Gel Chromatography (Total 375086 Papers Found)

Human erythropoietin, derived from urine of patients with aplastic anemia, has been purified to apparent homogeneity. The seven-step procedure, which included ion exchange chromatography, ethanol precipitation, gel filtration, and adsorption chromatography, yielded a preparation with a potency of 70,400 units/mg of protein in 21% yield. This represents a purification factor of 930. The purified hormone has a single electrophoretic component in polyacrylamide gels at pH 9, in the presence of sodi ...
The "prohormone thiol protease" (PTP) from adrenal medullary chromaffin granules has been demonstrated as a novel cysteine protease that converts the model enkephalin precursor, ([35S]Met)-preproenkephalin, to appropriate enkephalin related peptide products [Krieger, T. J., & Hook, V. Y. H. (1991) J. Biol. Chem. 266, 8376-8383; Kreiger, T. J., Mende-Mueller, L., & Hook, V. Y. H. (1992) J. Neurochem. 59, 26-31; Azaryan, A. V., & Hook, V. Y. H. (1994) FEBS Lett. 341, 197-202]. In this report, PTP ...
Nuclear factor-kappa B (NF-kappaB) is a universal transcription factor that participates in induction of a wide variety of cellular genes. In nonstimulated cells, NF-kappaB is sequestered in the cytoplasm. However, little is known about where NF-kappaB is located. We have studied the effect of inducing a reorganization of the actin filament system on NF-kappaB distribution, using normal and E1A+cHa-ras-transformed rat fibroblasts. This paper demonstrates that the p65/RelA subunit of NF-kappaB in ...
Two cysteine proteinase inhibitors I and II were purified from goat kidney using alkaline denaturation, ammonium sulphate fractionation, gel filtration on Sephadex G-75 and ion exchange chromatography on DEAE cellulose. The purified inhibitors were homogenous and showed a single band on SDS PAGE under reducing and non-reducing conditions with an apparent molecular mass of 67 kDa. The cystatin forms were stable in the range of pH 3-10 and up to 95 degrees C. Immunological identity with the sheep ...
In an effort to identify life-stage specific Boophilus microplus proteins that elicit a humoral response in cattle, soluble proteins were extracted from 10- to 14-day-old larvae and subsequently fractionated by size-exclusion chromatography and reverse-phase high pressure liquid chromatography. Several antigens were identified by Western blotting as potentially shared with other ixodid tick species since antibodies to these proteins were present in sera of calves not previously exposed to B. mic ...
Inter-strain and inter-species inhibition mediated by a bacteriocin-like inhibitory substance (BLIS) from a pathogenic Vibrio harveyi strain VIB 571 was demonstrated against four isolates of the same species, and one culture each of a Vibrio sp., Vibrio fischeri, Vibrio gazogenes and Vibrio parahaemolyticus. The crude BLIS, which was obtained by ammonium-sulphate precipitation of the cell-free supernatant of a 72 h broth culture of strain VIB 571, was inactivated by lipase, proteinase K, pepsin, ...
The levels of cytochrome P-450, cytochrome b5, aminopyrine N-demethylase, and benzo[a]pyrene hydroxylase were depressed in hepatic microsomes following treatment of mice with the interferon inducer poly rI.rC. The decrease in the hepatic mixed function oxidase system was accompanied by an increase in the incorporation of amino acids into total microsomal protein. Fractionation of solubilized microsomes using a Sephacryl S-200 gel filtration column demonstrated that the increase in amino acid inc ...
Steady-state analyses were performed on the proopiomelanocortin (POMC)-related end-products present in acid extracts of the pars intermedia of the anuran amphibian, Bombina orientalis. Sephadex G-75 gel filtration chromatography indicated that immunoreactive alpha-MSH-sized material and N-acetylated beta-endorphin-related material are the major POMC-related products present in this tissue. The alpha-MSH-sized immunoreactivity was further fractionated by reversed phase HPLC. The major peak of imm ...
Binding proteins for the polycyclic aromatic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) have been purified from C57B1/6J mouse liver. Following affinity chromatography on aminopyrene-Sepharose, a single polypeptide of 29,000 daltons was isolated. The photolabile compound 1-azidopyrene was developed as a photoaffinity labeling agent to identify the protein during its purification. 1-Azidopyrene was found to be a competitive inhibitor of [3H]B[a]P binding. Affinity labeling studies with [3H]-1- ...
A psychrotolerant bacterial strain of Serratia marcescens, originally isolated from a glacial site in Indian Himalayan Region (IHR), has been investigated for laccase production under different culture conditions. The bacterial strain was found to grow between 4 to 45°C (opt. 25°C) and 3 to 14 pH (opt. 5 pH) on prescribed growth medium, coinciding with production of laccase in laccase producing medium. However, the production of laccase was more consistent toward alkaline pH. Laccase enzyme wa ...
Cereal Chem. 78(4):442–446 In this study, 3% aqueous high-amylose maize starch (Hylon VII) dispersions were heated to temperatures of 140–165°C. The onset and rate of gel formation was observed using a small-strain oscillation rheometer as a function of temperature from 90 to 25°C. The gel formation clearly began earlier in high-amylose starch paste preheated at lower temperatures, but the rate of gelation was slower and the resulting gel was weaker in comparison with starch pastes preheat ...
The first bacterial nonheme type bromoperoxidase has been purified to homogeneity from the chlorotetracycline-producing actinomycete Streptomyces aureofaciens Tü 24. Purification was accomplished by (NH4)2SO4 precipitation, DEAE-cellulose chromatography at different pH-values, and molecular sieve chromatography. The purified enzyme has a molecular mass of 90 to 95 kDa based on ultracentrifugation and gel filtration. The enzyme is composed of three subunits of identical molecular mass (m = 31 kD ...
As a therapeutic agent in thrombosis the fibrinolytic enzymes are of interest and the search for a new enzyme continues. A novel fibrinolytic enzyme was produced from Rhizopus chinensis 120, which was screened from the starter for brewing rice wine in the South of China, by solid fermentation, and purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange and gel filtration chromatographies. The purified enzyme hydrolyzed fibrin, it cleaved the alpha-, beta- and gam ...
A high molecular form of bovine factor X has been isolated from freshly collected bovine blood by BaSO4 absorption, exhaustive washing with 0.001 M BaCl2 and chromatographed on DEAE-cellulose column employing a linear salt gradient. This isolated factor X showed a single protein band on analytical polyacrylamide gel disc electrophoresis. Only one single protein peak was observed in the chromatogram of DEAE-Sephadex A-50 chromatography conducted at 3 degrees C. Sedimentation equilibrium analysis ...
Seven fractions of GTP-binding proteins separated by gel filtration of an extract of epicotyls of Pisum sativum seedlings were partially characterized. Seven fractions of GTP-binding proteins tentatively designated GP1 to GP7 had the capacity to be ADP-ribosylated by pertussis toxin. Pooled fractions of GP2 to GP7 showed Km values 2, 20, 50, 10, 3 and 1 nM, respectively. The binding of [35S]GTP gamma S to GTP-binding proteins was prevented competitively in the presence of 0.1 mM GTP and also pre ...
one-third of this protein to be in molecular weight range 20,000-40,000 (fraction BJC). BJC, prepared from 9 liters of urine by gel filtration, was chromatographed on carboxymethylcellulose. Two proteins were eluted from the resin in pure form (as shown by zone and immunoelectrophoresis) in yields representing 8 and 3 mg/liter of urine: BjC1 and BJC2. Their amino acid compositions were identical. BJC1 conitained 61% carbohydrate (33% hexose, 11% sialic acid, 13% glucosamine, 5% galactosamine). R ...
Penicillin kills susceptible bacteria by specifically inhibiting the transpeptidase that catalyzes the final step in cell wall biosynthesis, the cross-linking of peptidoglycan. It was hypothesized (Tipper, D., and Strominger, J. (1965) Proc. Natl. Acad. Sei. U. S. A. 64,11331141) that 1) penicillin is a structural analog of the acylD-alanyl-D-alanine terminus of the pentapeptide side chains of nascent peptidoglycan, and that 2) penicillin, by virtue of its highly reactive /”ctam structure, irr ...
To investigate the effect of nanoparticulate impurities (NPIs) isolated from pharmaceutical-grade sucrose, on the stability of monoclonal antibodies (mAbs). NPIs were purified from pharmaceutical-grade sucrose and spiked into trastuzumab, rituximab, infliximab, and cetuximab formulations. The stability of the mAbs as a function of storage time, temperature, and NPI concentration was assessed by visual inspection, flow-imaging microscopy, nanoparticle tracking analysis, size-exclusion chromatogra ...
A unique property of human mast cell tryptase is its spontaneous inactivation, which may be relevant to the regulation of the activity of this enzyme in vivo. We have found, using size-exclusion chromatography, that the dissociation of the tetrameric active enzyme into the inactive monomer occurred immediately from the beginning of the inactivation process and at a rate significantly faster than that of the appearance of the inactive, tetrameric form. Eventually, a relatively long-lived state of ...
The potent platelet-activating factor isolated from the venom of Crotalus durissus cascavella is an acid-soluble multisubunit glycoprotein of Mr 72,000 built up of two types of subunits, alpha and beta, linked by disulphide bonds. The mean apparent Mr of the reduced complex was around 12,000 by gel filtration under denaturating conditions. The Mrs of the alpha and beta subunits, with an apparent ratio of 1/1, were 12,600 and 13,580 by SDS-PAGE respectively. The Mr 72,000 glycoprotein is thought ...
An endonuclease named DNase gamma was purified to apparent homogeneity from rat splenocyte nuclei and its properties were characterized. We also determined the NH2-terminal and partial amino acid sequences of the proteolytic internal peptides. The molecular mass of gamma DNase was 33,000 daltons as determined by SDS-polyacrylamide gel electrophoresis. A native molecular mass of 30,000 was estimated by gel filtration. Purified DNase gamma is active in the presence of both Ca2+ and Mg2+ or Mn2+ al ...
Using analytical PAAG-electrophoresis two specific proteins, D1 and D2, were found in decidual tissue of pregnant women (6-12 weeks). These proteins were isolated and purified by means of ammonium sulfate saturation, chromatography on Sepharose 6B and ion exchange chromatography on DEAE 52 cellulose. The degree of purification of both D1 and D2 proteins was 100% and 81%, respectively. The both proteins were free of DNA, RNA and polysaccharides. The pI values of D1 and D2 proteins were at pH 5.6- ...
OBJECTIVE To study the specific targeting cytotoxicity mediated by an anti-CD(3)/anti-CD(20) diabody. METHODS The diabody was purified by affinity chromatography and identified by Western blot assay, size exclusion chromatography, FACS and rosetting assay; the effect of the anti-CD(3)/anti-CD(20) diabody mediated lysis of CD(20)-expressing tumor cells was assayed by (51)Cr release assay in vitro and by human B cell tumor nude mice xenograft model in vivo. RESULTS The anti-CD(3)/anti-CD(20) d ...
The research on protein-polymer conjugates by grafting from the surface of proteins has gained significant interest in the last decade. While there are many studies with globular proteins, membrane proteins have remained untouched to the best of our knowledge. In this study, we established the conjugate formation with a class of transmembrane proteins and grow polymer chains from the ferric hydroxamate uptake protein component A (FhuA; a β-barrel transmembrane protein of Escherichia coli). As t ...
The glomerular filtration barrier (GFB) is commonly conceived as a negatively charged sieve to proteins. Recent studies, however, indicate that glomerular charge effects are small for anionic, carboxymethylated (CM) dextran vs. neutral dextran. Furthermore, two studies assessing the glomerular sieving coefficients (θ) for negative CM-Ficoll vs. native Ficoll have demonstrated an increased glomerular permeability for CM-Ficoll (Asgeirsson D, Venturoli D, Rippe B, Rippe C. Am J Physiol Renal Phys ...
When liver cytosol prepared from rats administered [14C]3'-methyl-N,N-dimethyl-4-aminoazobenzene was subjected to Sephadex gel chromatography, four peaks (I-IV) of radioactivity containing proteins and one peak (V) of radioactivity devoid of protein were obtained. Forty to fifty-five per cent of the radioactivity in the protein peaks was butanol-extractable. When the protein peaks were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, over 90% of the radioactivity was separ ...
Plasminogen activators from prostate tissue were purified to apparent homogeneity by a procedure involving reverse ammonium sulfate gradient solubilization, chromatography on gelatine-Sepharose, gel filtration on Sephadex G-150, and chromatography on Con A-Sepharose as a final step. Two activators were obtained. The predominant one exhibited physicochemical, immunochemical and functional properties indistinguishable from human urinary high molecular weight urokinase. The other one, which amounte ...
The Notch signaling pathway is essential for embryonic development, organogenesis, and tissue homeostasis. Aberrant Notch signaling is associated with several types of cancers. The active form of Notch receptor is its intracellular domain (NICD), which is released from the cell membrane by serial proteolytic cleavages following ligand binding. Dose-dependent effects of NICD on cellular phenotypes have been observed under several conditions although the underlying mechanisms have not been well st ...
The molecular size and stability of the nerve growth factor (NGF) secreted in culture by L cells have been studied by sedimentation and gel filtration chromatography. Results indicate that L cell NGF has a molecular weight of 160,000. It contains as part of its structure a protein component that is biologically, immunologically, and electrophoretically indistinguishable from the biologically active factor purified from mouse submandibular glands. However, unlike pure gland NGF, L cell NGF is hig ...
Non-starch polysaccharides of low (1.062 – 1.070) and high (1.102 – 1.106) specific gravity Netted Gem (Russet Burbank) potatoes and their fries were separated into high, intermediate and low molecular weight fractions by gel filtration and tested for their chemical composition. Polysaccharides in all three fractions were composed mainly of glucose (75.5 – 84.5%) and some fructose (9.5 – 14.5%) and galactose (6.0 – 11.0%). Results indicated that chemical composition of various molecula ...
The 67-kD laminin receptor (67LR) is a cell membrane-associated molecule exhibiting high affinity for the basement membrane glycoprotein, laminin. While export of the 67LR toward the extracellular matrix has been recently suggested by electron microscopy studies, there is to date no evidence of shedding of the 67LR from cells. Using two monoclonal antibodies directed against the 67LR, we developed a double-determinant radioimmunoassay that demonstrates that the 67LR is released from cancer cells ...
Human IgG changed molecular size upon mild reduction and alkylation as shown by HPLC gel filtration. IgG1, IgG2 and IgG4 proteins increased in molecular size while IgG3 proteins were decreased in molecular size by this treatment. Several proteins within each subclass covering different light chain types and Gm types were tested all showing the same effect. A plausible explanation was related to the hinge and to the CH2 region since Fab fragments experienced unchanged molecular size irrespective ...
Human platelets separated from platelet rich plasma (PRP) by two different methods: gel filtraton and centrifugation on albumin gradient have been compared for yield, cellular and protein contamination, ultra-structure, platelet populations, shape change, aggregability and functional preservation. A new method for the separation of platelets from total blood on metrizamide gradients has been established. This technique is rapid and easy; it avoids the initial centrifugation of the PRP where abou ...
Free Sm was purified by gel filtration and anti-Sm affinity chromatography. Using this purified antigen, an ELISA for anti-Sm was performed. Three hundred and fifty patients with various rheumatic diseases were studied with respect to immunoglobulin classes of anti-Sm by ELISA. A high frequency of IgG anti-Sm was specifically detected in patients with systemic lupus erythematosus (SLE), but IgA and IgM anti-Sm showed a low frequency and also was detected in other diseases. In patients with SLE, ...
beta-Galactosidase from T. cornutus was resolved into two activity peaks by gel filtration column chromatography. The pH optima of the two peaks designated P1 and P2, were 5.5 and 3.0, respectively, when p-nitrophenyl-beta-D-galactopyranoside was used as the substrate. The molecular weights of P1 and P2 were 700,000 +/- 70,000 and 78,000 +/- 7800, respectively, when estimated by gel filtration chromatography. The activities of both forms of the enzymes are stimulated by anions such as Cl-, Br- a ...
A persistent puzzle in our understanding of hemostasis has been the absence of hemorrhagic symptoms in the majority of patients with Hageman trait, the hereditary deficiency of Hageman factor (factor XII). One proposed hypothesis is that alternative mechanisms exist in blood through which plasma thromboplastin antecedent (PTA, factor XI) can become active in the absence of Hageman factor. In order to test this hypothesis, the effect of several proteolytic enzymes, among them thrombin, plasma kal ...
Major histocompatibility complex (MHC) class II molecules are heterodimers formed by noncovalent linkage of alpha and beta chains. It has been shown that the subunits of the MHC class II molecules IAd and IEk bind antigenic peptides as well as antigenic peptides labeled with fluorescent probes. Laser scanning fluorescence microscopy on SDS/polyacrylamide gels demonstrates that the subunit-peptide complexes of IAd are stable over a wide pH range. Below pH 5.3 the heterodimer of IAd dissociates in ...
The incorporation of L-[U-14C]lysine into the Q and /3 chains of hemoglobin has been compared in acetylphenylhydrazineinduced rabbit reticulocytes and normal and acetylphenylhydrazine-induced rabbit marrow. Evidence for a pool of Q chains and a/3 dimers in both reticulocytes and marrow cells was found in (a) an LY: /3 specific activity ratio of less than 1.0 in purified hemoglobin, (b) the relative losses of a! and /3 chain radioactivity on purification of the hemoglobin, (c) the radioactivity e ...
Continuous Vero cell lines are more suitable for large-scale production of rabies vaccine. The purification of Vero cell-derived rabies vaccine is critical because of the residual cellular DNA and serum proteins. The perfection of techniques using column chromatography with different matrix material, gel filtration and zonal centrifugation is of paramount importance for the optimal purification of rabies vaccine, leaving minimal residual cellular DNA, below the permissible level of 100 pg per do ...
Walker carcinoma cell lines sensitive or resistant to bifunctional alkylating agents have been found to contain multiple forms of cyclic AMP phosphodiesterase (3':5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17). These activities have been resolved using Sepharose 6B gel filtration and their apparent molecular weights have been estimated. The enzyme appears to occur in four active forms of apparent mol. wts of greater than 1 000 000, 430 000, 350 000 and 225 000, when assayed at low substrate ...
A neuropeptide Y (NPY)-like peptide has been localized in the frog brain by the direct immunofluorescence method. Subcellular localization of NPY has been studied by PAP immunocytochemistry and the immunogold technique. Using a specific radioimmunoassay technique, NPY-like peptide has been quantified in various brain areas and the immunoreactive material was characterized by Sephadex G-50 gel filtration and high performance liquid chromatography. The results indicate that frog NPY is closely rel ...
The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser des ...
Beta-amyloid (Abeta) is a major protein component of senile plaques in Alzheimer's disease, and is neurotoxic when aggregated. The size of aggregated Abeta responsible for the observed neurotoxicity and the mechanism of aggregation are still under investigation; however, prevention of Abeta aggregation still holds promise as a means to reduce Abeta neurotoxicity. In research presented here, we show that Hsp20, a novel alpha-crystallin isolated from the bovine erythrocyte parasite Babesia bovis, ...
One endopeptidase (EP1) and at least three aminopeptidases (AP1, AP2, and AP3) were discovered in the stroma of chloroplasts isolated from pea seedlings (Pisum sativum L.), and purified over 100-fold. EP1 requires added Mg(2+) or Ca(2+) for activity, may have an additional tightly bound metal atom, and is inhibited by sulfhydryl reagents but not by serine residue-directed inhibitors. It is reversibly inhibited by dithiothreitol. Its specificity is for the bond between two adjacent Ala or Gly res ...
Antenna polypeptides from two species of the family Ectothiorhodospiraceae have been investigated. By means of gel filtration and subsequent high-performance liquid chromatography, at least five polypeptides were isolated from each of Ectothiorhodospira halochloris and Ectothiorhodospira halophila. The majority of their primary structures was identified by Edman degradation. Comparison of these polypeptide sequences with the known primary structures of antenna polypeptides from various purple no ...
A GMP-compliant process is described for producing F5cys-PEG-lipid conjugate. This material fuses with preformed, drug-loaded liposomes, to form "immunoliposomes" that bind to HER2/neu overexpressing carcinomas, stimulates drug internalization, and ideally improves the encapsulated drug's therapeutic index. The soluble, single-chain, variable region antibody fragment, designated F5cys, was produced in E. coli strain RV308 using high-density cultures. Affinity adsorption onto horizontally tumbled ...
The properties were investigated of low molecular weight factors acting on the G1-S transition of baby rat hepatocytes. These factors were produced by incubating adult rat serum with trypsin or a 100,000 g liver microsomal fraction, and isolated by ultrafiltration. Enzyme degradation assays indicated that the active compound was in both cases a glycopeptide sensitive to pronase and papain and to the combination of neuraminidase and beta galactosidase. No loss of hepatocyte G1-S inhibitory activi ...
A highly purified preparation of a monoclonal antibody to vasoactive intestinal peptide (VIP) was analysed by gel filtration. Three peaks of VIP hydrolysing activity were observed, corresponding to the 150 kDa tetramer IgG, 80 kDa dimer of the heavy and light chains (H-L dimer) and 25 kDa L chain monomer. The hydrolytic activity of all three peaks was removed by adsorption on immobilized anti-mouse IgG. The specific activities (CPM hydrolysed/microgram protein) of the H-L dimer and the L chain m ...
An immunosuppressive peptide fraction was isolated by means of gel filtration, membrane partition, and ion-exchange chromatography from the sera of patients hospitalized for cancer. The resulting peptide fraction, which was heterogeneous as judged by high-voltage electrophoresis, was found to suppress both phytohemagglutinin-induced proliferation of lymphocytes in vitro and the in vivo induction of splenic plaque-forming cells in mice. The specific activity of the peptide fraction, which was iso ...