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PubMed2006

Magnetic bead-based solid phase for selective extraction of genomic DNA.

Magnetic bead-based solid phases are widely used for the separation of nucleic acids from complex mixtures. The challenge to selectively separate specific DNA molecules (via complementary hybridization) in a single step is the selection of a linker between the capture probe and the solid support that can be exposed to high temperatures in the presence of a high salt media. This article presents a general platform for the fabrication of a magnetic bead-based selective solid phase that can be used for subtractive hybridization or sequence capture applications. Phosphorus dendrimers are used for the first time as linkers in a magnetic bead-based selective solid phase for capture of genomic DNA. Aside from providing a high loading capacity, they render a stable bond between the capture probe and the surface under the high temperature and salt conditions required for denaturation and capture to proceed in a single step. The thermal stability of the solid phase under these conditions is first demonstrated by hybridizing a Cy3-labeled target. The selective capture of DNA targets in a single step is then demonstrated by subtractive hybridization of fragmented human genomic DNA. The specificity and selectivity of the solid phase are demonstrated by the recovery of adenovirus serotype 4 DNA spiked into the human DNA target. The effect of steric and electrostatic constraints was also investigated by using dendrimers of different generations that vary in their size and the number of branches. The results demonstrate that this platform can be used for single-step subtractive hybridization applications with better performance over the conventional two-step method using streptavidin-coated magnetic beads.

Adenoviridae↗
PubMed1979

Selective extraction of marker enzymes of bovine milk fat globule membrane by nonionic detergents.

Large amounts (66-97%) of marker enzymes such as alkaline phosphatase, 5'-nucleotidase, phosphodiesterase I, and gamma-glutamyl transpeptidase of bovine milk fat globule membrane (MFGM) were selectively solubilized by nonionic detergents such as Triton X-100, Tween 20, Nonidet P-40, Liponox NCK, and Emulgen 109-P. On the other hand, the extractability of MFGM protein with these detergents was less than 50%. Judging from the recovery of total activity, it is likely that alkaline phosphatase, phosphodiesterase I, and gamma-glutamyl transpeptidase are activated by nonionic detergents, whereas 5'-nucleotidase is somewhat inhibited by the detergents, except for Tween 20, and acid phosphatase is strongly inhibited by all detergents. In addition, solubilization of the protein with the nonionic detergents was found to be somewhat selective by SDS-polyacrylamide gel electrophoresis. There was no appreciable difference between the five brands of nonionic detergents used as regards the extractability of protein and the enzymatic activity of the extracted marker enzymes of MFGM, except that the solubilizing ability of Tween 20 was relatively low.

Acid Phosphatase↗
PubMed1988

Selective extraction of haemagglutinin and matrix protein from Sendai virions by employing trifluoperazine as a detergent.

Incubation of trifluoperazine, a local anaesthetic, at concentrations higher than the cmc with Sendai virus particles produces the selective solubilization of the haemagglutinin neuraminidase (HN) and matrix (M) proteins. This phenomenon involves aggregation of the Sendai virions and therefore the separation of HN and M from the rest of the particle can be performed by bench centrifugation. The supernatant contains the HN and M proteins and HN, once inserted into liposomes, elicits its own biological activities. Therefore, the method seems suitable for purifying large amounts of HN.

Animals↗
PubMed1983

Selective extraction with Sarkosyl and repolymerization in vitro of cytoskeleton proteins from Giardia.

Sarkosyl has been used to dissociate structures in cytoskeletons isolated from Giardia lamblia. Results from sodium dodecyl sulphate/polyacrylamide gel electrophoresis and from electron microscopy of insoluble residues show that the solubilization of components is partly selective. At low concentrations of detergent (less than 0.3%), microribbons and microtubules of the disc cytoskeleton disappear, but doublet microtubules from axonemes resist extraction. Consequently, the microribbon protein giardin is extracted into solution more completely than tubulin. Soluble proteins in 0.1% Sarkosyl have been fractionated by gel filtration chromatography in Bio-Gel P300. Giardin elutes in two positions: as a low molecular weight subunit, and in early fractions corresponding to a larger particle size in which subunits might be forming oligomers. Supernatants prepared in 0.5% Sarkosyl were diluted with 0.1 M-KCl or 0.1 mM-MgCl2 to bring about reaggregation of the cytoskeleton proteins. Reassembled structures seen in negatively stained preparations were polymorphic. Some tubulin ribbons of 5 nM protofilaments were identifiable' also there were large fibres and some flat sheets of very thin filaments. Electron micrographs of sheets have been analysed by optical diffraction. The transforms show that the lateral separation of the fine filaments is about 2.5 nm. Axial periodicities from features spaced along filaments were weak. A 3.75 nm layer-line has been detected, corresponding to a similar periodicity found earlier in microribbons.

Animals↗
PubMed2005

Selective extraction, preconcentration and transport studies of thorium(IV) using octa-functionalized calix[4]resorcinarene-hydroxamic acid.

A new calix[4]resorcinarene bearing eight hydroxamic acid groups (C4RAHA) has been synthesized and characterized by FT-IR, 1H-NMR and elemental analysis. Its analytical properties were investigated, and showed high affinity and selectivity toward thorium(IV) in the presence of large quantities of associated metal ions. Thorium(IV) was extracted from an ethyl acetate solution of C4RAHA at pH 4.5. The lambdamax and molar absorptivity (epsilon) for thorium(IV) were 341 nm and 7120 l mol(-1) cm(-1). The complexation of thorium(IV) with C4RAHA has a 4:1 metal:ligand stoichiometry, which is relatively rare. The extracts were directly aspirated in the plasma for ICP-AES measurements for thorium(IV) in the presence of a diversified matrix. The system obeyed Beer's law over the range 0.1 - 6.5 microg ml(-1) of thorium(IV) with a Sandell sensitivity of 0.0325 microg cm(-2). The preconcentration factor and overall stability constant evaluated at 25 degrees C for thorium(IV) were 133 and 15.86, respectively. The complexation was characterized by a favorable enthalpy change. A liquid-membrane transport study of thorium(IV) was carried out from the source to the receiving phase under controlled conditions, and a mechanism of transport proposed. To check the validity of the proposed method, thorium(IV) was determined in monazite sand, rare earth sand and water samples.

Journal Article↗
PubMed2003

Terpyridine-based silica supports prepared by ring-opening metathesis polymerization for the selective extraction of noble metals.

The synthesis of a terpyridine-based sorbent for solid-phase extraction (SPE) of noble metal ions is described. For this purpose, 4'-(norborn-2-en-5-ylmethylenoxy)terpyridine was copolymerized with norborn-2-ene via Mo(N-2,6-i-Pr2-C6H3)(=CHCMe2Ph) (OC(CH3)(CF3)2)2-catalyzed ring-opening metathesis polymerization (ROMP) to give a poly(norbornene900(-b-4'-(norborn-2-en-5-ylmethylenoxy)terpyridine60) block-copolymer. This block-copolymer was used for the preparation of polymer-coated silica 60 (4.8 wt.% coating), which was investigated for its extraction capabilities for Cr(III), Mn(II), Re(II), Fe(III), Ru(III), Co(II), Rh(III), Ir(III), Ni(II), Pd(II), Pt(II), Cu(II), Ag(I), Au(III), Zn(II), Cd(II) and Hg(II), at different pH. Under competitive conditions and at pH < 0.6, the selectivity order was Pd approximately = Ag approximately = Au approximately = Pt > Re > Ir > Rh > Ru > Fe > Cr approximately = Mn approximately = Cd approximately = Zn. Enhanced selectivity was observed at pH = 3.5, the order was Au > Hg > Pd approximately = Ag > Rh > Pt > Ir approximately = Re > Cu > Co approximately = Zn approximately = Cd approximately = Ni > Cr > Mn. The maximum metal loading that was achieved under non-competitive conditions was > 6 mg/g for Au(III), Hg(II), Pd(II) and Ag(I). Even under competitive conditions, loadings of > 6 mg/g were realized for Au(III) and Hg(II). Quantitative recoveries > 97% were observed for all metals in case loading was stopped before reaching the point of breakthrough.

Magnetic Resonance Spectroscopy↗
PubMed2001

Selective extraction of subunit D of the Na(+)-translocating methyltransferase and subunit c of the A(1)A(0) ATPase from the cytoplasmic membrane of methanogenic archaea by chloroform/methanol and characterization of subunit c of Methanothermobacter thermoautotrophicus as a 16-kDa proteolipid.

Chloroform/methanol was applied to cytoplasmic membranes of the thermophilic methanogens Methanothermobacter thermoautotrophicus and Methanothermobacter marburgensis as well as to the mesophile Methanosarcina mazei Gö1. In any case, the chloroform/methanol extraction yielded only two proteins, subunit D (MtrD) of the Na(+)-translocating methyltetrahydromethanopterin:coenzyme M methyltransferase and the proteolipid of the A(1)A(0) ATPase. Both polypeptides are assumed to be directly involved in ion translocation in their respective enzymes, but have not been studied in detail due to lack of simple isolation procedures. The rapid and selective isolation by chloroform/methanol offers a new way to obtain the large quantities of material required for biochemical analyses. As a first result, molecular and biochemical data suggest that the proteolipid from M. thermoautotrophicus is a duplication of the 8-kDa proteolipid usually present in other archaea, but it retained the conserved glutamate involved in proton translocation in every copy. This is the first 16-kDa proteolipid found in archaea.

Adenosine Triphosphatases↗
PubMed2004

Selective extraction of U(VI), Th(IV), and La(III) from acidic matrix solutions and environmental samples using chemically modified Amberlite XAD-16 resin.

A new grafted polymer has been developed by the chemical modification of Amberlite XAD-16 (AXAD-16) polymeric matrix with [(2-dihydroxyarsinoylphenylamino)methyl]phosphonic acid (AXAD-16-AsP). The modified polymer was characterized by a combination of (13)C CPMAS and (31)P solid-state NMR, Fourier transform-NIR-FIR-Raman spectroscopy, CHNPS elemental analysis, and thermogravimetric analysis (TGA). The distribution studies for the extraction of U(VI), Th(IV), and La(III) from acidic solutions were performed using an AXAD-16-AsP-packed chromatographic column. The influences of various physiochemical parameters on analyte recovery were optimized by both static and dynamic methods. Accordingly, even under high acidities (>4 M), good distribution ratio (D) values (10(2)-10(4)) were achieved for all the analytes. Metal ion desorption was effective using 1 mol L(-1) (NH(4))(2)CO(3). From kinetic studies, a time duration of <15 min was sufficient for complete metal ion saturation of the resin phase. The maximum metal sorption capacities were found to be 0.25, 0.13, and 1.49 mmol g(-1) for U(VI); 0.47, 0.39, and 1.40 mmol g(-1) for Th(IV); and 1.44, 1.48, and 1.12 mmol g(-1) for La(III), in the presence of 2 mol L(-1) HNO(3), 2 mol L(-1) HCl, and under pH conditions, respectively. The analyte selectivity of the grafted polymer was tested in terms of interfering species tolerance studies. The system showed an enrichment factor of 365, 300, and 270 for U(VI), Th(IV), and La(III), and the limit of analyte detection was in the range of 18-23 ng mL(-1). The practical applicability of the polymer was tested with synthetic nuclear spent fuel and seawater mixtures, natural water, and geological samples. The RSD of the total analytical procedure was within 4.9%, thus confirming the reliability of the developed method.

Journal Article↗
PubMed2000

Calix[4]arenes as selective extracting agents. An NMR dynamic and conformational investigation of the lanthanide(III) and thorium(IV) complexes.

The lanthanide and Th4+ complexes with calix[4]arene ligands substituted either on the narrow or at the wide rim by four coordinating groups behave totally differently as shown by an NMR investigation of the dia- and paramagnetic complexes. Solutions of complexes were prepared by reacting anhydrous metal perchlorate salts with the ligands in dry acetonitrile (CAUTION). Relaxation time T1 titrations of acetonitrile solutions of Gd3+ by calixarenes indicate that ligands subsituted on the narrow rim form stable 1:1 complexes whether they feature four amide groups (1) or four phosphine oxide functions. In contrast, a ligand substituted by four (carbamoylmethyl)-diphenylphosphine oxide moieties on the wide rim (3) and its derivatives from polymeric species even at a 1:1 ligand/metal concentration ratio. Nuclear magnetic relaxation dispersion (NMRD) curves (relaxation rates 1/T1 vs magnetic field strength) of Gd3+, Gd3+.1 and Gd3+.3 perchlorates in acetonitrile are analyzed by an extended version of the Solomon-Bloembergen-Morgan equations. A comparison of the calculated rotational correlation times tau r shows that ligand 3 forms oligomeric Gd3+ species. The chelates of ligand 1 are axially symmetric (C4 symmetry), and the paramagnetic shifts induced by the Yb3+ ion are accounted for quantitatively. The addition of water or of nitrate ions does not modify the geometry of the complex. The metal chelates of 3 and its derivatives adopt a C2 symmetry, and the paramagnetic shifts are interpreted on a semiquantitative basis only. Water and NO3- ions completely labilize the complexes of the heavy lanthanides. The very high selectivity of ligand 3 through the lanthanide series stems from a complex interplay of factors.

Journal Article↗
PubMed1989

Reestimations of the protein concentrations of birch pollen allergen extracts selected as candidates for the international standard (IS) preparation.

The total protein concentrations of six selected candidates for an international standard (IS) of birch pollen were previously estimated (Ann Allergy 1987;58:71-77). The results of analyses showed large variations depending both on the method and the reference protein used. More than 2-fold variations in the protein content were obtained using Bradford's protein binding method. In the present communication the protein contents of these birch IS-candidates were reestimated by Lowry, a modified Lowry technique, Bradford's protein-dye binding, bicinchoninic acid reagent and amino acid compositions. Great variations were obtained for the protein contents, depending on the technique employed, emphasizing the difficulties of choosing a proper method of protein analyses of pollen allergens. The Lowry and the bicinchoninic acid reagent methods gave the highest obtained protein estimates, while the modified Lowry and Bradford's protein-dye binding methods showed the lowest values. The amino acid analysis gave concentrations similar to those obtained by Lowry and bicinchoninic acid reagent methods. The Lowry method seemed to be adequate for the analysis of plant proteins. The amino acid analysis is currently the most precise method for estimation of the protein concentration irrespective of additional prosthetic groups. A serious shortcoming of the method is that it requires an amino acid analyser or other liquid chromatrographic systems, which are not commonly in laboratory use.

Amino Acids↗
PubMed2002

A rapid selective extraction procedure for the outer membrane protein (OmpF) from Escherichia coli.

Porins are essential pore-forming proteins found in the outer membrane of several gram-negative bacteria. Investigating the relationships between molecular structure and function involves an extremely time-consuming and labor-intensive purification procedure. We report a method for rapid extraction of the outer membrane protein, OmpF, from freeze-dried Escherichia coli cells using valeric acid, alleviating the effort and time in sample preparation. Extraction results in a highly enriched fraction containing OmpF as 76% of the total protein content. The apparent molecular mass determined by SDS-PAGE mobility was 38,900, similar to that of the monomeric form of OmpF. N-terminal sequencing yielded 23 amino acids with 100% identity to the published OmpF sequence. The trimeric form of OmpF was observed in unheated samples run on SDS-PAGE and analysis of these samples by periodic acid/silver staining revealed the presence of unbound lipopolysaccharides. Furthermore, this method should prove useful for isolating other outer membrane proteins.

Amino Acid Sequence↗
PubMed1987

Mechanized techniques for the selective extraction of enzymes from plant epidermal glands.

Many plant products are biosynthesized and accumulated in epidermal glands. For investigations on the metabolism of these compounds it is most convenient to obtain cell-free preparations enriched in gland contents. Two simple mechanized procedures have been developed for gently abrading the plant surface in order to efficiently extract glandular enzymes in high purity. These methods allow rapid processing of large quantities of plant material and yield extracts largely uncontaminated with materials from underlying tissue. The use of these procedures for isolating several enzymes of terpenoid metabolism is described. These techniques work especially well for microsomal enzymes and may be useful not only for enzymes found in epidermal glands but also for other enzymes localized in or near the epidermis. With simple modification, these procedures can be adapted for use with a variety of different types of plant tissues.

Cell-Free System↗