtain N Shapira M Qimron U 《Applied and environmental microbiology》2004,70(12):7156-7160
We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete lambdaPR or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage lambda Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding beta-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures. 相似文献