Journal articles: 'Separations of proteins mixture'

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Hassan, Sammer-ul. "Microchip Electrophoresis." Encyclopedia 1, no. 1 (December 23, 2020): 30–41. http://dx.doi.org/10.3390/encyclopedia1010006.

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Microchip electrophoresis (MCE) is a miniaturized form of capillary electrophoresis. Electrophoresis is a common technique to separate macromolecules such as nucleic acids (DNA, RNA) and proteins. This technique has become a routine method for DNA size fragmenting and separating protein mixtures in most laboratories around the world. The application of higher voltages in MCE achieves faster and efficient electrophoretic separations.

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Zagst, Holger, Christin Elgert, Sönke Behrends, and Hermann Wätzig. "Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures." Analytical and Bioanalytical Chemistry 414, no. 4 (December 6, 2021): 1699–712. http://dx.doi.org/10.1007/s00216-021-03797-4.

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AbstractTwo-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified. Graphical abstract .

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Pathak, Karishma. "Preliminary Phytochemical Screening and Thin Layer Chromatography of Different Solvent Extract of Bark of Ficus lacor (Pakar)." Journal of Drug Discovery and Development 5, no. 1 (February 24, 2022): 12–16. http://dx.doi.org/10.24321/2581.6861.202102.

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Medicinal plant used for many disease treatments in the form of extract. This study performed for confirmation the different solvents extract such as alcoholic, hydroalcoholic, aqueous. An herbal plant Ficus lacor uses different parts for treating various diseases such a seed, leaves, fruits, roots, berries and bark. In this research perform the Phytochemical activities and also perform thin layer chromatography of extract were performed at SHEAT college laboratory. Phytochemical screening resultant a various chemical constituents are found such as: Carbohydrates, Tannins and Phenolic, Alkaloids, Anthraquinones glycoside, Amino Acid, proteins, Reducing Sugar. Thin layer chromatography for analyzed the mixture by separations of a compounds this test are successfully performed for the expected out come as antiulcer activity.

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McDonald, W. Hayes, and John R. Yates. "Shotgun Proteomics and Biomarker Discovery." Disease Markers 18, no. 2 (2002): 99–105. http://dx.doi.org/10.1155/2002/505397.

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Coupling large-scale sequencing projects with the amino acid sequence information that can be gleaned from tandem mass spectrometry (MS/MS) has made it much easier to analyze complex mixtures of proteins. The limits of this “shotgun” approach, in which the protein mixture is proteolytically digested before separation, can be further expanded by separating the resulting mixture of peptides prior to MS/MS analysis. Both single dimensional high pressure liquid chromatography (LC) and multidimensional LC (LC/LC) can be directly interfaced with the mass spectrometer to allow for automated collection of tremendous quantities of data. While there is no single technique that addresses all proteomic challenges, the shotgun approaches, especially LC/LC-MS/MS-based techniques such as MudPIT (multidimensional protein identification technology), show advantages over gel-based techniques in speed, sensitivity, scope of analysis, and dynamic range. Advances in the ability to quantitate differences between samples and to detect for an array of post-translational modifications allow for the discovery of classes of protein biomarkers that were previously unassailable.

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Sirkisoon, Leona R., Honest C. Makamba, Shingo Saito, and Christa L. Colyer. "Carbon Dot-Mediated Capillary Electrophoresis Separations of Metallated and Demetallated Forms of Transferrin Protein." Molecules 24, no. 10 (May 18, 2019): 1916. http://dx.doi.org/10.3390/molecules24101916.

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Carbon dots (CDs) are fluorescent nanomaterials used extensively in bioimaging, biosensing and biomedicine. This is due in large part to their biocompatibility, photostability, lower toxicity, and lower cost, compared to inorganic quantum dots or organic dyes. However, little is known about the utility of CDs as separation adjuvants in capillary electrophoresis (CE) separations. CDs were synthesized in-house according to a ‘bottom-up’ method from citric acid or other simple carbon precursors. To demonstrate the applicability of CDs as separation adjuvants, mixtures of holo- (metallated) and apo- (demetallated) forms of transferrin (Tf, an iron transport protein) were analyzed. In the absence of CDs, the proteins were not resolved by a simple CE method; however, upon addition of CDs to the separation buffer, multiple forms of Tf were resolved indicating that CDs are valuable tools to facilitate the separation of analytes by CE. CE parameters including sample preparation, buffer identity, ionic strength, pH, capillary inside diameter, and temperature were optimized. The results suggest that dots synthesized from citric acid provide the best resolution of various different forms of Tf and that CDs are versatile and promising tools to improve current electrophoretic separation methods, especially for metalloprotein analysis.

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Jun, Sei-Hwan, and Eli Ruckenstein. "Separation of a Multicomponent Mixture of Proteins by Potential Barrier Chromatography (PBC)." Separation Science and Technology 21, no. 2 (April 1986): 111–38. http://dx.doi.org/10.1080/01496398608058369.

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Hasan, Farhana, Punprabhashi Vidanapathirana, Susmita Das, Vivian E. Fernand, Noureen Siraj, Jack N. Losso, and Isiah M. Warner. "Ionic liquids as buffer additives in ionic liquid-polyacrylamide gel electrophoresis separation of mixtures of low and high molecular weight proteins." RSC Advances 5, no. 85 (2015): 69229–37. http://dx.doi.org/10.1039/c5ra11559k.

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Zaccheria, Federica, Matteo Mariani, Nicola Scotti, Rinaldo Psaro, and Nicoletta Ravasio. "Catalytic upgrading of lactose: a rest raw material from the dairy industry." Green Chemistry 19, no. 8 (2017): 1904–10. http://dx.doi.org/10.1039/c7gc00741h.

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Lactose, a residue from the separation of high value-added proteins from whey, was converted into an equimolar mixture of sorbitol and dulcitol through a one-step cascade hydrolysis plus hydrogenation process.

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Guerrera, Ida Chiara, and Oliver Kleiner. "Application of Mass Spectrometry in Proteomics." Bioscience Reports 25, no. 1-2 (February 4, 2005): 71–93. http://dx.doi.org/10.1007/s10540-005-2849-x.

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Mass spectrometry has arguably become the core technology in proteomics. The application of mass spectrometry based techniques for the qualitative and quantitative analysis of global proteome samples derived from complex mixtures has had a big impact in the understanding of cellular function. Here, we give a brief introduction to principles of mass spectrometry and instrumentation currently used in proteomics experiments. In addition, recent developments in the application of mass spectrometry in proteomics are summarised. Strategies allowing high-throughput identification of proteins from highly complex mixtures include accurate mass measurement of peptides derived from total proteome digests and multidimensional peptide separations coupled with mass spectrometry. Mass spectrometric analysis of intact proteins permits the characterisation of protein isoforms. Recent developments in stable isotope labelling techniques and chemical tagging allow the mass spectrometry based differential display and quantitation of proteins, and newly established affinity procedures enable the targeted characterisation of post-translationally modified proteins. Finally, advances in mass spectrometric imaging allow the gathering of specific information on the local molecular composition, relative abundance and spatial distribution of peptides and proteins in thin tissue sections.

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Haribabu, Malavika, Dave E. Dunstan, Gregory J. O. Martin, Malcolm R. Davidson, and Dalton J. E. Harvie. "Simulating the ultrafiltration of whey proteins isolate using a mixture model." Journal of Membrane Science 613 (November 2020): 118388. http://dx.doi.org/10.1016/j.memsci.2020.118388.

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Hidayat, Rachmat, and Patricia Wulandari. "Western Blotting (WB) Technique Guideline for Separation and Isolation of Protein." Bioscientia Medicina : Journal of Biomedicine and Translational Research 5, no. 2 (January 29, 2021): 359–72. http://dx.doi.org/10.32539/bsm.v5i2.229.

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A B S T R A C TWestern blotting is an important technique used in cell and molecularbiology. Using the western blot, researchers can identify specific proteinsfrom the complex mixture of proteins extracted from cells. This techniqueuses three elements to accomplish this task: (1) separation by size, (2)transfer to a solid support, and (3) marking target proteins using appropriateprimary and secondary antibodies to visualize. This paper will attempt toexplain the techniques and theory behind western blot, and offer severalways to solve the problem

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Hidayat, Rachmat, and Patricia Wulandari. "Western Blotting (WB) Technique Guideline for Separation and Isolation of Protein." Bioscientia Medicina : Journal of Biomedicine and Translational Research 5, no. 4 (January 29, 2021): 367–80. http://dx.doi.org/10.37275/bsm.v5i4.229.

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A B S T R A C TWestern blotting is an important technique used in cell and molecularbiology. Using the western blot, researchers can identify specific proteinsfrom the complex mixture of proteins extracted from cells. This techniqueuses three elements to accomplish this task: (1) separation by size, (2)transfer to a solid support, and (3) marking target proteins using appropriateprimary and secondary antibodies to visualize. This paper will attempt toexplain the techniques and theory behind western blot, and offer severalways to solve the problem

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Ostrihoňová, Marta, Jana Adamíková, Tomáš Molnár, Monika Antošová, and Milan Polakovič. "Recombinant human erythropoietin separation using a cation-exchange multimodal adsorbent." Acta Chimica Slovaca 12, no. 1 (April 1, 2019): 103–7. http://dx.doi.org/10.2478/acs-2019-0015.

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Abstract This work deals with the capture of human recombinant erythropoietin (rhEPO) from a mixture of proteins in a concentrated postcultivation supernatant. Cation-exchange multimodal adsorbent Capto MMC ImpRes was selected as potential chromatographic separation material. Its equilibrium properties were investigated in batch adsorption experiments. The effect of pH in the range of 5.5—7.5 and NaCl concentration in the range of 0—300 mM on the adsorption of rhEPO and contaminant proteins was examined. Optimal conditions found in these equilibrium experiments were applied to rhEPO adsorption in a chromatographic column. Several experiments were carried out at different elution conditions to optimize the rhEPO yield and selectivity.

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Pecha, Jiří, Jakub Husár, Miloš Jelínek, Lubomír Šánek, and Karel Kolomazník. "Optimization of lupine hydrolyzate separation." MATEC Web of Conferences 292 (2019): 01026. http://dx.doi.org/10.1051/matecconf/201929201026.

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Lupine hydrolyzate is a promising source of proteins. Hydrolysis of lupine flour was studied under various conditions and their influence on reaction mixture separation by means of filtration was assessed. A mathematical model describing separation process was suggested and verified. This model was used in further calculations and process simulations. It was shown, that the filtration largely depends on the molar mass distribution, respectively the degree of hydrolysis. In addition, an approach enabling optimizing filtration was presented. The time of filtration performed at optimal conditions was almost ten times decreased.

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Tsybin, Youri O., Per Håkansson, Magnus Wetterhall, Karin E. Markides, and Jonas Bergquist. "Capillary Electrophoresis and Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Peptide Mixture and Protein Digest Analysis." European Journal of Mass Spectrometry 8, no. 5 (October 2002): 389–95. http://dx.doi.org/10.1255/ejms.514.

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Recent advances in peptide fragmentation techniques and mass spectrometry have opened up the possibility of combining peptide separation techniques, such as capillary electrophoresis (CE) and capillary liquid chromatography, with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and electron capture dissociation (ECD) in order to characterize peptide mixtures and protein digests. The results presented in this study show that CE/ECD-FT-ICR MS can be employed for peptide characterization in mixtures of standard peptides and in peptides resulting from the enzymatic digestion of proteins. Furthermore, the technique has potential for the identification and localization of post-translational modifications in peptides and proteins.

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Stan-Lotter, Helga, and Philip D. Bragg. "Electrophoretic determination of sulfhydryl groups and its application to complex protein samples, in vitro protein synthesis mixtures, and cross-linked proteins." Biochemistry and Cell Biology 64, no. 2 (February 1, 1986): 154–60. http://dx.doi.org/10.1139/o86-024.

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Without prior fractionation, the number of sulfhydryl groups of individual polypeptides in a protein mixture can be determined, provided their molecular weights and approximate isoelectric points are known. Urea-denatured protein samples are reacted with iodoacetamide and iodoacetate in a modified version of Creighton's procedure. After separation by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and isoelectric focusing, the number of sulfhydryl groups is determined by counting the protein bands which have additional negative charges. This method requires little material and provides an additional parameter, besides the molecular weight and isoelectric point, for the identification and characterization of a protein. The sensitivity may be enhanced for nonradioactive proteins by using 14C-labeled iodoacetamide and iodoacetate. The procedure has been applied to prokaryotic in vitro protein synthesis mixtures, bacterial membrane protein, and trypsin-cleaved or chemically cross-linked subunits of the F1 ATPase from Escherichia coli.

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TENG, M., S. LIN, C. WU, and R. JUANG. "Factors affecting selective rejection of proteins within a binary mixture during cross-flow ultrafiltration." Journal of Membrane Science 281, no. 1-2 (September 15, 2006): 103–10. http://dx.doi.org/10.1016/j.memsci.2006.03.019.

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Bossa, Guilherme, Sean Gunderson, Rachel Downing, and Sylvio May. "Role of Transmembrane Proteins for Phase Separation and Domain Registration in Asymmetric Lipid Bilayers." Biomolecules 9, no. 8 (July 25, 2019): 303. http://dx.doi.org/10.3390/biom9080303.

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It is well known that the formation and spatial correlation of lipid domains in the two apposed leaflets of a bilayer are influenced by weak lipid–lipid interactions across the bilayer’s midplane. Transmembrane proteins span through both leaflets and thus offer an alternative domain coupling mechanism. Using a mean-field approximation of a simple bilayer-type lattice model, with two two-dimensional lattices stacked one on top of the other, we explore the role of this “structural” inter-leaflet coupling for the ability of a lipid membrane to phase separate and form spatially correlated domains. We present calculated phase diagrams for various effective lipid–lipid and lipid–protein interaction strengths in membranes that contain a binary lipid mixture in each leaflet plus a small amount of added transmembrane proteins. The influence of the transmembrane nature of the proteins is assessed by a comparison with “peripheral” proteins, which result from the separation of one single integral protein into two independent units that are no longer structurally connected across the bilayer. We demonstrate that the ability of membrane-spanning proteins to facilitate domain formation requires sufficiently strong lipid–protein interactions. Weak lipid–protein interactions generally tend to inhibit phase separation in a similar manner for transmembrane as for peripheral proteins.

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Xia, Hongjun, Huaiming Wang, Jianshan Wang, Lin Wang, Lin Jin, and Quan Bai. "Preparation of Core–Shell Silica Microspheres with Controllable Shell Thickness for Fast High Performance Liquid Chromatography Separation of Alkyl Benzenes and Intact Proteins." Journal of Biomedical Nanotechnology 17, no. 3 (March 1, 2021): 439–46. http://dx.doi.org/10.1166/jbn.2021.3042.

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As it is difficult to prevent secondary nucleation and agglomeration during the preparation of core–shell silica microspheres, these issues have been successfully resolved in this study using template-dissolution-induced redeposition. The non-porous particles are transformed into core–shell silica microspheres (CSSMs) in the presence of cetyltrimethylammonium bromide and octyltrimethylammonium bromide under basic conditions. The shell thickness and pore sizes of the CSSMs are controlled by adjusting the etching time and molar ratio of the template, respectively. The CSSMs are modified using octadecyltrimethylammonium chloride to separate the mixture of alkyl benzenes, and a high column separation efficiency is achieved within two minutes. The CSSMs are used for the separation and analysis of proteins and the digests of bovine serum albumin. The chromatographic column packed with core–shell particles affords a significantly higher separation efficiency than the commercial column. Therefore, as a chromatographic stationary phase, these core–shell particles can potentially be used for the fast separation of proteins, small solutes, and complex samples.

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Abdelrasoul, Amira, Ning Zhu, and Ahmed Shoker. "Investigation on Human Serum Protein Depositions Inside Polyvinylidene Fluoride-Based Dialysis Membrane Layers Using Synchrotron Radiation Micro-Computed Tomography (SR-μCT)." Membranes 13, no. 1 (January 16, 2023): 117. http://dx.doi.org/10.3390/membranes13010117.

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Hemodialysis (HD) membrane fouling with human serum proteins is a highly undesirable process that results in blood activations with further severe consequences for HD patients. Polyvinylidene fluoride (PVDF) membranes possess a great extent of protein adsorption due to hydrophobic interaction between the membrane surface and non-polar regions of proteins. In this study, a PVDF membrane was modified with a zwitterionic (ZW) polymeric structure based on a poly (maleic anhydride-alt-1-decene), 3-(dimethylamino)-1-propylamine derivative and 1,3-propanesultone. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), and zeta potential analyses were used to determine the membrane’s characteristics. Membrane fouling with human serum proteins (human serum albumin (HSA), fibrinogen (FB), and transferrin (TRF)) was investigated with synchrotron radiation micro-computed tomography (SR-μCT), which allowed us to trace the protein location layer by layer inside the membrane. Both membranes (PVDF and modified PVDF) were detected to possess the preferred FB adsorption due to the Vroman effect, resulting in an increase in FB content in the adsorbed protein compared to FB content in the protein mixture solution. Moreover, FB was shown to only replace HSA, and no significant role of TRF in the Vroman effect was detected; i.e., TRF content was nearly the same both in the adsorbed protein layer and in the protein mixture solution. Surface modification of the PVDF membrane resulted in increased FB adsorption from both the protein mixture and the FB single solution, which is supposed to be due to the presence of an uncompensated negative charge that is located at the COOH group in the ZW polymer.

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Mazzei, Rosalinda, Anna Maria Szymczak, Enrico Drioli, Mohamed Al-Fageeh, Mohammed A. Aljohi, and Lidietta Giorno. "High Purity of α-Lactalbumin from Binary Protein Mixture by Charged UF Membrane Far from the Isoelectric Point to Limit Fouling." Applied Sciences 11, no. 19 (October 2, 2021): 9167. http://dx.doi.org/10.3390/app11199167.

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Separation and high recovery factor of proteins similar in molecular mass is a challenging task, and heavily studied in the literature. In this work, a systematic study to separate a binary protein mixture by charged ultrafiltration membranes without affecting membrane performance was carried out. α-lactalbumin (ALA, 14.4 kDa) and β-lactoglobulin (BLG, 18.4 kDa) were used as a binary model system. These two proteins are the main proteins of whey, a very well-known byproduct from the dairy industry. Initially, a systematic characterization of individual proteins was carried out to determine parameters (protein size and aggregation, zeta potential) which could influence their passage through a charged membrane. Then, the influence of operating parameters (such as initial protein concentration, pH, and critical pressure) on the UF process was investigated, so as to identify conditions that limit membrane fouling whilst maximizing protein recovery factor and purity. The study permitted to identify process conditions able to fully separate ALA from BLG, with high purity (95%) and recovery factor (80%), in a single UF step. Compared to studies reported in literature, here, the main approach used was to carry out a charged UF process far from proteins isoelectric point (pI) to limit protein aggregation and membrane fouling.

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Calinoiu, Lavinia Florina, Dan Cristian Vodnar, and Carmen Socaciu. "The Reactivity and Allergenic Potential of Hazelnut Peptides." Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Food Science and Technology 70, no. 1 (November 13, 2013): 25. http://dx.doi.org/10.15835/buasvmcn-fst:9506.

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The aim of this paper was to focus on proteins present in some food products, like hazelnuts and to investigate their allergenic potential. Several techniques were used to characterize these extracted proteins, with respect to their composition, degradability by digestive proteolytic enzymes and their reactivity with specific antibodies. It was important to analyse which proteins were present in the hazelnuts, to see if there were proteins present to trigger an allergic reaction and if the digestion enzymes trypsin and pepsin influence the presence of the (allergic) protein compounds. Allergies to tree nuts and seeds can cause life-threatening and sometimes fatal reactions. To examine the properties of Hazelnut protein it was important to solubilize it by extraction. After extraction, it was investigated how hazelnut protein can be modified by proteases and what the effect was on the immune reaction. The Bradford method is a fast and sensitive method to determine the concentration of soluble protein. When the Bradford reagent (Coomassie Brilliant Blue) binds to the protein, the colour changes from red to purple and the absorption maximum changes from 495 to 595 nm. The value obtained as the final concentration of proteins was 7.3495. SDS-PAGE is a method to separate mixtures of proteins by electrophoresis. Protein molecules are negatively charged by binding of SDS molecules; subsequently they are separated in an electric field. Their differences in size (molecular weight) leads to separation. In this case the method is used to follow proteolytic degradation of hazelnut proteins (allergens) by intestinal proteases (trypsin, pepsin). A different, more specific and sensitive method is immunoblotting (Western Blot) in which the SDS-PAGE separated proteins are transferred from the gel to a membrane and specific antibodies are used in a series of reactions to visualize specific allergens on this membrane. The remarked spots represented a positive identification of allergenic proteins. This means that peptide fragments of various size, produced during the digestion of a protein can still be immunological active. As it was shown there was still reactivity between proteins and specific antibodies. The Dot Blot is a simple immunoblotting technique used to detected specific proteins in a mixture of different proteins and/or other molecules. No separation technique prior to the actual immuno-detection is necessary. Also, Dot Blot confirmed the presence of allergenic proteins made visible through the light spots on the membrane.

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Bandara, Asanga, Afra Panahi, George A. Pantelopulos, Tetsuro Nagai, and John E. Straub. "Exploring the impact of proteins on the line tension of a phase-separating ternary lipid mixture." Journal of Chemical Physics 150, no. 20 (May 28, 2019): 204702. http://dx.doi.org/10.1063/1.5091450.

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Kussmann, Martin, and Peter Roepstorff. "Characterisation of the covalent structure of proteins from biological material by MALDI mass spectrometry ‣ possibilities and limitations." Spectroscopy 14, no. 1 (1998): 1–27. http://dx.doi.org/10.1155/1998/710163.

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Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) has become a primary tool for the detailed characterisation of the covalent structure of proteins isolated from biological material, mainly because of its following potentials: high sensitivity and specificity, speed of analysis, appropriateness for mixture analysis, high tolerance towards contaminants, and compatibility with separation techniques, e.g., gel electrophoresis. These characteristics enable the structural analysis of proteins even if they are only available in limited amounts and/or in mixtures, and even if the protein preparations contain large amounts of salts, buffers, detergents and denaturants. Additionally, structural data can be generated within a relatively short time.Whereas X-ray crystallography and multidimensional NMR techniques can provide “absolute” structural data, i.e., a three-dimensional “picture” of the protein of interest, MALDI-MS-especially in combination with selective protein chemistry – yields information on particular aspects of the entire protein structure, e.g., primary structure, active site(s), binding sites, and posttranslational modifications, all of which are often of crucial interest for the understanding of the protein function. Taking into account that protein crystallography and protein NMR studies require large quantities of highly purified sample, MALDI-MS can be even more regarded as a powerful complement in protein structure analysis.This review aims at describing the state-of-the-art of MALDI-MS for characterisation of proteins from biological material by evaluating its potential and limitations.

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Ali, Ashraf, Sarah Alharthi, Nora Hamad Al-Shaalan, and Eman Y. Santali. "Development of Narrow-Bore C18 Column for Fast Separation of Peptides and Proteins in High-Performance Liquid Chromatography." Polymers 14, no. 13 (June 25, 2022): 2576. http://dx.doi.org/10.3390/polym14132576.

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Separation with high efficiency and good resolution is constantly in demand in the pharmaceutical industry. The fast and efficient separation of complex samples such as peptides and proteins is a challenging task. To achieve high efficiency with good resolution, chromatographers are moving towards small particles packed into narrow-bore columns. Silica monolith particles (sub-2 µm) were derivatized with chlorodimethyl octadecyl silane (C18) and packed into stainless steel columns (100 mm × 1.8 mm i.d) by a slurry-packing method. The developed columns were used for the separation of peptides and proteins. A separation efficiency (N) of 40,000 plates/column (400,000 plates/m) was achieved for the mixture of five peptides. Similarly, the fast separation of the peptides was carried out using a high flow rate, and the separation of the five peptides was achieved in one minute with high efficiency (N ≅ 240,000 plates/m). The limit of detection (DL) and the limit of quantification (QL) for each analyte were determined by developing a linear regression curve with relatively very low concentrations of the target compound. The average values of the QL for the peptide and proteins were 0.55 ng and 0.48 ng, respectively, using short C18 column (1.8 mm × 100 mm) UV (at 214 nm). The fast analysis of peptides and proteins with such high efficiency and good resolution has not been reported in the literature yet. Owing to high efficiency, these home-made columns could be used as an alternative to the expensive commercial columns for peptide and protein separation.

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Winterbourn, C. C., and A. L. Molloy. "Susceptibilities of lactoferrin and transferrin to myeloperoxidase-dependent loss of iron-binding capacity." Biochemical Journal 250, no. 2 (March 1, 1988): 613–16. http://dx.doi.org/10.1042/bj2500613.

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Apolactoferrin and apotransferrin lost their ability to subsequently bind iron when exposed to an excess of either HOCl or myeloperoxidase plus H2O2 and Cl-. Apolactoferrin, however, was more resistant than apotransferrin. By oxidizing a mixture of the two proteins, then separating them by immunoprecipitation, the difference in susceptibility was shown to be due to the greater reactivity of transferrin iron-binding groups, rather than protective groups on the lactoferrin molecule. The iron-saturated proteins were much more resistant to oxidative modification than the apoproteins. The greater resistance of apolactoferrin should be advantageous for maintaining its iron binding capacity when co-released with myeloperoxidase and reactive oxygen species from stimulated neutrophils.

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Sajjadi, Sayyed Hashem, Shang-Jung Wu, Vitalijs Zubkovs, Hossein Ahmadzadeh, Elaheh K. Goharshadi, and Ardemis Anoush Boghossian. "A Simple Micropreparative Gel Electrophoresis Technique for Purification of Nanoscale Materials." ECS Meeting Abstracts MA2022-01, no. 12 (July 7, 2022): 849. http://dx.doi.org/10.1149/ma2022-0112849mtgabs.

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Many biochemical, biomedical, and material applications hinge on the ability to effectively separate and purify nanoscale materials. Though this need is largely addressed with biological macromolecules using a variety of chromatographic and electrophoretic purification techniques, such techniques are usually laborious, time-consuming, and often require complex and costly instalments that are inaccessible to most laboratories. Synthetic nanoparticles face similar purification challenges, often relying on techniques that are material-specific. In this work, we introduce a versatile micro-preparative (MP) method based on polyacrylamide gel electrophoresis (PAGE) to purify biological samples containing proteins, nucleic acids, and complex bioconjugates, as well as synthetic nanoparticles based on graphene quantum dots (GQDs). Using a conventional vertical slab PAGE, we demonstrate the extraction of purified DNA, proteins, and DNA-protein bioconjugates from their respective mixtures using MP-PAGE. We apply this system to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using specialized commercial devices. We also demonstrate the purification of folded enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, comparable to purities achieved using a two-step size exclusion and immobilized metal-ion affinity chromatography purification procedure. Moreover, we demonstrate the successful isolation of an EYFP-DNA bioconjugate that otherwise could not be processed using the two-step chromatography procedure. Finally, the technique was further extended to demonstrate size-dependent separation of a commercial mixture of GQDs into three different fractions with distinct optical properties. MP-PAGE thus offers a rapid and versatile means of purifying biological and synthetic nanomaterials without the need for specialized equipment.

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Fang, Mei, Rui Zhao, Li Shen, Tianshen Su, and Guoquan Liu. "SEPARATION OF FOUR BASIC PROTEINS IN THE MIXTURE BY CAPILLARY ELECTROPHORESIS WITH A NEW CHEMICAL MODIFICATION COLUMN." Analytical Letters 35, no. 2 (February 13, 2002): 397–411. http://dx.doi.org/10.1081/al-120002538.

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Talian, Ivan, Veronika Kováčová, Marián Petrovič, and Ján Sabo. "Impact of un-polymerized acrylamide monomer residues onto protein identification by MALDI TOF MS." Open Chemistry 10, no. 4 (August 1, 2012): 1073–78. http://dx.doi.org/10.2478/s11532-012-0024-3.

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AbstractPolyacrylamide gel electrophoresis is a powerful tool for protein mixture separation frequently used in proteomic studies. This article describes the problems which can arise during the identification process of separated proteins by matrix assisted laser desorption/ionization mass spectrometry. Presence of residual acrylamide monomers in the peptide samples leads to a significant peptide signal suppression. In the case of protein prohibitin, isolated from the MCF 7 breast cancer cell line, the peptide signal suppression was observed in 50% of all detected peptides.

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Adamovic, Milan, Horea Samanc, Danijela Kirovski, Ivan Vujanac, and Olivera Valcic. "Effects of buffering mineral mixtures on milk yield, milk composition, rumen pH and some blood biochemical parameters in heat stressed dairy cows." Veterinarski glasnik 68, no. 1-2 (2014): 31–42. http://dx.doi.org/10.2298/vetgl1402031a.

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The objective of the work was to investigate the influence of partial substitution of magnesium oxide with natural bentonite in feed mixtures used in feeding of cows during their exposure to heat stress. The investigation lasted 30 days and was carried out during last ten days of may and first twenty days of June when average air temperature in stables was 36.6?2.5oC. In the experiment there were included 30 cows of Holstein breed in first phase of lactation, which were divided into two groups of 15 cows: control (C) and experimental (E). Group C was fed with experimental mineral mixture that contained 60% of magnesium oxide during the whole investigation period. Group E was fed with experimental mineral mixture that contained 40% of magnesium oxide as well as 20% of natural bentonite. Remaining ingredients in both control and experimental mineral mixtures were the same and also contained 20% of sodium bicarbonate and 20% of zeolite in the same quantities. The control and experimental mineral mixtures were mixed into complete feed mixture (18% UP) in the amount of 1%. At tne end of the investigation period, on the 30th day, there were taken samples of rumens contents for determining pH, and after that blood samples, in which, after the separation of blood serum, were determined glucose concentration, total proteins, albumin, globulin, urea, HDL cholesterol, LDL cholesterol, total bilirubin, calcium and phosphorus, as well as the activity of ALT and AST. By computation there was calculated the ratio between albumin and globulin, ALT and AST, and the ratio between calcium and phosphorus. Daily allowance and milk chemical composition ( percentage of fat, proteins and dry substance) were determined at the end of the investigation period, that is on the 30th day of lactation, for each cow individually. Partial substitution of magnesium oxide with bentonite influenced milk production increase, but it was statistically insignificant. Besides that, in E group of cows, percentage of fat and dry substance in milk was significantly increased (p<0.05 and 0.01 respectively), while percentage of protein increase was insignificant. The substitution of magnesium oxide with bentonite had no impact on the values of examined parameters of metabolic profile, but it led to statistically significant increase of rumen contents pH values (p<0.05). From the obtained results it can be concluded that substitution of magnesium oxide with bentonite in feed mixtures that are used for feeding cows during summer period, can prevent rumen acidosis, which high yielding cows incline to under the conditions of elevated external temperatures. Besides that, this kind of substitution leads to improvement of milk composition, especially when percentage of fat and dry substance in milk is concerned.

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Konuspayev, Saparkali, Batiha Kassenova, Zauresh Akhatova, and Roza Nurbaeva. "Alkaline hydrolysis of wool fat (lanolin) in a medium of proton and aprotic solvents." Chemical Bulletin of Kazakh National University, no. 1 (March 30, 2018): 4–9. http://dx.doi.org/10.15328/cb978.

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The raw material being studied is the woolen fat of the sheep of the Edilbay fine-fleece and Kazakh arkharomeric fine-fleece, which is excreted when washing wool in primary wool processing plants (PWP) in the regions of Kazakhstan, such as Semipalatinsk, Aktyubinsk, Zhambyl and Tokmak. Earlier we obtained anhydrous lanolin from the fat of various factories of the PWP. In both cases, positive results were obtained and a certificate of compliance of anhydrous lanolin FS RK was obtained. In terms of its chemical composition, wool fat is a mixture of C10-C16 carboxylic acid esters with aliphatic, terpenic, triterpene and sterol alcohols. It also contains vitamins, proteins, sterols and other physiologically active compounds. In the hydrolysis of wool fat, a mixture of sterol alcohols, triterpene alcohols and fatty acid salts are assumed. Valuable among them are sterol alcohols, which constitute up to 29% of the sum of all alcohols. Cholesterol and its derivatives are the raw materials for the synthesis of steroid drugs. Salts of fatty acids are used as an emulsifier in pharmacy and cosmetology. The aim of this paper is to complete the saponification of wool fat and the separation of a mixture of sterol alcohols. We show the patterns of alkaline hydrolysis of wool fat in the liquid phase in the presence of mixtures of various solvents. As a solvent, the ethanol-water, isopropanol-water system in which wool fat is only partially dissolved has been studied. In the wool fat-alcohol-water-NaOH system, a stable emulsion is formed. Ways that prevent the formation of an emulsion are proposed.

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Fare, Charlotte M., Alexis Villani, Lauren E. Drake, and James Shorter. "Higher-order organization of biomolecular condensates." Open Biology 11, no. 6 (June 2021): 210137. http://dx.doi.org/10.1098/rsob.210137.

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A guiding principle of biology is that biochemical reactions must be organized in space and time. One way this spatio-temporal organization is achieved is through liquid–liquid phase separation (LLPS), which generates biomolecular condensates. These condensates are dynamic and reactive, and often contain a complex mixture of proteins and nucleic acids. In this review, we discuss how underlying physical and chemical processes generate internal condensate architectures. We then outline the diverse condensate architectures that are observed in biological systems. Finally, we discuss how specific condensate organization is critical for specific biological functions.

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Syaubari, Syaubari, and S. Nurdin. "Numerical Solution Of Electrokinetics Mass Transfer Model For Protein Recovery Through Membrane Electrofilter." REAKTOR 7, no. 02 (June 19, 2017): 66. http://dx.doi.org/10.14710/reaktor.7.02.66-69.

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Separation based on electrophoresis and electroosmosis (electrokinetics) of binary mixture of proteins (bovine serum albumin-hemoglobin) was studied on a membrane electrofilter. The mixture was separated using ionic polycarbonate membrane with variable studied consist of voltage, current, protein diffusivity, and electrophoresis mobility. Operation parameters were varied to investigate hemoglobin concentration, which pass semi permeable membrane. A model was been derived based on mass transfer principle for the case of unsteady state. For simplification, the model has been modified using Cramer Method with pseudo steady state approach to give the dimentionless form. A program for computer simulation has een written in C/C+ + language. This programming language was shown to have more effective computing ability. Furthermore, using a model and simulation on computer, the result indicates that initial mechanism of electrofilter can also be used to separate and to concentrate protein on their buffer solution.Keywords : electrophoresis, electroosmosis, protein, membrane, electrofilter

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Wolny, Anna, and Anna Chrobok. "Ionic Liquids for Development of Heterogeneous Catalysts Based on Nanomaterials for Biocatalysis." Nanomaterials 11, no. 8 (August 10, 2021): 2030. http://dx.doi.org/10.3390/nano11082030.

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The development of effective methods of enzyme stabilization is key for the evolution of biocatalytic processes. An interesting approach combines the stabilization process of proteins in ionic liquids and the immobilization of the active phase on the solid support. As a result, stable, active and heterogeneous biocatalysts are obtained. There are several benefits associated with heterogeneous processes, as easy separation of the biocatalyst from the reaction mixture and the possibility of recycling. Accordingly, this work focused on the supported ionic liquid phases as the efficient enzyme stabilization carriers, and their application in both continuous flow and batch biocatalytic processes.

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Eudier, H., S. Ben-Harb, J. P. Lorand, F. Duthil, M. Negahban, J. M. Saiter, and M. Chan-Huot. "Properties Of An Analogue Cheese Obtained From Raw Peanut." Peanut Science 47, no. 2 (May 28, 2020): 81–88. http://dx.doi.org/10.3146/ps20-1.1.

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ABSTRACT The focus is on a peanut suspension in which starch is added and that exhibits specific mechanical characteristics relevant for food products. The mixture is composed of water, lipids, starch, and proteins. The process consists of blending together the different constituents, and the study changes the experimental conditions to tune the mechanical behavior of the mixture. The rheological properties (viscosity, indentation) and physical parameters such as color, dry extract, and particle size distribution were measured. The matrix behavior was studied after a centrifugation step necessary to determine stability of the emulsion, and for varying shearing durations. Short shearing duration induce a maximum of firmness, observed by measuring indentation resistance, and a maximum of spreadability, evaluated by shear rheometry. On the contrary, long shearing durations destabilize the matrix emulsion by increasing the oil separation capacity. This study observes structural changes in the rheological behavior of this analogue artificial cheese that correlates with the extent of shearing.

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Udeani, George O., Joeby Bass, and Thomas P. Johnston. "Compatibility of Oral Morphine Sulfate Solution with Enteral Feeding Products." Annals of Pharmacotherapy 28, no. 4 (April 1994): 451–55. http://dx.doi.org/10.1177/106002809402800404.

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OBJECTIVE: Patients with terminal cancer often receive continuous enteral nutrition and oral medications concomitantly through nasogastric or gastrostomy feeding tubes. This study evaluated in vitro the compatibility of morphine sulfate (MS) solution 2 mg/mL (Roxane Laboratories) with three enteral nutrition products (Jevity [J], Osmolite-HN [O], and Pulmocare [P]) at 22 and 37 °C for 48 hours and J alone at 50 °C for 48 hours. METHODS: Mixtures containing 1 mg/mL MS were prepared with each enteral product: J, O, and P (Ross Laboratories). Serial samples (1 mL) were collected from each mixture and analyzed for morphine by reverse-phase HPLC. An analog pH meter was used to measure the pH of each mixture at scheduled intervals. RESULTS: The addition of MS 2 mg/mL (MS2) to J caused an immediate decrease in pH from 6.24 ± 0.01 to 4.96 ± 0.05 and a noticeable precipitate/phase separation. No phase separation was observed with a 1 mg/mL mixture of MS and J, O, and P when they were prepared with a more concentrated MS solution (20 mg/mL, MS20) (Roxanol Intensol). The pH declined linearly for all three enteral feeding products as the MS20 concentration was increased from 0 to 1 mg/mL. The precipitate observed in the mixture of MS2 with J was probably caused by the decrease in pH, which was caused by the greater volume fraction of MS solution. The concentration of morphine in the supernatant of a MS2/J solution was 0.83 mg/mL, and the concentration of MS in a homogeneous MS20/J solution was 0.86 mg/mL. At 48 hours, there was negligible (<2 percent) morphine decomposition in all MS admixtures at all temperatures. No microbial growth was observed in any admixture at 24 hours. Electrophoretic analysis demonstrated equal protein migration and molecular weight distribution for J and MS/J solutions. CONCLUSIONS: MS is stable in enteral feeding solutions at temperatures from 22 to 50 °C. MS2 is associated with a pH-dependent protein precipitation (but not destruction of the proteins), resulting in disproportionate concentrations of morphine in the supernatant and precipitate. This incompatibility may adversely affect enteral feeding analgesic delivery. We, therefore, recommend MS 20 mg/mL for this method of oral MS delivery, because of its superior compatibility and stability with enteral feeding products.

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Ratnaningsih, Enny, Reynard Reynard, Khoiruddin Khoiruddin, I. Gede Wenten, and Ramaraj Boopathy. "Recent Advancements of UF-Based Separation for Selective Enrichment of Proteins and Bioactive Peptides—A Review." Applied Sciences 11, no. 3 (January 25, 2021): 1078. http://dx.doi.org/10.3390/app11031078.

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Proteins are one of the primary building blocks that have significant functional properties to be applied in food and pharmaceutical industries. Proteins could be beneficial in their concentrated products or isolates, of which membrane-based filtration methods such as ultrafiltration (UF) encompass application in broad spectra of protein sources. More importantly, selective enrichment by UF is of immense interest due to the presence of antinutrients that may dominate their perspicuous bioactivities. UF process is primarily obstructed by concentration polarization and fouling; in turn, a trade-off between productivity and selectivity emerges, especially when pure isolates are an ultimate goal. Several factors such as operating conditions and membrane equipment could leverage those pervasive contributions; therefore, UF protocols should be optimized for each unique protein mixture and mode of configuration. For instance, employing charged UF membranes or combining UF membranes with electrodialysis enables efficient separation of proteins with a similar molecular weight, which is hard to achieve by the conventional UF membrane. Meanwhile, some proposed strategies, such as utilizing ultrasonic waves, tuning operating conditions, and modifying membrane surfaces, can effectively mitigate fouling issues. A plethora of advancements in UF, from their membrane material modification to the arrangement of new configurations, contribute to the quest to actualize promising potentials of protein separation by UF, and they are reviewed in this paper.

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Vladimirov, Vasiliy I., Viktoriia E. Baksheeva, Irina V. Mikhailova, Ramis G. Ismailov, Ekaterina A. Litus, Natalia K. Tikhomirova, Aliya A. Nazipova, Sergei E. Permyakov, Evgeni Yu Zernii, and Dmitry V. Zinchenko. "A Novel Approach to Bacterial Expression and Purification of Myristoylated Forms of Neuronal Calcium Sensor Proteins." Biomolecules 10, no. 7 (July 10, 2020): 1025. http://dx.doi.org/10.3390/biom10071025.

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N-terminal myristoylation is a common co-and post-translational modification of numerous eukaryotic and viral proteins, which affects their interaction with lipids and partner proteins, thereby modulating various cellular processes. Among those are neuronal calcium sensor (NCS) proteins, mediating transduction of calcium signals in a wide range of regulatory cascades, including reception, neurotransmission, neuronal growth and survival. The details of NCSs functioning are of special interest due to their involvement in the progression of ophthalmological and neurodegenerative diseases and their role in cancer. The well-established procedures for preparation of native-like myristoylated forms of recombinant NCSs via their bacterial co-expression with N-myristoyl transferase from Saccharomyces cerevisiae often yield a mixture of the myristoylated and non-myristoylated forms. Here, we report a novel approach to preparation of several NCSs, including recoverin, GCAP1, GCAP2, neurocalcin δ and NCS-1, ensuring their nearly complete N-myristoylation. The optimized bacterial expression and myristoylation of the NCSs is followed by a set of procedures for separation of their myristoylated and non-myristoylated forms using a combination of hydrophobic interaction chromatography steps. We demonstrate that the refolded and further purified myristoylated NCS-1 maintains its Ca2+-binding ability and stability of tertiary structure. The developed approach is generally suited for preparation of other myristoylated proteins.

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Cuartas, B., M. I. Alcaina, and E. Soriano. "Separation of Mineral Salts and Lactose Solutions through Nanofiltration Membranes." Food Science and Technology International 10, no. 4 (August 2004): 255–62. http://dx.doi.org/10.1177/1082013204045883.

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Whey is a highly polluting by-product obtained from the elaboration of cheese. Its high content in organic matter, mainly proteins and lactose, makes it a valuable candidate for multiple applications in the food industry. However, mineral salts have to be previously eliminated. The main objective of this work was to eliminate mineral salts from model solutions with different nanofiltration membranes: NF200, NF270, Ds-5 DK and Ds-5 DL polyamide. Although all four membranes showed fairly good results in the demineralisation of the used samples, the most promising results in terms of good permeate flux and low solute retention were obtained with the NF200 and Ds-5 DL membranes, respectively. The effect of the salt mixture concentration on permeate flux and solute retention was further evaluated for Ds-5 DL membrane.

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Liaw, Der-Jang, Dmitry I. Zybin, Anna I. Prostyakova, Elena Yu Yagudaeva, Alexander A. Vikhrov, Anatoly A. Ishchenko, Vitaly P. Zubov, and Dmitry V. Kapustin. "STATIC AND DYNAMIC SORPTION OF NUCLEIC ACIDS AND PROTEINS ON SURFACE OF SORBENTS MODIFIED WITH NANOLAYERS OF POLYMERS." IZVESTIYA VYSSHIKH UCHEBNYKH ZAVEDENIY KHIMIYA KHIMICHESKAYA TEKHNOLOGIYA 61, no. 1 (December 21, 2017): 4. http://dx.doi.org/10.6060/tcct.20186101.5694.

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Sorption properties of composite silica sorbents modified with nanolayers of fluoropolymers; polyanilines and polyaramides containing aromatic nitrogen; fluorine; as well as donor and acceptor fragments; with respect to nucleic acids (DNA and RNA) and proteins differing in molecular weight and pI are considered. The use of the investigated sorbents in the sample preparation for molecular diagnostics (in particular; in the PCR analysis) not only provides a one-step isolation of nucleic acids; but also allows the isolation and simultaneous purification of protein compounds from the impurities presented in the initial mixture. For the first time; the properties of these materials are compared in the static sorption regime using the compact spin-columns and in the regime of dynamic sorption by the method of spectral-correlation interferometry. The effect of the chemical composition; morphology; and surface charge of these polymer coatings on their sorption properties was studied. Possible mechanisms of sorption of biopolymers on the investigated sorbents are discussed. The use of the developed approaches to the analysis of properties of the sorbents as well as the obtained data open new possibilities for the synthesis of composite sorbents with specific properties. PANI and polyaramides were shown to demonstrate the similar sorption properties when interacting with nucleic acids; but they differ in a various extent in the retention of various proteins. In a neutral aqueous medium (optimal for separation of biopolymers) polyaramides; although did not retain DNA; had a weaker affinity to proteins as compared to PANI. Since the recovery of DNA passed through the PANI-coated silica was the maximal among the particulate adsorbents; the PANI-modified composites were preferred as the carriers for the single-step isolation of nucleic acids from complex biological mixtures. The article cites the results of systematic studies of the authors in the development of sorbents for one-step separation and isolation of biopolymers from complex biological mixtures.Forcitation:Liaw D.-J.; Zybin D.I.; Prostyakova A.I.; Yagudaeva E.Yu.; Vikhrov A.A.; Ishchenko A.A.; Zubov V.P.; Kapustin D.V. Static and dynamic sorption of nucleic acids and proteins on surface of sorbents modified with nanolayers of polymers. Izv. Vyssh. Uchebn. Zaved. Khim. Khim. Tekhnol. 2018. V. 61. N 1. P. 4-22

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Miguet, Laurent, Luc-Matthieu Fornecker, Claire Felden, Carine Gervais, Raoul Herbrecht, Sarah Sanglier, Alain van Dorsselaer, and Laurent Mauvieux. "Proteomic Analysis of Chronic Malignant B-Cell Derived Microparticles Reveals CD148 as a Potential Antigenic Marker for Mantle Cell Lymphoma Diagnosis." Blood 112, no. 11 (November 16, 2008): 1766. http://dx.doi.org/10.1182/blood.v112.11.1766.1766.

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Abstract The diagnosis of mature B-cell neoplasms remains difficult in a number of cases, especially leukemic phases of non Hodgkin lymphomas, for which discriminating criteria or marker are often lacking. In order to identify new surface markers, we developed an original proteomic approach based on mass spectrometry analysis of plasma membrane microparticles derived from chronic B-cell lymphoproliferations: chronic lymphocytic leukemia/ small cell lymphoma (CLL/SLL) and mantle cell lymphoma (MCL). The approach consisted of protein extraction from plasma membrane microparticles (MPs), generated following actinomycin D stimulation and recovered using differential centrifugation. The complex protein mixture was further separated using 1D-gel electrophoresis separation and gel bands were systematically cutted at 2 mm intervals, trypsin digested and the resulting peptide mixtures was subsequently analysed by tandem mass spectrometry prior to protein identification. The lists of the proteins obtained for each pathology were then examined with respect to the membrane localization of the proteins in order to fulfill biological requirements needed for its further clinical use. Comparison of the lists of the proteins obtained for each pathology identified CD148, a membrane receptor with phosphatase activity, as a candidate for a discriminating marker detected in MCL but not in CLL in this approach. Flow cytometry studies, performed on 55 patients and 10 controls, showed that an anti-CD148 antibody stained significantly higher MCL (n=22) than CLL/SLL (n=33) circulating cells (p<0.0001). Even if the reason of such a regulated expression of CD148 remains unknown, our results indicate that a medium or high CD148 expression level may exclude the diagnosis of CLL/SLL and high CD148 expression levels may be strongly in favor of MCL diagnosis. Figure Figure

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Wu, Mengxi, Yingshi Ouyang, Zeyu Wang, Rui Zhang, Po-Hsun Huang, Chuyi Chen, Hui Li, et al. "Isolation of exosomes from whole blood by integrating acoustics and microfluidics." Proceedings of the National Academy of Sciences 114, no. 40 (September 18, 2017): 10584–89. http://dx.doi.org/10.1073/pnas.1709210114.

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Exosomes are nanoscale extracellular vesicles that play an important role in many biological processes, including intercellular communications, antigen presentation, and the transport of proteins, RNA, and other molecules. Recently there has been significant interest in exosome-related fundamental research, seeking new exosome-based biomarkers for health monitoring and disease diagnoses. Here, we report a separation method based on acoustofluidics (i.e., the integration of acoustics and microfluidics) to isolate exosomes directly from whole blood in a label-free and contact-free manner. This acoustofluidic platform consists of two modules: a microscale cell-removal module that first removes larger blood components, followed by extracellular vesicle subgroup separation in the exosome-isolation module. In the cell-removal module, we demonstrate the isolation of 110-nm particles from a mixture of micro- and nanosized particles with a yield greater than 99%. In the exosome-isolation module, we isolate exosomes from an extracellular vesicle mixture with a purity of 98.4%. Integrating the two acoustofluidic modules onto a single chip, we isolated exosomes from whole blood with a blood cell removal rate of over 99.999%. With its ability to perform rapid, biocompatible, label-free, contact-free, and continuous-flow exosome isolation, the integrated acoustofluidic device offers a unique approach to investigate the role of exosomes in the onset and progression of human diseases with potential applications in health monitoring, medical diagnosis, targeted drug delivery, and personalized medicine.

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Shkrigunov, Timur, Pavel Pogodin, Victor Zgoda, Olesya Larina, Yulia Kisrieva, Maria Klimenko, Oleg Latyshkevich, Peter Klimenko, Andrey Lisitsa, and Natalia Petushkova. "Protocol for Increasing the Sensitivity of MS-Based Protein Detection in Human Chorionic Villi." Current Issues in Molecular Biology 44, no. 5 (May 9, 2022): 2069–88. http://dx.doi.org/10.3390/cimb44050140.

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An important step in the proteomic analysis of missing proteins is the use of a wide range of tissues, optimal extraction, and the processing of protein material in order to ensure the highest sensitivity in downstream protein detection. This work describes a purification protocol for identifying low-abundance proteins in human chorionic villi using the proposed “1DE-gel concentration” method. This involves the removal of SDS in a short electrophoresis run in a stacking gel without protein separation. Following the in-gel digestion of the obtained holistic single protein band, we used the peptide mixture for further LC–MS/MS analysis. Statistically significant results were derived from six datasets, containing three treatments, each from two tissue sources (elective or missed abortions). The 1DE-gel concentration increased the coverage of the chorionic villus proteome. Our approach allowed the identification of 15 low-abundance proteins, of which some had not been previously detected via the mass spectrometry of trophoblasts. In the post hoc data analysis, we found a dubious or uncertain protein (PSG7) encoded on human chromosome 19 according to neXtProt. A proteomic sample preparation workflow with the 1DE-gel concentration can be used as a prospective tool for uncovering the low-abundance part of the human proteome.

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Aliprantis, Antonios O., David S. Weiss, Justin D. Radolf, and Arturo Zychlinsky. "Release of Toll-Like Receptor-2-Activating Bacterial Lipoproteins in Shigella flexneri Culture Supernatants." Infection and Immunity 69, no. 10 (October 1, 2001): 6248–55. http://dx.doi.org/10.1128/iai.69.10.6248-6255.2001.

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ABSTRACT Shigella spp. cause dysentery, a severe form of bloody diarrhea. Apoptosis, or programmed cell death, is induced during Shigella infections and has been proposed to be a key event in the pathogenesis of dysentery. Here, we describe a novel cytotoxic activity in the sterile-culture supernatants of Shigella flexneri. An identical activity was identified in purified S. flexneri endotoxin, defined here as a mixture of lipopolysaccharide (LPS) and endotoxin-associated proteins (EP). Separation of endotoxin into EP and LPS revealed the activity to partition exclusively to the EP fraction. Biochemical characterization of S. flexneri EP and culture supernatants, including enzymatic deactivation, reverse-phase high-pressure liquid chromatography analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a Toll-like receptor-2 (TLR2) activation assay, indicates that the cytotoxic component is a mixture of bacterial lipoproteins (BLP). We show that biologically active BLP are liberated into culture supernatants of actively growing S. flexneri. In addition, our data indicate that BLP, and not LPS, are the component of endotoxin of gram-negative organisms responsible for activating TLR2. The activation of apoptosis by BLP shed from S. flexneri is discussed as a novel aspect of the interaction of bacteria with the host.

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CASSERON, FLORENCE, GUIDO RYCHEN, XIMO RUBERT-ALEMAN, GERARD JEAN MARTIN, and FRANÇOIS LAURENT. "15N enrichments of casein and plasma protein amino acids in cows ingesting 15N-labelled ammonium sulphate." Journal of Dairy Research 64, no. 3 (August 1997): 367–76. http://dx.doi.org/10.1017/s0022029997002306.

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The aim of this work was to determine by ion-exchange liquid chromatography and isotope ratio mass spectrometry the specific 15N enrichment of amino acids in casein and plasma proteins in cows receiving three successive daily oral doses (300, 150 and 150 g) of (15NH4)2SO4 (10 atom per cent isotopic enrichment) and to examine the 15N enrichments obtained with regard to nitrogen transport and metabolism in the lactating cow. To investigate the 15N distribution in amino acids in casein and in plasma proteins, samples of 15N-labelled casein and plasma proteins were extracted either from a pool of several milkings (36–96 h after starting to administer the tracer) or from pooled venous blood (removed on the fourth day after the start of administration) from the four lactating cows. 15N enrichments of the proteins studied, expressed as atoms percent excess, were 0·2509 for casein and 0·0577 for plasma protein. Chromatographic fractionation of the amino acid mixture (protein hydrolysates) resulted in nine groups containing between one and four amino acids: Asp, Ser and Thr; Glu; Pro; Gly, Ala, Val and Met; Ileu and Leu; Tyr; Phe; His and Lys; and Arg. High 15N incorporation was demonstrated in all individual or groups of amino acids studied. In both proteins, Glu appeared to be the most enriched amino acid, Phe and Arg the least enriched. Most aliphatic molecules with a single amino group were highly enriched. The much lower (3·5–7·7-fold) enrichments in plasma protein compared with casein suggest considerable intracellular dilution at the site of liver protein synthesis. Finally, the amino acid separation methods are discussed and suggestions for improving them considered.

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De Corato, Marco, and Marino Arroyo. "A theory for the flow of chemically responsive polymer solutions: Equilibrium and shear-induced phase separation." Journal of Rheology 66, no. 5 (September 2022): 813–35. http://dx.doi.org/10.1122/8.0000475.

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Chemically responsive polymers are macromolecules that respond to local variations of the chemical composition of the solution by changing their conformation, with notable examples including polyelectrolytes, proteins, and DNA. The polymer conformation changes can occur in response to changes in the pH, the ionic strength, or the concentration of a generic solute that interacts with the polymer. These chemical stimuli can lead to drastic variations of the polymer flexibility and even trigger a transition from a coil to a globule polymer conformation. In many situations, the spatial distribution of the chemical stimuli can be highly inhomogeneous, which can lead to large spatial variations of polymer conformation and of the rheological properties of the mixture. In this paper, we develop a theory for the flow of a mixture of solute and chemically responsive polymers. The approach is valid for generic flows and inhomogeneous distributions of polymers and solutes. To model the polymer conformation changes introduced by the interactions with the solute, we consider the polymers as linear elastic dumbbells whose spring stiffness depends on the solute concentration. We use Onsager’s variational formalism to derive the equations governing the evolution of the variables, which unveils novel couplings between the distribution of dumbbells and that of the solute. Finally, we use a linear stability analysis to show that the governing equations predict an equilibrium phase separation and a distinct shear-induced phase separation whereby a homogeneous distribution of solute and dumbbells spontaneously demix. Similar phase transitions have been observed in previous experiments using stimuli-responsive polymers and may play an important role in living systems.

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Jones, D. A. C., R. J. Henderson, and J. Riley. "Preliminary characterization of the lipid and protein components of the protective surface membranes of a pentastomid Porocephalus crotali." Parasitology 104, no. 3 (June 1992): 469–78. http://dx.doi.org/10.1017/s0031182000063733.

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All instars of the pentastomid Porocephalus crotali, in the tissues of rat intermediate hosts and the lung of rattlesnake definitive hosts, are covered by a vesicular or stacked membranous secretory product which is synthesized in sub-parietal cells (SPC) and channelled to the cuticle via multitudinous chitin-lined ducts. In rats this enveloping foam of vesicles survives for the duration of a cuticle. We have purified and partially characterized the lipid and protein composition of lamellate droplets from SPC of infective nymphs. Lipids in the droplets comprised a mixture of neutral and polar lipid classes with cholesterol being the major neutral lipid and phosphatidylcholine the dominant polar lipid. In addition, Triton X-114 phase separation of the protein component of droplets partitioned these into an aqueous phase (with a major band at 60 kDa), a detergent phase with two hydrophobic polypeptides (24 and 16 kDa) and an insoluble pellet containing several minor proteins and major bands at 31 and 107 kDa. Western blotting, with rabbit anti-lamellate droplet antiserum, strongly label only the 60 kDa and the two hydrophobic proteins. Significantly no proteins at all label with serum from infected rats from 50–150 days post-infection, a finding endorsed by indirect fluorescent antibody tests on sectioned V–VII stage nymphs. Possible immunomodulatory functions of SPC-derived surface membranes are discussed and, in this regard, they are compared with pulmonary surfactant, a product of alveolar Type II cells which lines all vertebrate lungs.

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Banach, T., Ł. Adaszek, D. Wyłupek, M. Winiarczyk, and S. Winiarczyk. "Applicability of 2D gel electrophoresis and liquid chromatography in proteomic analysis of urine using mass spectrometry MALDI-TOF." Polish Journal of Veterinary Sciences 16, no. 3 (September 1, 2013): 587–92. http://dx.doi.org/10.2478/pjvs-2013-0083.

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AbstractProteomics including the studies of the structure, function and dependences between proteins is more and more extensively applied in human medicine and veterinary medicine. The analysis of protein profiles of tissues and body fluid from healthy and ill individuals allows to identify diagnostic, prognostic and predictive markers in various pathological states in people and animals. This paper presents preparation of urine samples for analysis in the mass spectrometer MALDI-TOF (Ultraflextreme, Bruker, Bremen, Germany) by means of two methods: liquid chromatography based on the system Nano-LC (PROTEINER FC II, Bruker Daltonics, Bremen Germany). and two-direction electrophoresis 2DE (GE Healthcare, United Kingdom). Both methods enable separation of the mixture under consideration into individual fractions of high purity indispensable for obtaining readable mass spectra. The purpose of this paper is to determine applicability of these methods in analysis of protein composition of urine samples.

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Mykhaliuk, V. V., and V. V. Havryliak. "Obtaining human hair keratin-based films and their characteristics." Studia Biologica 15, no. 1 (2021): 27–36. http://dx.doi.org/10.30970/sbi.1501.643.

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Background. Keratins are natural biopolymers with a wide range of applications in the field of biotechnology. Materials and Methods. Extraction of keratins was performed by a modified Nakamura method using 250 mM DTT. The protein concentration in the supernatant was determined by Bradford method. The protein composition was studied by their electrophoretic separation in a polyacrylamide gel in the presence of sodium dodecyl sulfate. The films were made by casting. The surface characteristics of the films were determined using a scanning electron microscope REMMA-102. The elemental composition of the films was determined using an X-ray microanalyzer. Results. The protein concentration in the supernatant was 3.75 mg/mL. After using dithiothreitol in the extraction mixture, we obtained proteins of intermediate filaments with a molecular weight of 40–60 kDa and a low Sulfur content. In the low molecular weight region, we obtained keratin-associated proteins with a molecular weight of 10–30 kDa and a high content of Sulfur. These proteins belong to fibrillar proteins, which can be used as a matrix for the creation of new keratin-containing biocomposites with a wide range of applications in reparative medicine and tissue engineering. Based on the obtained keratin extract, polymer films with and without the addition of glycerol were made. Scanning electron microscopy revealed that glycerol provided the film structure with homogeneity and plasticity due to the accumulation of moisture after the fixation by water vapor. The X-ray microanalysis of films revealed such elements as Sodium, Silicon, Sulfur, Potassium. Among the detected elements, Sulfur has the largest share that is due to the large number of disulfide bonds in the keratin molecule. Conclusions. The polymer keratin films with the addition of glycerol demonstrated better mechanical properties and can be used in biomedicine.

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Polotskaya, Galina, Alexandra Pulyalina, Mikhail Goikhman, Irina Podeshvo, Iosif Gofman, Sergey Shugurov, Valeriia Rostovtseva, et al. "Asymmetric Membranes Based on Copolyheteroarylenes with Imide, Biquinoline, and Oxazinone Units: Formation and Characterization." Polymers 11, no. 10 (September 22, 2019): 1542. http://dx.doi.org/10.3390/polym11101542.

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Modern ultrafiltration requires novel perfect membranes with narrow pore size, high porosity, and minimal pore tortuosity to achieve high separation performance. In this work, copolyamic acid (co-PAA) was synthesized and used for the preparation of asymmetric porous membranes by phase inversion technique. Several co-PAA membranes were heated up to 250 °C; during heating, they undergo solid-phase transformation into co-polybenzoxazinoneimide (co-PBOI) via dehydration and cyclization. Comparative characterization of both co-PAA and co-PBOI membranes was realized by scanning electron microscopy, mechanical testing, thermogravimetric analysis, and ultrafiltration experiments. Membrane calibration was carried out using a mixture of seven proteins with different molecular weights. During heat treatment, the molecular weight cut-off of the membranes decreased from 20 × 103 g/mol (co-PAA) to 3 × 103 g/mol (co-PBOI). Abnormally low dispersions of rejection (0.3 for co-PAA and 0.45 for co-PBOI) were observed for the studied membranes; this fact indicates that the membranes possess enhanced resolving power.

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Journal articles on the topic 'Separations of proteins mixture'

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