Academic literature on the topic 'Selective isolation and concentration of proteins'
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Journal articles on the topic "Selective isolation and concentration of proteins"
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Stratford, C. A., and S. S. Brown. "Isolation of an actin-binding protein from membranes of Dictyostelium discoideum." Journal of Cell Biology 100, no. 3 (March 1, 1985): 727–35. http://dx.doi.org/10.1083/jcb.100.3.727.
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We prepared a probe of radiolabeled, glutaraldehyde cross-linked filamentous actin (F-actin) to study binding of actin to membranes of Dictyostelium discoideum. The probe bound to membranes or detergent extracts of membranes with a high affinity and in a saturable manner. The binding could be reduced by boiling of either the actin probe or the membranes, or by addition of excess native F-actin, but not by addition of an equivalent amount of bovine serum albumin, to the assay. The probe labeled several proteins when used to overlay sodium dodecyl sulfate gels of Dictyostelium membranes. One of these labeled proteins was a 24,000-mol-wt protein (p24), which was soluble only in the presence of a high concentration of sodium deoxycholate (5%, wt/vol) at room temperature or above. The p24 was purified by selective detergent extraction and column chromatography. When tested in a novel two-phase binding assay, p24 bound both native monomeric actin (G-actin) and F-actin in a specific manner. In this assay, G-actin bound p24 with a submicromolar affinity.
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Bouvier, M., S. D. Miszczycha, X. Guillarme, C. Tadla, L. Collinet, and D. Thevenot Sergentet. "Comparative Evaluation of a Novel Phage Protein Ligand Assay and Immunomagnetic Separation Method To Isolate the Seven Top Serogroups of Escherichia coli (O157, O26, O103, O145, O111, O45, and O121) in Foods at Risk." Journal of Food Protection 80, no. 12 (November 13, 2017): 1973–79. http://dx.doi.org/10.4315/0362-028x.jfp-16-479.
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ABSTRACT The presence of Shiga toxin–producing Escherichia coli (STEC) in food is a major concern for food safety authorities and industries. Methods for detecting these pathogenic bacteria are crucial. Enrichment of foods for STEC identification has been optimized, but selective concentration of bacteria before isolation still needs to be improved. In the present study, we tested the performance of the VIDAS ESPT detection method against that of the immunomagnetic separation (IMS) method. A preenrichment inoculation was performed to provide a realistic scenario of the contamination that occurs in foods, and the methods were then compared. Results obtained were then confirmed in naturally contaminated foods. Preenrichment inoculation assays revealed that the novel concentration method using phage recombinant proteins or the selective capture of the target top seven STEC serogroups is as specific and sensitive as IMS. Subsequent evaluation of naturally contaminated samples confirmed that the novel concentration method and IMS are equivalent in performance under the conditions tested.
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Athauda, Senarath B. P., Katsuji Yoshioka, Tadayoshi Shiba, and Kenji Takahashi. "Isolation and characterization of recombinant Drosophila Copia aspartic proteinase." Biochemical Journal 399, no. 3 (October 13, 2006): 535–42. http://dx.doi.org/10.1042/bj20060800.
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The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe–Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 °C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15−–Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects.
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Haddad, F., R. R. Roy, H. Zhong, V. R. Edgerton, and K. M. Baldwin. "Atrophy responses to muscle inactivity. I. Cellular markers of protein deficits." Journal of Applied Physiology 95, no. 2 (August 2003): 781–90. http://dx.doi.org/10.1152/japplphysiol.00317.2003.
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The goal of this study was to use the model of spinal cord isolation (SI), which blocks nearly all neuromuscular activity while leaving the motoneuron muscle-fiber connections intact, to characterize the cellular processes linked to marked muscle atrophy. Rats randomly assigned to normal control and SI groups were studied at 0, 2, 4, 8, and 15 days after SI surgery. The slow soleus muscle atrophied by ∼50%, with the greatest degree of loss occurring during the first 8 days. Throughout the SI duration, muscle protein concentration was maintained at the control level, whereas myofibrillar protein concentration steadily decreased between 4 and 15 days of SI, and this was associated with a 50% decrease in myosin heavy chain (MHC) normalized to total protein. Actin relative to the total protein was maintained at the control level. Marked reductions occurred in total RNA and DNA content and in total MHC and actin mRNA expressed relative to 18S ribosomal RNA. These findings suggest that two key factors contributing to the muscle atrophy in the SI model are 1) a reduction in ribosomal RNA that is consistent with a reduction in protein translational capacity, and 2) insufficient mRNA substrate for translating key sarcomeric proteins comprising the myofibril fraction, such as MHC and actin. In addition, the marked selective depletion of MHC protein in the muscles of SI rats suggests that this protein is more vulnerable to inactivity than actin protein. This selective MHC loss could be a major contributor for the previously reported loss in the functional integrity of SI muscles. Collectively, these data are consistent with the involvement of pretranslational and translational processes in muscle atrophy due to SI.
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Farhana, Mashuri Nurul, Huey Ling Tan, Ying Pei Lim, and Siti Noor Suzila Maqsood-Ul-Haque. "Isolation of Antimicrobial Peptide from Food Protein Hydrolysates: An Overview." Key Engineering Materials 797 (March 2019): 168–76. http://dx.doi.org/10.4028/www.scientific.net/kem.797.168.
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Antimicrobial resistance (AMR) is one of the most serious public health threats that results mostly from the selective pressure exerted by antibiotic use and abuse. AMR has become a major problem with global human deaths due to antibiotic resistant infections predicted to reach 10 million by 2050. Antimicrobial peptides (AMPs) has been discovered to have capabilities to kill microorganisms and can take other roles as an alternative to antibiotics which is favorable to the need of minimizing the usage of antibiotics as they lead to the increasing of AMR. AMP can be found naturally in almost all domains of life as part of the innate immune system to combat virus, bacteria, fungi and even cancer cells. It can also be extracted from food proteins using enzymatic hydrolysis. High antimicrobial properties/activities are depend on the degree of hydrolysis (DH) of peptides, peptide source/origin, and type of enzyme used for the hydrolysis process. There are several other variables that can be manipulated to optimum condition to obtain high DH. Variables such as temperature, pH, enzyme concentration and hydrolysis time have proven to bring impact to the DH of peptides. Other bioactive peptides that have been discovered during the process have great potential to bring benefit in medicinal and nutraceutical areas.
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Sjolin, C., and C. Dahlgren. "Isolation by calcium-dependent translation to neutrophil-specific granules of a 42-kD cytosolic protein, identified as being a fragment of annexin XI." Blood 87, no. 11 (June 1, 1996): 4817–23. http://dx.doi.org/10.1182/blood.v87.11.4817.bloodjournal87114817.
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Secretion of neutrophil granules is dependent on calcium, but at the same time this process is regulated differently for each type of granules. We attempted to find calcium-regulated proteins that selectively translocate from the cytosol to the membranes of the neutrophil granules. An in vitro calcium-dependent translocation assay was designed by mixing cytosol with different neutrophil organelles isolated by subcellular fractionation. Immunoblotting using an anti- cytosol antiserum revealed a cytosolic protein of 42 kD that selectively binds to the specific granules of human neutrophils. It was neither associated with the azurophil granules nor with the secretory vesicles/plasma membrane. The protein was translocated at a calcium concentration of 100 micromol/L and binding was further increased by 1 mmol/L calcium. The 42-kD protein was partially purified from neutrophil cytosol by using its affinity for specific granules and by ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partly purified protein allowed the 42-kD band to be excised and subjected to tryptic peptide mapping. Peptides from three peaks were N-terminally sequenced. Searching among known proteins, each one of the amino acid sequences was found to share sequence similarity to annexin XI.
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Platt, R. M., G. G. Geesey, J. D. Davis, and D. C. White. "Isolation and partial chemical analysis of firmly bound exopolysaccharide from adherent cells of a freshwater sediment bacterium." Canadian Journal of Microbiology 31, no. 8 (August 1, 1985): 675–80. http://dx.doi.org/10.1139/m85-128.
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Cells of a freshwater sediment bacterium produced firmly bound extracellular polymers in laboratory cultures which, at the ultrastructural level, resembled those produced by natural sediment bacterial populations. Production of the exopolymers during subculture was maintained by using as a source of inoculum the population of cells which adhered to each other and to the wall of the glass culture vessel. The exopolymers were selectively released from the cells by blending and centrifugation in the presence of EDTA. Evaluation of glucose-6-phosphate dehydrogenase activity and 2-keto-3-deoxyoctonate concentration indicated that only small amounts of intracellular and cell wall components were released from the cells during exopolymer removal. Chemical analysis of the isolated crude exopolymer material indicated that it contained protein, polysaccharide, and DNA. The treatment promoted the selective isolation of firmly bound polymers from the surface of adherent cells.
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Franke, C. A., C. M. Rice, J. H. Strauss, and D. E. Hruby. "Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants." Molecular and Cellular Biology 5, no. 8 (August 1985): 1918–24. http://dx.doi.org/10.1128/mcb.5.8.1918.
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The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.
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Franke, C. A., C. M. Rice, J. H. Strauss, and D. E. Hruby. "Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants." Molecular and Cellular Biology 5, no. 8 (August 1985): 1918–24. http://dx.doi.org/10.1128/mcb.5.8.1918-1924.1985.
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The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.
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Balmayor, Elizabeth R., Iva Pashkuleva, Ana M. Frias, Helena S. Azevedo, and Rui L. Reis. "Synthesis and functionalization of superparamagnetic poly- ɛ -caprolactone microparticles for the selective isolation of subpopulations of human adipose-derived stem cells." Journal of The Royal Society Interface 8, no. 59 (January 5, 2011): 896–908. http://dx.doi.org/10.1098/rsif.2010.0531.
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There has been a growing interest in using biofunctionalized magnetic particles for cell isolation. This paper describes the synthesis and characterization of magnetite-polymer (Fe 3 O 4 -poly- ɛ -caprolactone, magnetite-PCL) microparticles surface functionalized with amino and epoxy groups allowing easy covalent attachment of specific antibodies and subsequent ability to bind target cells. Particles with different sizes (4–135 µm), spherical shape and superparamagnetic behaviour (magnetite content of about 13 wt%) were obtained. The functionalized microparticles presented high protein-binding capacity (coupling efficiency of 47% for epoxy- and 71% for amino-functionalized particles) with a low level of non-specific binding. We have further investigated the influence of initial protein concentration, pH, ionic strength, temperature and incubation time on the capacity of amino-functionalized particles to bind protein molecules. The results showed that maximum protein coupling is rapidly achieved (≤5 h) at pH 5.5 and low ionic strength (0.05 M NaCl). Furthermore, when cultured in direct contact with osteoblast-like cells (Saos-2) or human-derived adipose stem cells (ASCs), the amino-functionalized particles did not affect the proliferation and morphology of the cells. As a proof of principle for the application of magnetic microparticles for cell isolation, CD105 (endoglin) antibody was coupled to the magnetic particle surface to bind subpopulations of human ASCs expressing the CD105 antigen. The isolation of CD105+ ASCs from a heterogeneous cell population was confirmed by flow cytometry analysis. Given the demonstrated potential of functionalized magnetite-PCL microparticles for selective cell isolation, we expect that these particles may be further applied in immuno-magnetic cell separation owing to their versatility and ease of surface modification.
Dissertations / Theses on the topic "Selective isolation and concentration of proteins"
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Lin, Shu-Ling. "Electric Field Gradient Focusing-UV Detection for Protein Analysis." Diss., CLICK HERE for online access, 2006. http://contentdm.lib.byu.edu/ETD/image/etd1372.pdf.
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Behrend, Frank Gerhard. "Selective isolation of linear plasmids from Streptomyces spp. by concentration at a phenol-buffer interface." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ28916.pdf.
Full textBook chapters on the topic "Selective isolation and concentration of proteins"
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Ruissen, M. A., C. A. J. Helderman, J. Schipper, and J. W. L. Van Vuurde. "Selective Isolation and Concentration of Phytopathogenic Bacteria on Immunoaffinity Columns." In Plant Pathogenic Bacteria, 882. Dordrecht: Springer Netherlands, 1987. http://dx.doi.org/10.1007/978-94-009-3555-6_190.
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Mizutani, Akihiro, Katsuhiro Kawaai, Chihiro Hisatsune, Hideaki Ando, Takayuki Michikawa, and Katsuhiko Mikoshiba. "Isolation of Inositol 1,4,5-Trisphosphate Receptor-Associating Proteins and Selective Knockdown Using RNA Interference." In Methods in Molecular Biology, 133–41. Totowa, NJ: Humana Press, 2010. http://dx.doi.org/10.1007/978-1-60327-175-2_9.
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González, L. J., E. Torres, Y. García, L. H. Betancourt, G. Moya, V. Huerta, V. Besada, and G. Padrón. "A New Method for the Isolation of the C-Terminal Peptide of Proteins by the Combination of Selective Blocking of Proteolytic Peptides and Cation Exchange Chromatography." In Proteome and Protein Analysis, 175–87. Berlin, Heidelberg: Springer Berlin Heidelberg, 2000. http://dx.doi.org/10.1007/978-3-642-59631-5_12.
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Matthews, K. S., and R. Matthews. "Selective Chemical Deuteration of Aromatic Amino Acids: A Retrospective." In Biological NMR Spectroscopy. Oxford University Press, 1997. http://dx.doi.org/10.1093/oso/9780195094688.003.0021.
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In 1970 when we began post-doctoral work in the laboratory of Professor Oleg Jardetzky, selective deuteration of proteins to limit the number of protons present in the system for subsequent analysis was a newly developed and effective technique for NMR exploration of protein structure (Crespi et al., 1968; Markley et al., 1968). This approach allowed more facile assignment of specific resonances and generated the potential to follow the spectroscopic behavior of protons for a specific amino acid sidechain over a broad range of conditions. The primary method for labeling at that time involved growth of microorganisms (generally bacteria or algae) in D2O, followed by isolation of the deuteratedamino acids from a cellular protein hydrolysate. The amino acids isolated were, therefore, completely deuterated. Selective deuteration of a target protein was achieved by growing the producing organism on a mixture of completely deuterated and selected protonated amino acids under conditions that minimized metabolic interconversion of the amino acids. In one-dimensional spectra, aromatic amino acid resonances occur well downfield of the aliphatic resonances, and this region can therefore be examined somewhat independently by utilizing a single protonated aromatic amino acid to simplify the spectrum of the protein. However, the multiple spectral lines generated by aromatic amino acids can be complex and overlapping, precluding unequivocal interpretation. To address this complication, chemical methods were developed to both completely and selectively deuterate side chains of the aromatic amino acids, thereby avoiding the costly necessity of growing large volumes of microorganisms in D2O and subsequent tedious isolation procedures. In addition, selective deuteration of the amino acids simplified the resonance patterns and thereby facilitated assignment and interpretation of spectra. The methods employed were based on exchange phenomena reported in the literature and generated large quantities of material for use in growth of microorganisms for subsequent isolation of selectively labeled protein (Matthews et al., 1977a). The target protein for incorporation of the selectively deuterated aromatic amino acids generated by these chemical methods was the lactose repressor protein from Escherichia coli, and greatly simplified spectra of this 150,000 D protein were produced by this approach.
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Deamer, David W. "Self-Assembly Processes Were Essential for Life’s Origin." In Assembling Life. Oxford University Press, 2019. http://dx.doi.org/10.1093/oso/9780190646387.003.0010.
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In the absence of self-assembly processes, life as we know it would be impossible. This chapter begins by introducing self-assembly then focuses on the primary functions of membranes in living cells, most of which depend on highly evolved proteins embedded in lipid bilayers. These serve to capture light energy in photosynthesis and produce ion concentration gradients from which osmotic energy can be transduced into chemical energy. Although lipid bilayer membranes provide a permeability barrier, they cannot be absolutely impermeable because intracellular metabolic functions depend on external sources of nutrients. Therefore, another set of embedded proteins evolved to form transmembrane channels that allow selective permeation of certain solutes. The earliest life did not have proteins available, so in their absence what was the primary function of membranous compartments in prebiotic conditions? There are three possibilities. First, the compartments would allow encapsulated polymers to remain together as random mixtures called protocells. Second, populations of protocells that vary in composition would be subject to selective processes and the first steps of evolution. Even though any given protocell would be only transiently stable, certain mixtures of polymers would tend to stabilize the surrounding membrane. Such an encapsulated mixture would persist longer than the majority that would be dispersed and recycled, and these more robust protocells would tend to emerge as a kind of species. Last and perhaps most important, there had to be a point in early evolution at which light energy began to be captured by membranous structures, just as it is today. Bilayer membranes are not necessarily composed solely of amphiphilic molecules. They can also contain other nonpolar compounds that happen to be pigments capable of capturing light energy. This possibility is almost entirely unexplored, but the experiments are obvious and would be a fruitful focus for future research. Questions to be addressed: What is meant by self-assembly? Why is self-assembly important for the origin of life? What compounds can undergo self-assembly processes? How can mixtures of monomers and lipids assemble into protocells? We tend to think of living cells in terms of directed assembly.
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Reed, Ian, and Duncan Mackay. "Clarification techniques." In Protein Purification Techniques. Oxford University Press, 2001. http://dx.doi.org/10.1093/oso/9780199636747.003.0008.
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Proteins can be produced by a number of different routes such as fermentation, tissue culture, and by extraction from plasma or plants. Whatever route is chosen, the raw protein-bearing stream is likely to be a complex mixture containing both dissolved species and particulate material. The target protein will be present at very low concentration and with a host of contaminants such as cells or cell debris, DNA, proteins and polysaccharides, and a large quantity of water. Such a mixture is very difficult to treat using the highly selective processes that are required to obtain the target product at high purity since the presence of particulate material impairs their function. The first challenge of protein purification is therefore to convert the complex fermentation broth which is a mixture of dissolved and suspended solids into a form that is amenable to further purification. Although there is much interest in direct recovery of protein from such materials, the most frequent first step currently is to clarify the raw protein source to remove suspended matter. It is then possible to use a range of highly selective techniques to purify the target protein. There are a number of clarification techniques that can be adopted and the choice of which to use depends on both the source of raw feed and the scale of operation. There are two main classes of process; sedimentation and filtration. Sedimentation can be carried out under normal gravity conditions or, as is almost always the case for biological streams, using a centrifuge. Filtration can be performed using either conventional filter media or using membrane filters for removal of finer particles. The aim of this chapter is to describe these methods, and their underlying principles, the advantages of each are discussed, and examples of equipment are presented. Practical advice is presented on how and when to use each technique. Sedimentation processes operate primarily on the basis of density differences between the various components of a mixture. They are most commonly applied to suspensions of solid in liquid, but also to disengage immiscible liquids. If there is no density difference between particulates and the suspending medium, sedimentation cannot occur.
Conference papers on the topic "Selective isolation and concentration of proteins"
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Krasnoshtanova, Alla, and Alesya Yudina. "PRODUCTION OF ANTIBODIES FROM POULTRY YOLK (IgY) AND INVESTIGATION OF THEIR IMMUNOCHEMICAL PROPERTIES." In GEOLINKS Conference Proceedings. Saima Consult Ltd, 2021. http://dx.doi.org/10.32008/geolinks2021/b1/v3/17.
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"A particularly important aspect of immunology is to develop non-invasive methods of obtaining antibodies which could be a great alternative to traditional ones that based on the harmful procedure of isolation of immunoglobulins from animal blood sera. That’s why the extraction of antibodies from poultry egg yolks (IgY) is the most promising. Due to the fact of variation of IgY structural features that determine the definite immunochemical properties, yolk antibodies in comparison with mammalian immunoglobulins (IgG) does not interact with rheumatoid factor (Rf), contribute to the activation of the complement system, bind to the Fc-receptor (FcR), and also has weak cross-reactivity, which confirms the possibility of their widespread use in medicine and food. Also the presence of phylogenetic distance between chickens and mammalians guarantees immune response against conservative mammalian protein molecules which is highly important for the creation of new generation test systems. The aim of this work is to develop a selective method of producing high-purity immunoglobulin Y preparations from the yolk of chicken eggs. There were adopted selective conditions of isolation of IgY under spontaneous thawing procedure at the room temperature of firstly frozen yolk solution in a sodium-phosphate buffer mixed with water (pH 5.0) in a ratio of 1:6, which leads to receiving a water-soluble fraction further precipitated with the sodium chloride at a concentration of 10% of the solution mass and subsequently concentrated using ultrafiltration with membrane UAM-10, that allows achieving the content of IgY not less than 95% per dry substance in immunoglobulin fraction. It is possible to produce a protein fraction with a protein content of at least 9 g/l. The purity of the immunoglobulin fraction was verified using polyacrylamide gel electrophoresis. The presence of a light chain in the IgY solution was proved to be a low-molecular compound using the method of gel-filtration-chromatography. The immunological activity of IgY was studied with respect to bovine serum albumin (BSA) as an antigen. The enzymatic resistance of IgY against proteolytic enzymes was tested in area of the gastrointestinal tract."
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Nguyen, Thai Huu, and Qiao Lin. "An Aptamer-Functionalized Microfluidic Platform for Biomolecular Purification and Sensing." In ASME 2009 7th International Conference on Nanochannels, Microchannels, and Minichannels. ASMEDC, 2009. http://dx.doi.org/10.1115/icnmm2009-82142.
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Aptamers are oligonucleotides (DNA or RNA) that bind to chemical and biological analyte targets via affinity interactions. Through an in vitro synthetic process, aptamers can be developed for an extremely broad spectrum of analytes, such as small molecules, proteins, cells, viruses, and bacteria. Target recognition by aptamers is highly selective, as affinity interactions result in secondary aptamer conformational structures that specifically fit the target. The aptamer-target binding is also reversible and depends strongly on external stimuli such as pH and temperature. The specificity and stimuli-responsiveness of aptamers are highly attractive to biological purification and sensing, which generally involve isolating minute quantities of targets from complex samples with non-specific molecules and impurities present at orders-of-magnitude higher concentrations. We present an aptamer-functionalized microfluidic platform that by design exploits the specificity and temperature-dependent reversibility of aptamers to enable biomolecular purification and sensing. Using the specificity of aptamers, we demonstrate highly selective capture and enrichment of biomolecules. Employing thermally induced, reversible disruption of aptamer-target binding, we accomplish isocratic elution of the captured analytes and regeneration of the aptamer surfaces, thereby eliminating the use of potentially harsh reagents. Using integrated microfluidic control, the eluted analytes are detected in a label-free fashion by mass spectrometric methods.
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Toti, F., A. Stierlé, M. L. Wiesel, A. Schwartz, J. M. Freyssinet, and J. P. Cazenave. "PRODUCTION OF ANTIBODIES TO HUMAN VON WILLEBRAND FACTOR IN LAYING HENS. ISOLATION OF IMMUNOGLOBULINS AND APPLICATIONS TO THE DETECTION OF MOLECULAR DEFECTS OF VON WILLEBRAND FACTOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644084.
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Von Willebrand disease (vWD) is an inherited disorder of primary hemostasis caused by deficiency or structural abnormalities of von Willebrand factor (vWF). VWF circulates in plasma and is also present in platelets. Plasma vWF, the carrier protein for factor VIII, is a large multimeric glycoprotein composed of identical subunits linked by disulfide bridges. Plasma and platelet vWF display distinct multimeric electrophoretic patterns. The different vWD subtypes can be classified either by the determination of vWFantigen (vWFíAg) and/or by multimer distribution. Antibodies to human vWF were raised in laying hens by intramuscular injections of purified human vWF. Immunoglobulins were isolated from egg yolks by selective polyethylene glycol and ammonium sulfate precipitations. These antibodies appeared to be monospecific, as they did not react with the plasma proteins of a patient with severe vWD. The pullets received weekly 50 μg vWF for 4 weeks and then had monthly injections. The antibodies occurred as early as the third injection, the yield being 300 to 500 mg of immunoglobulin per week (6-7 eggs). The titre could be constant over periods greater than 1 year. These immunoglobulins to vWF were tested in vWFíAg electroimmunoassays and for the multimer analysis of plasma and platelet vWF by electrophoresis and immunoblotting techniques. In no case could a difference be detected between assays performed with rabbit monospecific antiserum or with yolk immunoglobulins to human vWF. Ten to 12 multimers could be revealed for normal plasma vWF and up to 12 to 14 bands for normal platelet vWF (1.7% agarose). In the case of vWD, the electrophoresis patterns were identical with both antibodies. Thus, antibodies to vWF raised in laying hens are a suitable tool to detect and to characterize vWD. Although they do not interact with protein A, yolk antibodies are certainly advantageous to produce, as they do not contain IgM or IgA. Immunoglobulin fractions can contain up to 10 % of specific antibodies. Since they are available in larger quantities and are easy to isolate, larger homogeneous batches of antibodies can be obtained. This method may easily be applied to develop antibodies to a variety of antigens.
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Abdel Samad, Rim, Zulfa Al Disi, Mohammad Ashfaq, and Nabil Zouari. "The use of Principle Component Analysis and MALDI-TOF MS for the differentiation of mineral forming Virgibacillus and Bacillus species isolated from Sabkhas." In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0069.
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Occurrence of mineral forming and other bacteria in mats is well demonstrated. However, their high diversity shown by ribotyping was not explained, although it could explain the diversity of formed minerals. Common biomarkers as well as phylogenic relationships are useful tools to clustering the isolates and predict their potential role in the natural niche. In this study, combination of MALDI-TOF MS with PCA was shown a powerful tool to categorize 35 mineral forming bacterial strains isolated from Dohat Fshaikh sabkha, at northwest of Qatar (23 from decaying mats and 12 from living ones). 23 strains from decaying mats belong to Virgibacillus genus as identified by ribotyping and are shown highly involved in formation of protodolomite and a diversity of minerals. They were used as internal references in categorization of sabkha bacteria. Combination of isolation of bacteria on selective mineral forming media, their MALDI TOF MS protein profiling and PCA analysis established their relationship in a phyloproteomic based on protein biomarkers including m/z 4905, 3265, 5240, 6430, 7765, and 9815. PCA analysis clustered the studied strains into 3 major clusters, showing strong correspondence to the 3 phyloproteiomic groups that were established by the dendrogram. Both clustering analysis means have evidently demonstrated a relationship between known Virgibacillus strains and other related bacteria based on profiling of their synthesized proteins. Thus, larger populations of bacteria in mats can be easily screened for their potential to exhibit certain activities, which is of ecological, environmental and biotechnological significance.