TY - JOUR. T1 - Immobilization of histidine-tagged proteins by magnetic nanoparticles encapsulated with nitrilotriacetic acid (NTA)-phospholipids micelle. AU - Lim, Yong Taik. AU - Lee, Kun Yeong. AU - Lee, Kwangyeol. AU - Chung, Bong Hyun. N1 - Funding Information: This work was supported by Grant No. RTI04-03-06 from the Regional Technology Innovation Program of the Ministry of Commerce, Industry and Energy (MOCIE), the KETI Research Program (Grant No. 10023796-2005-01), and the KRIBB Research Initiative Program. Copyright: Copyright 2008 Elsevier B.V., All rights reserved.. PY - 2006/6/9. Y1 - 2006/6/9. N2 - We described the development of functionalized magnetic nanoparticles (MNPs) with PEG-modification, a phospholipids micelle coating, and their use in manipulating histidine-tagged proteins. Highly monodisperse MNPs were synthesized in an organic solvent and could be phase-transferred into an aqueous solution by encapsulating the nanoparticles with a phospholipids micelle. The ...

The Cu2+-nitrilotriacetic acid complex improves loading of ?-helical double histidine site for precise distance measurements by pulsed ESR Publication

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Citation: Loveday, K.S., Lugo, M.H., Resnick, M.A., Anderson, B., and Zeiger, E. Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro II: Results with 20 chemicals Environ. Molec. Mutagen. Vol. 13 (1989) 60- ...

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You can now access full text articles from research journals published by CSIR-NISCAIR! Full text facility is provided for all nineteen research journals viz. ALIS, AIR, BVAAP, IJBB, IJBT, IJCA, IJCB, IJCT, IJEB, IJEMS, IJFTR, IJMS, IJNPR, IJPAP, IJRSP, IJTK, JIPR, JSIR & JST. NOPR also hosts three Popular Science Magazines viz. Science Reporter (SR), Vigyan Pragati (VP) & Science Ki Duniya (SKD) and a Natural Products Repository (NPARR ...

Sigma-Aldrich offers abstracts and full-text articles by [Mei Liu, Shu-Jie Li, Yong-Ning Xin, Shu-Sheng Ji, Rui-Jin Xie, Shi-Ying Xuan].

Principal Investigator：HAMAZAKI Shuji, Project Period (FY)：1994 - 1995, Research Category：Grant-in-Aid for General Scientific Research (C), Research Field：Experimental pathology

p,A carboxyfluorescein (CF)-enveloping soybean phosphatidylcholine liposome was used as a model of physicochemical damage of biomembranes. The liposomes were exposed to a metal-chelate complex [2 mM of ferric nitrilotriacetate (FeNTA) or cupric nitrilotriacetate (CuNTA)] plus a reductant (2 mM of ascorbate or various concentrations of reduced glutathione), and CF release from damaged liposomal membranes and the generation of thiobarbituric acid-reactive substances (TBARS) were measured. In the presence of a reducing agent, both FeNTA and CuNTA stimulated markedly CF release and an increase in the TBARS level, while in the absence of a reducing agent both of the chelate complexes showed little CF release and TBARS. The effects of H2O2 addition to the reaction system containing liposome with FeNTA or CuNTA plus ascorbate were also examined. The CF release was slightly increased by the addition of a smaller dose (0.5 mM) of H2O2 and it was inhibited by 8 mM of H2O2. A similar result was obtained in ...

p,A carboxyfluorescein (CF)-enveloping soybean phosphatidylcholine liposome was used as a model of physicochemical damage of biomembranes. The liposomes were exposed to a metal-chelate complex [2 mM of ferric nitrilotriacetate (FeNTA) or cupric nitrilotriacetate (CuNTA)] plus a reductant (2 mM of ascorbate or various concentrations of reduced glutathione), and CF release from damaged liposomal membranes and the generation of thiobarbituric acid-reactive substances (TBARS) were measured. In the presence of a reducing agent, both FeNTA and CuNTA stimulated markedly CF release and an increase in the TBARS level, while in the absence of a reducing agent both of the chelate complexes showed little CF release and TBARS. The effects of H2O2 addition to the reaction system containing liposome with FeNTA or CuNTA plus ascorbate were also examined. The CF release was slightly increased by the addition of a smaller dose (0.5 mM) of H2O2 and it was inhibited by 8 mM of H2O2. A similar result was obtained in ...

It has been reported that Cu(II) ions in human blood are bound mainly to serum albumin (HSA), ceruloplasmin (CP), alpha-2-macroglobulin (α2M) and His, however, data for α2M are very limited and the thermodynamics and kinetics of the copper distribution are not known. We have applied a new LC-ICP MS-based approach for direct determination of Cu(II)-binding affinities of HSA, CP and α2M in the presence of competing Cu(II)-binding reference ligands including His. The ligands affected both the rate of metal release from Cu•HSA complex and the value of KD. Slow release and KD = 0.90 pM was observed with nitrilotriacetic acid (NTA), whereas His showed fast release and substantially lower KD = 34.7 fM (50 mM HEPES, 50 mM NaCl, pH 7.4), which was explained with formation of ternary His•Cu•HSA complex. High mM concentrations of EDTA were not able to elicit metal release from metallated CP at pH 7.4 and therefore it was impossible to determine the KD value for CP. In contrast to earlier inconclusive

TY - JOUR. T1 - DNA-enzyme conjugate with a weak inhibitor that can specifically detect thrombin in a homogeneous medium. AU - Shimada, Josui. AU - Maruyama, Tatsuo. AU - Kitaoka, Momoko. AU - Kamiya, Noriho. AU - Goto, Masahiro. PY - 2011/7/1. Y1 - 2011/7/1. N2 - We present the DNA-assisted control of enzymatic activity for the detection of a target protein using a new type of DNA-enzyme conjugate. The conjugate is composed of an enzyme inhibitor to regulate enzyme activity and a DNA aptamer to be responsive toward the analyte protein. Glutathione S-transferase (GST) and thrombin were selected as a model enzyme and an analyte protein. A hexahistidine tag was genetically attached to the C terminus of the GST, and the 5′ end of an oligonucleotide was conjugated with nitrilotriacetic acid (NTA) for the site-specific conjugation of the DNA with the GST based on a Ni2+ complex interaction. We found that fluorescein acted as a weak inhibitor of GST and succeeded in the regulation of GST activity by ...

A supreme vaccine for Hepatitis C virus (HCV) infection should elicit strong Th1-oriented cellular responses. In the absence of a Th1-specific adjuvant, immunizations by protein antigens generally induce Th2-type and weak cellular responses.To evaluate the adjuvant effect of BCG in comparison with nonionic copolymer-Pluronic F127 (F127) as a classic adjuvant in the formulation of HCV core protein (HCVcp) as a candidate vaccine for induction of Th1 immune responses.Expression of N-terminally His-Tagged HCVcp (1-122) by pIVEX2.4a-core vector harboring the corresponding gene under the control of arabinose-inducible (araBAD) promoter was achieved in BL21-AI strain of E.coli and purified through application of nitrilotriacetic acid (Ni-NTA) chromatography. Mice were immunized subcutaneously (s.c.) in base of the tail with 100 μl of immunogen (F127+HCVcp or BCG+HCVcp; 5 μgHCVcp/mouse/dose) or control formulations (PBS, BCG, F127) at weeks 0, 3, 6. Total and subtypes of IgG, as well as cellular immune

We describe the self-assembly of discrete SiO2 nanofibers via grafting of silicatein side chains to a polymer backbone. The covalent binding of silicatein to the backbone of the polymer is based on the affinity of the nitrilotriacetic acid (NTA) side chain, which serves as a ligand for the immobilization of

Iron has been implicated in the pathophysiology of several models of acute and chronic renal disease. In this study, energy-dispersive x-ray spectrometry was used to quantify and localize iron in rat remnant kidneys (RK) and normal kidneys (NK) and to determine its pathophysiologic significance. Substantial iron accumulation occurred in proximal tubular cell secondary lysosomes of RK (P , 0.001 versus NK) and reached a plateau at 8 wk after partial nephrectomy. In NK, minor increases of iron also occurred with aging (P , 0.02). Proximal tubular iron accumulation correlated independently with protein excretion (r = 0.90) and impairment of GFR (r = 0.70) and was associated with tubular damage and phosphate accumulation (both P , 0.001). Iron nitrilotriacetate (1 mg/kg ip) increased tubular lysosomal iron accumulation and tubular damage (P , 0.001 versus nitrilotriacetate) in NK, comparable to levels seen in untreated RK, and increased cortical cytosolic malondialdehyde, consistent with reactive ...

RhlA purification and assay.The expression of recombinant RhlA protein with an N-terminal His-tag encoded by plasmid pKZ002 was induced with isopropyl-1-thio-β-d-galactopyranoside (IPTG) in E. coli BL21(DE3). Cells were collected by centrifugation, resuspended in MCAC buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 10% glycerol) and lysed with a French press. Soluble proteins were applied to a Ni2+-nitrilotriacetic acid agarose (Qiagen) column and washed with MCAC buffer plus 40 mM imidazole. His-tagged RhlA was eluted with MCAC buffer containing 200 mM imidazole. The fractions containing most of the RhlA protein were pooled, concentrated, and applied to a Superdex S200 column (GE Healthcare) to purify RhlA to homogeneity in a buffer of 20 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol, 50 mM EDTA. Intact MS gave a molecular weight of 34,964, positively identifying the protein as His-tagged RhlA lacking the N-terminal fMet amino acid.. The RhlA activity was determined by measuring the formation of ...

To evaluate liposome formulations for use as intracellular sustained-release drug depots, we have compared the uptake and degradation in rat liver and spleen of liposomes of various compositions, containing as their bulk phospholipid an ether-linked phospholipid or one of several ester-linked phospholipids, by perturbed angular correlation spectroscopy. Multilamellar and small unilamellar vesicles (MLVs and SUVs), composed of egg phosphatidylcholine, sphingomyelin, distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC) or its analog dihexadecylglycerophosphorylcholine (DHPC), and cholesterol plus phosphatidylserine, and containing 111In complexed to nitrilotriacetic acid, were injected intravenously in rats. Recovery of 111In-labeled liposomes in blood, liver, and spleen was assessed at specific time points after injection and the percentage of liposomes still intact in liver and spleen was determined by measurement of the time-integrated angular perturbation factor ...

Uptake kinetics of cadmium and zinc in gram-positive bacteria, Bacillus firmus, isolated from Hong Kong sediments were examined in the present study. The metal uptake by the bacteria was measured at different ambient free metal ion concentrations (10(-12)-10(-6) M Cd(2+) and 10(-10)-10(-6) M Zn(2+)) by adding different concentrations of total dissolved Cd and Zn and hydrophilic organic ligands (ethylenedinitrilotetraacetic acid, nitrilotriacetic acid, and citrate). Our data suggest that Cd and Zn uptake by B. firmus is best predicted by Cd(2+) and Zn(2+) activities. Free metal ions were complexed with the active sites on the bacterial surface, and an equilibrium between the free metal ion and surface-metal complex was reached quickly. After binding, the metal ions were then biologically transported into the bacteria. In addition, with the presence of lipophilic organic ligands (diethyldithiocarbamate and oxine), the lipophilic metal complex was internalized rapidly into B. firmus by passive ...

Evaluates the carcinogenic risk to humans posed by 20 individual compounds and three groups of compounds (cyclamates, saccharin and its salts, and nitrilotriacetic acid and its salts) that are known to induce tumours of the kidney or urinary bladder in rodents. As data on cancer in humans were judged inadequate for all compounds and groups of compounds, the evaluations draw on recent guidelines for determining whether the mechanisms by which a chemical exerts its carcinogenic effects in rodents are relevant to humans. Evaluations also draw on findings from studies of metabolic fate, toxic effects, reproductive and developmental effects, and genetic and related effects. Some of the compounds covered in the volume are widely used as chemical intermediates, pesticides, artificial sweeteners, and food additives. The volume also includes evaluations of the analgesic and antipyretic, paracetamol, and several naturally occurring substances in food. All but two of the compounds (meta-dichlorobenzene and ...

BL21/pES2KI pellets were subjected to ammonium sulfate precipitation (30-40%), resuspended in buffer A (30 mM NaCl and 20 mM Tris-Cl, pH 8.0), and applied to a Fractogel column (Merck, USA). The fraction. was eluted by a NaCl gradient (30 mM-1.4 M). After purification through a P-100 size-exclusion column (BioRad, USA), the CaroS2K fractions were CB-5083 pooled and concentrated using an Amicon centriprep-50 column (Millipore, USA) and dissolved in buffer A. BL21/pES2I pellets were precipitated by ammonium sulfate (70-100%) and resuspended in buffer A. CaroS2I purification involved a similar chromatographic procedure using the Amicon centriprep-3 column (Millipore, USA). The concentration of protein was determined by the Bradford assay (Amresco, USA). In vitro determination of Carocin S2 activity Total RNA was treated with calf intestinal alkaline phosphatase (Promega, USA) at 55°C for 30 min as recommended by the manufacturer. The reaction was arrested by adding 5 mM nitrilotriacetic acid, and ...

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Purpose : ARPE-19 is currently used as an in vitro model for retinal diseases such as age-related degeneration (AMD). Up to now few studies show morphological and genetic expression differences between ARPE-19 and foetal or adult human retinal pigment epithelial (hRPE) cells. This study aims to compare ARPE-19 to hRPE cells derived from induced pluripotent stem cells (iPS) cells in both normal and oxidative stress condition (Fe-NTA treatment), the study hypothesis being to consider that hRPE-iPS cells are more able than ARPE-19 to model AMD in vitro. Methods : iPS cell obtained from peripheral venous blood samples of individuals aged more than 60 years were differenciated into hRPE cells (N=4). hRPE-iPS cells were characterized by morphology, immunofluorescence, FACS and RTqPCR. In basal condition, β-galactosidase activity was measured to quantify senescent cells. Cell count was performed manually with ZEN software. Fe-NTA added to the culture media was used to mimic oxidative environment. In ...

NF-B-inducing kinase (NIK) is a central component in the non-canonical NF-B signaling pathway. cells (Invitrogen) grown at 27 C for 72 h in ESF 921 insect cell medium (Expression Systems). Cells were microfluidized, and the supernatant was loaded on an affinity column made up of nickel-nitrilotriacetic acid beads for His-tagged protein (Qiagen) and eluted with … [Read more…]. ...

1GVC: The Mechanism of Iron Uptake by Transferrins: The X-Ray Structures of the 18 kDa Nii Domain Fragment of Duck Ovotransferrin and its Nitrilotriacetate Complex

The proteins were purified by nickel affinity chromatography. The cell pellet ( ~ 1 g) of each clone was solubilized in 5 ml ofbuffer A (6 M guanidine hydrochloride, 0.1 M NaH2P04, 0.01 M Tris, pH 8.0). The suspension was centrifuged at 8000 X g for 15 min at 4°C and the supernatant containing the recombinant protein was mixed with Ni-NT A resin (Nickel-Nitrilotriacetic acid equilibrated with buffer A) and kept for gentle end-to-end shaking for 1 hat RT. The resin was loaded on a column and washed with 10 bed-volumes of buffer A. The column was subsequently washed with 5 bed-volumes each of buffers B, and C, which contained 8 M urea, 0.1 M NaH2P04 and 0.01 M Tris and had successively reducing pH values of 8.0 and 6.3 respectively. The protein was eluted with buffers D and E (composition same as buffer B) in which the pH was further reduced to 5.9 and 4.5 respectively. Five fractions of 4 ml each were collected during elution with buffer D and buffer E respectively. The eluted proteins were ...

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Molecular Cloning, also known as Maniatis, has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential.

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TALON Superflow is specifically designed for quick and effective purification of polyhistidine-tagged proteins at high flow rates and medium pressure (up to 150 psi)-its ideal for FPLC applications.

TALON Superflow is specifically designed for quick and effective purification of polyhistidine-tagged proteins at high flow rates and medium pressure (up to 150 psi)-its ideal for FPLC applications.

very strange indeed. what size column? what is buffer? what is pI of His-protein? what is pH of buffer? what is amount of protein loaded? Jim Bassuk U Washington Seattle On 6 Mar 1998, Rob wrote: , Hi , Has anyone experienced his-tagged proteins apparently disappearing on , FPLC gel filtration columns (eg Superose 6 or 12 columns). I assume theyre , coming out of solution, but its happened now for the last 2 his-tagged , proteins Ive tried to purify but doesnt happen for the GST-tagged , equivalents.... Very strange! , Thanks in advance , Rob , , ...

Construction, expression, and selective purification of polyhistidine-tagged pro-urokinase. (Upper part) In the BamHI- XhoI excised pcDNAneoI, a double stran

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The purpose of this protocol is to provide a detailed method for the creation of an NTA surface on top of a carboxymethylated dextran chip. This protocol specifically focuses on the BiaCore CM5 chip, but could be adapted to other similar chips. ...

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Can be used at low concentrations (20 mM) to inhibit non-specific binding and, at higher concentrations (,100 mM), to elute the 6xHis-tagged protein from the Ni-NTA ...

0091]The pyrophosphate salts are described in more detail in Kirk & Othmer, Encyclopedia of Clinical Technology Third Edition, Volume 17, Wiley-Interscience Publishers (1982), incorporated herein by reference in its entirety, including all references incorporated into Kirk & Othmer. Additional anticalculus agents include pyrophosphates or polyphosphates disclosed in U.S. Pat. No. 4,590,066 issued to Parran & Sakkab on May 20, 1986; polyacrylates and other polycarboxylates such as those disclosed in U.S. Pat. No. 3,429,963 issued to Shedlovsky on Feb. 25, 1969 and U.S. Pat. No. 4,304,766 issued to Chang on Dec. 8, 1981; and U.S. Pat. No. 4,661,341 issued to Benedict & Sunberg on Apr. 28, 1987; polyepoxysuccinates such as those disclosed in U.S. Pat. No. 4,846,650 issued to Benedict, Bush & Sunberg on Jul. 11, 1989; ethylenediaminetetraacetic acid as disclosed in British Patent No. 490,384 dated Feb. 15, 1937; nitrilotriacetic acid and related compounds as disclosed in U.S. Pat. No. 3,678,154 ...

We report the expression and purification of recombinant rodent malarialPlasmodium yoeliicircumsporozoite surface protein (PyCSP) inEscherichia coli.To facilitate purification of the recombinant protein, the PyCSP was expressed as an amino-terminal fusion protein to glutathioneS-transferase and as a carboxy-terminal fusion protein to a hexahistidyl tag. The expression of the fusion protein was controlled by the inducibletacpromoter. Under optimal conditions the immunoreactive PyCSP represented approximately 0.04% of the total cell lysate. Western blot analysis probing with an anti-PyCSP antibody revealed a wide array of immunoreactive bands. Material isolated by affinity purification on glutathione-Sepharose 4B resin also contained multiple bands indicative of premature termination or carboxyl-terminal degradation. Analysis of protein retained on a nickel nitrilotriacetic acid resin revealed evidence of amino-terminal deleted material. Combining the two mild affinity purifications resulted in ...

GoldiBlot™ His-Tag Western Blot Kit uses Ni-NTA (nickel-nitrilotriacetic acid)-functionalized gold nanoparticles to specifically and directly bind to His-tagged proteins1-6. With autometallographic amplification subsequently applied to the gold nanoparticles, GoldiBlot™ allows the direct visualization of His-tagged proteins. GoldiBlot™ generates specific, purple-colored metallic bands or dots, which do not fade and will not dissolve in water and organic solvents. The GoldiBlot™ His-Tag Western Blot Kit can detect nanogram levels of purified His-tagged proteins, and detects His-tagged proteins in crude extract as well. The entire procedure takes about 1 hour. ...

TRILON A92R (NTA 3Na) CAS No. 5064-31-3. ชื่อทางเคมี trisodium nitrilotriacetate. ลักษณะภายนอก ... ... Read more ...

Unda 600 UNDA Numbered Compounds are unique remedies synergistically formulated with plant constituents that target and carry potentized metals to specific organs where enzymatic and metabolic functions are carried out on a cellular level helping to facilitate the drainage of toxins. UNDA Numbered Compounds are the onl

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In article ,Pine.OSF.3.96.980325220537.21753J-100000 at saul9.u.washington.edu,, James Bassuk ,bassuk at u.washington.edu, wrote: , nonsense. his-tag wont by itself cause nothing more than an increase , in MW of 600. , , but, being a recombinant protein, all kinds of screwy things can happen. , your question is too general because every protein would have an , emperical answer. , , Jim Bassuk , U Washington , Seattle , , , On Tue, 10 Mar 1998 ct133 at columbia.edu wrote: , , , Hi, , , , , Does anybody know whether his-tagged protein expressed from pRSET , , vector (Invitrogen) can migrate aberrantly on SDS-PAGE? , , , , Thanks, , , , , C. Tunyaplin , , , , It is NOT nonsense. I also experienced with several different proteins that adding a his6-tag can cause the protein to migrate at a higher molecular mass in SDS-PAGE. I estimate the difference to about 2-3 kDA. Matthias Engel, University of Saarland, Homburg -- Thomas Seib Kantstr. 12 66292 Riegelsberg ...

5 nm gold particles functionalized with nickel (II) nitraloacetic acid (NTA) chelates. Use for localizing and detecting polyhistidine-tagged targets in protein complexes, cells or tissues. Go directly to EM without silver or gold enhancement ...

The present study was designed to evaluate the protective effects of hesperidin, a flavonoid on DEN initiated and Fe‐NTA promoted renal carcinogenesis in Wistar rats. Renal cancer was initiated by a single i.p. injection of DEN (200 mg/kg b.wt.) and promoted with Fe‐NTA (9 mg Fe/kg b.wt. i.p.) twice a week for 16 weeks. Rats were simultaneously administered with hesperidin (100 and 200 mg/kg b.wt ...

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Fingerprint Dive into the research topics of Biochemical characterization of a new thymic antigen recognized by a monoclonal NTA from NZB mice. Together they form a unique fingerprint. ...

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Mechanical- & chemical-stable Nickel-immobilized resin for direct purification of His-tagged proteins from eukaryotic cell culture samples. Compatible with EDTA, DTT and β-ME.

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