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Sucrose gradient centrifugation is a very useful technique for isolating specific membrane types based on their size and density. This is especially useful for detecting fatty acids and lipid molecules that are targeted to specialized membranes. Without fractionation, these types of molecules could be below the levels of detection after being diluted out by the more abundant lipid molecules with a more ubiquitous distribution throughout the various cell membranes. Isolation of specific membrane types where these lipids are concentrated allows for their detection and analysis. We describe herein our synaptic membrane isolation protocol that produces excellent yield and clear resolution of five major membrane fractions from a starting neural tissue homogenate: P1 (nuclear), P2 (cytoskeletal), P3 (neurosynaptosomal), PSD (post-synaptic densities), and SV (synaptic vesicle).

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A wide range of applications are available for kraft lignin (KL). However, the dark color and wide size distribution of KL make it challenging to use in cosmetics and nanoparticle preparation. In this study, we fractionated KL from a paper-making enterprise using ultrafiltration membrane fractionation, and obtained four kinds of lignin with different molecular weights, namely ultrafiltration lignin (UL). Following that, lignin nanoparticles (ULNPs) were formed by self-assembly from four types of UL. Analyzing the UL and ULNP properties, the low molecular weight lignin, such as ULA, exhibited good antioxidant properties (89.47 %, 5 mg/mL), high brightness (ISO% = 7.55), high L⁎ value (L⁎ = 72.3) and low polydispersity index (PDI = 1.41). The ULNP showed a narrow size distribution (0.8-1.4 m) and high dispersibility in sunscreen. When ULNP was added to sunscreen with 5 % load, its sun protection factor (SPF) value increased from 14.93 to 63.74. Therefore, this study offered an effective way for the comprehensive utilization of pulping waste KL.

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Mango peel is rich in nutritional and functional compounds, such as carbohydrates, dietary fibers, proteins, and phenolic compounds, with high potential to be applied in the food industry. Most of the investigation about recovery of bioactive compounds from fruit bioproducts involves extraction techniques and further separation of target compounds. There is still a lack of information about the potential of membrane processes to recover the nutritive/functional compounds present in aqueous extracts of those bioproducts. This research is addressed to study the performance of ultrafiltration (UF), followed by nanofiltration (NF) of UF permeates, to fractionate the compounds present in aqueous extracts of mango peel. Both UF and NF concentration processes were carried up to a volume concentration factor of 2.0. Membranes with molecular weight cut-offs of 25 kDa and 130 Da were used in the UF and NF steps, respectively. UF and NF concentrates showed antioxidant activity, attributed to the presence of phenolic compounds, with rejections of about 75% and 98.8%, respectively. UF membranes totally rejected the higher molecular weight compounds, and NF membranes almost totally concentrated the fermentable monosaccharides and disaccharides. Therefore, it is envisaged that NF concentrates can be utilized by the food industry or for bioenergy production.

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Gram-negative diderm bacteria are characterized by a tripartite cell envelope, composed of an inner membrane (IM) and a lipopolysaccharide (LPS)-containing outer membrane (OM), separated by an aqueous space where the peptidoglycan is embedded. LPS is a peculiar glycolipid endowed with several biological activities. The biosynthesis and transport of LPS to its final location take place in every compartment of the cell envelope. Proteins and protein machineries with different subcellular localization are involved in this process to facilitate the trafficking of LPS across subcellular compartments that differ in their physicochemical proprieties. The fractionation of bacterial cell envelopes can give information on the status of the LPS biogenesis by allowing the analysis of LPS profiles and of the localization of proteins involved in the transport. Here, we describe a standardized protocol for membrane fractionation in Escherichia coli using sucrose density gradient centrifugation that separates the IM from the OM cellular fractions. Bacterial cells are first converted into spheroplasts and lysed; then the membrane fractions are collected by ultracentrifugation and separated at high speed by exploiting the differences in membrane density. The fractions obtained are analyzed for LPS total amount and electrophoretic profile.

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Organic matter (OM) is the most important factor influencing the effectivity and efficiency of micropollutant (MP) ozonation in wastewater effluents. The importance of the quantity of OM is known, because of this, total organic carbon (TOC) is generally used to determine the required ozone dose for any water sample. Still, the effect of OM type on MP ozonation is not well understood. In this study, effluents from five wastewater treatment plants were collected and the organic matter in these effluents was fractionated using membranes (F1-4) and resin (HI, HOA, HON and HOB). Fractions were diluted to the same TOC concentration, spiked with MPs and ozonated at three ozone doses. Our results show that all five effluents had comparable OM compositions and similar MP removal, confirming the suitability of OM quantity (TOC) to compare the ozone requirements for wastewater effluents. From the 19 analysed MPs, three groups were identified that showed similar removal behaviour. The strongest differences between the groups were observed around MP ozone reactivities of 102, 104 and 106 M-1 s-1. This indicates the presence of three OM groups in the samples that interfere with the removal of different MPs. MP removal in the resin fraction HON were higher for MPs with high and medium ozone reactivity, indicating a low interference of OM in this fraction with MP ozonation. OM in the resin fractions HOA and HI showed higher interference with MP ozonation. Therefore, removing the HOA and HI fractions prior to ozonation would result in a lower required ozone dose and a more efficient removal of the MPs. MP removal correlated with the OM characteristics A300, SR and fluorescence component comp 2. These characteristics can be used as inline tools to predict the required ozone dose in water treatment plants.

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The study of subcellular membrane structure and function facilitates investigations into how biological processes are divided within the cell. However, work in this area has been hampered by the limited techniques available to fractionate the different membranes. Free Flow Electrophoresis (FFE) allows for the fractionation of membranes based on their different surface charges, a property made up primarily of their varied lipid and protein compositions. In this study, high-resolution plant membrane fractionation by FFE, combined with mass spectrometry-based proteomics, allowed the simultaneous profiling of multiple cellular membranes from the leaf tissue of the plant Mesembryanthemum crystallinum. Comparisons of the fractionated membranes' protein profile to that of known markers for specific cellular compartments sheds light on the functions of proteins, as well as provides new evidence for multiple subcellular localization of several proteins, including those involved in lipid metabolism.

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The fractionation of enough membrane protein from limited samples is challenging for MS-based quantitative targeted absolute proteomics (QTAP) of drug metabolizing enzymes (DMEs) and transporters. This study evaluated differential detergent fractionation (DDF) of membrane protein from progressively smaller numbers of primary mouse hepatocytes (5 million down to 50,000 cells) and limited liver tissue (25-50 mg) in quantifying select DMEs and transporters by QTAP. Two non-ionic detergents, digitonin and Triton-X-100, were applied in sequence to permeabilize cells and extract membrane proteins. Comparison was made with a membrane protein extraction kit and with homogenization in hypotonic buffer and subsequent differential centrifugation (DC). DDF produced linear membrane protein yields with increasing hepatocyte numbers and better permeabilization evidenced by the higher ratio of cytosolic to membrane protein yields. DDF produced 5-times more membrane protein from liver tissue than DC. The concentration of DMEs and transporters remained consistent in the fractions prepared by DDF from progressively smaller numbers of hepatocytes, but declined in kit fractions. In liver tissue, the concentrations were comparatively higher in DDF versus kit and DC. In conclusion, sequential digitonin and Triton-X-100 fractionation of membrane protein from limited samples is efficient, reproducible and cost-effective for QTAP of DMEs and transporters.

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Bacteriophages use a large number of different bacterial cell envelope structures as receptors for surface attachment. As a consequence, bacterial surfaces represent a major control point for the defense against phage attack. One strategy for phage population control is the production of outer membrane vesicles (OMVs). In Gram-negative host bacteria, O-antigen-specific bacteriophages address lipopolysaccharide (LPS) to initiate infection, thus relying on an essential outer membrane glycan building block as receptor that is constantly present also in OMVs. In this work, we have analyzed interactions of Salmonella (S.) bacteriophage P22 with OMVs. For this, we isolated OMVs that were formed in large amounts during mechanical cell lysis of the P22 S. Typhimurium host. In vitro, these OMVs could efficiently reduce the number of infective phage particles. Fluorescence spectroscopy showed that upon interaction with OMVs, bacteriophage P22 released its DNA into the vesicle lumen. However, only about one third of the phage P22 particles actively ejected their genome. For the larger part, no genome release was observed, albeit the majority of phages in the system had lost infectivity towards their host. With OMVs, P22 ejected its DNA more rapidly and could release more DNA against elevated osmotic pressures compared to DNA release triggered with protein-free LPS aggregates. This emphasizes that OMV composition is a key feature for the regulation of infective bacteriophage particles in the system.

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For the utilization of each lignin fraction in the lignin liquors, the development of separation strategies to fractionate the lignin streams by molecular weight ranges constitutes a timely challenge to be tackled. Herein, membrane filtration was applied to the refining of lignin streams obtained from a lignin-first biorefining process based on H-transfer reactions catalyzed by Raney Ni, by using 2-PrOH as a part of the lignin extraction liquor and as an H-donor. A two-stage membrane cascade was considered to separate and concentrate the monophenol-rich fraction from the liquor. Building on the results, an economic evaluation of the potential of membrane filtration for the refining of lignin streams was undertaken. In this proof-of-concept report, a detailed analysis is presented of future developments in the performance required for the utilization of membrane filtration for lignin refining and, more aspiringly, solvent reclamation.

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Tuna protein hydrolysate (TPH) was prepared by hydrolysis with Prolyve BS and fractionated by membranes process. The antioxidant activities of recovered peptide fractions were evaluated. Four novel antioxidant peptides that were isolated from nanofiltration retentate exhibited the highest antioxidant activity, using gel chromatography and reversed phase high-performance liquid chromatography. The amino acid sequences of isolated peptides were identified as Tyr-Glu-Asn-Gly-Gly (P2), Glu-Gly-Tyr-Pro-Trp-Asn (P4), Tyr-Ile-Val-Tyr-Pro-Gly (P7) and Trp-Gly-Asp-Ala-Gly-Gly-Tyr-Tyr (P8) with molecular weights of 538.46, 764.75, 710.78 and 887.85 Da, respectively. P2, P4, P7 and P8 exhibited good scavenging activities on hydroxyl radical (IC50 0.41, 0.327, 0.17 and 0.042 mg/ml), DPPH radical (IC50 0.666, 0.326, 0.451 and 0.377 mg/ml) and superoxide radical (IC50 0.536, 0.307, 0.357 and 0.115 mg/ml). P7 was effective against lipid peroxidation in the model system. The isolated peptides might be useful used as natural food additive in food industry and formulation of nutritional products.

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Positive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients. This revealed that SFV infection strongly modifies cellular membranes. We removed soluble proteins, the Golgi membranes, and most of the mitochondria, but plasma membrane, endoplasmic reticulum (ER), and late endosome markers were retained in the membrane fraction that contained viral RNA synthesizing activity, replicase proteins, and minus- and plus-strand RNA. Electron microscopy revealed that the purified membranes displayed spherule-like structures with a narrow neck. This membrane enrichment was specific to viral replication, as such a distribution of membrane markers was only observed after infection. Besides the plasma membrane, SFV infection remodeled the ER, and the cofractionation of the RC-carrying plasma membrane and ER suggests that SFV recruits ER proteins or membrane to the site of replication. The purified RCs were highly active in synthesizing both genomic and subgenomic RNA. Detergent solubilization destroyed the replication activity, demonstrating that the membrane association of the complex is essential. Most of the newly made RNA was in double-stranded replicative molecules, but the purified complexes also produced single-stranded RNA as well as released newly made RNA. This indicates that the purification established here maintained the functionality of RCs and thus enables further structural and functional studies of active RCs.IMPORTANCE Similar to all positive-strand RNA viruses, the arthropod-borne alphaviruses induce membranous genome factories, but little is known about the arrangement of viral replicase proteins and the presence of host proteins in these replication complexes. To improve our knowledge of alphavirus RNA-synthesizing complexes, we isolated and purified them from infected mammalian cells. Detection of viral RNA and in vitro replication assays revealed that these complexes are abundant and highly active when located on the plasma membrane. After multiple purification steps, they remain functional in synthesizing and releasing viral RNA. Besides the plasma membrane, markers for the endoplasmic reticulum and late endosomes were enriched with the replication complexes, demonstrating that alphavirus infection modified cellular membranes beyond inducing replication spherules on the plasma membrane. We have developed here a gentle purification method to obtain large quantities of highly active replication complexes, and similar methods can be applied to other positive-strand RNA viruses.

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Sucrose gradient centrifugation is a very useful technique for isolating specific membrane types based on their size and density. This is especially useful for detecting fatty acids and lipid molecules that are targeted to specialized membranes. Without fractionation, these types of molecules could be below the levels of detection after being diluted out by the more abundant lipid molecules with a more ubiquitous distribution throughout the various cell membranes. Isolation of specific membrane types where these lipids are concentrated allows for their detection and analysis. We describe herein our synaptic membrane isolation protocol that produces excellent yield and clear resolution of five major membrane fractions from a starting neural tissue homogenate: P1 (Nuclear), P2 (Cytoskeletal), P3 (Neurosynaptosomal), PSD (Post-synaptic Densities), and SV (Synaptic Vesicle).

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The enzymatic hydrolysis using Prolyve BS coupled to membrane process (Ultrafiltration (UF) and nanofiltration (NF)) is a means of biotransformation of tuna protein waste to Tuna protein hydrolysate (TPH) with higher added values. This method could be an effective solution for the production of bioactive compounds used in various biotechnological applications and minimizing the pollution problems generated by the seafood processing industries. The amino acid composition, functional and antioxidant properties of produced TPH were evaluated. The results show that the glutamic acid, aspartic acid, glycine, alaline, valine and leucine were the major amino acids detected in the TPH profile. After membrane fractionation process, those major amino acids were concentrated in the NF retentate (NFR). The NFR and NF permeate (NFP) have a higher protein solubility (>95 %) when compared to TPH (80 %). Higher oil and water binding capacity were observed in TPH and higher emulsifying and foam stability was found in UF retentate. The NFP showed the highest DPPH radical scavenging activity (65 %). The NFR contained antioxidant amino acid (30.3 %) showed the highest superoxide radical and reducing power activities. The TPH showed the highest iron chelating activity (75 %) compared to other peptide fractions. The effect of the membrane fractionation on the molecular weight distribution of the peptide and their bioactivities was underlined. We concluded that the TPH is a valuable source of bioactive peptides and their peptide fractions may serve as useful ingredients for application in food industry and formulation of nutritional products.

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Protein interaction networks at gap junction plaques are increasingly implicated in a variety of intracellular signaling cascades. Identifying protein interactions of integral membrane proteins is a valuable tool for determining channel function. However, several technical challenges exist. Subcellular fractionation of the bait protein matrix is usually required to identify less abundant proteins in complex homogenates. Sufficient solvation of the lipid environment without perturbation of the protein interactome must also be achieved. The present chapter describes the flotation of light and heavy liver tissue membrane microdomains to facilitate the identification and analysis of endogenous gap junction proteins and includes technical notes for translation to other integral membrane proteins, tissues, or cell culture models. These procedures are valuable tools for the enrichment of gap junction membrane compartments and for the identification of gap junction signaling interactomes.

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Photosystem II (PSII), a large multisubunit membrane protein complex found in the thylakoid membranes of cyanobacteria, algae and plants, catalyzes light-driven oxygen evolution from water and reduction of plastoquinone. Biogenesis of PSII requires coordinated assembly of at least 20 protein subunits, as well as incorporation of various organic and inorganic cofactors. The stepwise assembly process is facilitated by numerous protein factors that have been identified in recent years. Further analysis of this process requires the development or refinement of specific methods for the identification of novel assembly factors and, in particular, elucidation of the unique role of each. Here we summarize current knowledge of PSII biogenesis in cyanobacteria, focusing primarily on the impact of methodological advances and innovations. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux.

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