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Cold Spring Harbor Protocols Dec 2021Most analytical electrophoreses of proteins are achieved by separation in polyacrylamide gels under conditions that ensure dissociation of proteins into individual...
Most analytical electrophoreses of proteins are achieved by separation in polyacrylamide gels under conditions that ensure dissociation of proteins into individual polypeptide subunits and minimize aggregation. Most commonly, the anionic detergent sodium dodecyl sulfate (SDS) is used in combination with a reducing agent (β-mercaptoethanol or dithiothreitol) and with heating to dissociate proteins before loading onto the gel. SDS binding denatures the polypeptides and imparts a negative charge that masks their intrinsic charge. The amount of SDS bound is generally sequence-independent and proportional to molecular weight; at saturation, approximately one SDS molecule is bound per two amino acids, or ∼1.4 g of SDS per gram of polypeptide. Therefore, the migration of SDS-polypeptide complexes in an electric field is proportional to the relative size of the polypeptide chain, and its molecular weight can be estimated by comparison to protein markers of known molecular weight. However, hydrophobicity, highly charged sequences, and certain posttranslational modifications such as glycosylation or phosphorylation may also influence migration. Thus, the apparent molecular weight of modified proteins does not always accurately reflect the mass of the polypeptide chain. This protocol describes preparation and running of SDS-PAGE gels, followed by staining to detect proteins using Coomassie Brilliant Blue. Finally, the stained SDS-PAGE gel may be scanned to an image or preserved by drying.
Topics: Electrophoresis, Polyacrylamide Gel; Gels; Molecular Weight; Peptides; Proteins; Sodium Dodecyl Sulfate
PubMed: 34853120
DOI: 10.1101/pdb.prot102228 -
Journal of Visualized Experiments : JoVE Jun 2019Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a...
Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.
Topics: Calibration; Chromatography, Gel; Dynamic Light Scattering; Light; Molecular Weight; Proteins; Scattering, Radiation
PubMed: 31282880
DOI: 10.3791/59615 -
ACS Macro Letters Dec 2022Controlled radical polymerization techniques enable the synthesis of polymers with predetermined molecular weights, narrow molecular weight distributions, and controlled...
Controlled radical polymerization techniques enable the synthesis of polymers with predetermined molecular weights, narrow molecular weight distributions, and controlled architectures. Moreover, these polymerization approaches have been routinely shown to result in retained end-group functionality that can be reactivated to continue polymerization. However, reactivation of these end groups under conditions that instead promote depropagation is a viable route to initiate depolymerization and potentially enable closed-loop recycling from polymer to monomer. In this report, we investigate light as a trigger for thermal depolymerization of polymers prepared by reversible-addition-fragmentation chain-transfer (RAFT) polymerization. We study the role of irradiation wavelength by targeting the → π* and π → π* electronic transitions of the thiocarbonylthio end-groups of RAFT-generated polymers to enhance depolymerization via terminal bond homolysis. Specifically, we explore depolymerization of polymers with trithiocarbonate, dithiocarbamate, and -substituted dithiobenzoate end groups with the purpose of increasing depolymerization efficiency with light. As the wavelength decreases from the visible range to the UV range, the rate of depolymerization is dramatically increased. This method of photoassisted depolymerization allows up to 87% depolymerization efficiency within 1 h, results that may further the advancement of recyclable materials and life-cycle circularity.
Topics: Polymerization; Molecular Weight; Polymers
PubMed: 36469937
DOI: 10.1021/acsmacrolett.2c00603 -
Methods in Molecular Biology (Clifton,... 2019Currently, gelatin nanoparticles are gaining more grounds in drug and gene delivery throughout all the available several routes of administration. Yet, the homogenous...
Currently, gelatin nanoparticles are gaining more grounds in drug and gene delivery throughout all the available several routes of administration. Yet, the homogenous and less disperse preparation of this type of nanoparticles is still a challenging task due to the variation of the gelatin quality according to its source and due to its variable molecular weight. Accordingly, several methods were proposed from which the double-desolvation method has been proven to yield optimum results regarding particle size and homogeneity. Thereby, we describe in this chapter a detailed procedure of this method. We also introduce our protocols of the cationization of this kind of nanoparticles as it is extensively needed in case of loading genetic materials or proteins. Additionally, FITC labeling of gelatin nanoparticles that is usually utilized for purposes of imaging or bio-distribution studies is also introduced step by step.
Topics: Drug Delivery Systems; Gelatin; Molecular Weight; Nanoparticles; Particle Size
PubMed: 31148009
DOI: 10.1007/978-1-4939-9516-5_6 -
Chemical Communications (Cambridge,... Jun 2023Reversible Addition-Fragmentation Chain Transfer (RAFT) step-growth polymerization is an emerging method that synergistically combines the benefits of RAFT... (Review)
Review
Reversible Addition-Fragmentation Chain Transfer (RAFT) step-growth polymerization is an emerging method that synergistically combines the benefits of RAFT polymerization (functional group and user-friendly nature) and step-growth polymerization (versatility of the polymer backbone). This new polymerization method is generally achieved by using bifunctional reagents of monomer and Chain Transfer Agent (CTA), that efficiently yield Single Monomer Unit Insertion (SUMI) adducts under stoichiometrically balanced conditions. This review covers a brief history of the RAFT-SUMI process and its transformation into RAFT step-growth polymerization, followed by a comprehensive discussion of various RAFT step-growth systems. Furthermore, characterizing the molecular weight evolution of step-growth polymerization is elaborated based on the Flory model. Finally, a formula is introduced to describe the efficiency of the RAFT-SUMI process, assuming rapid chain transfer equilibrium. Examples of reported RAFT step-growth and SUMI systems are then categorized based on the driving force.
Topics: Polymerization; Polymers; Molecular Weight
PubMed: 37287313
DOI: 10.1039/d3cc01087b -
Chemical Society Reviews May 2018Self-assembled peptide nanostructures have been increasingly exploited as functional materials for applications in biomedicine and energy. The emergent properties of... (Review)
Review
Self-assembled peptide nanostructures have been increasingly exploited as functional materials for applications in biomedicine and energy. The emergent properties of these nanomaterials determine the applications for which they can be exploited. It has recently been appreciated that nanomaterials composed of multicomponent coassembled peptides often display unique emergent properties that have the potential to dramatically expand the functional utility of peptide-based materials. This review presents recent efforts in the development of multicomponent peptide assemblies. The discussion includes multicomponent assemblies derived from short low molecular weight peptides, peptide amphiphiles, coiled coil peptides, collagen, and β-sheet peptides. The design, structure, emergent properties, and applications for these multicomponent assemblies are presented in order to illustrate the potential of these formulations as sophisticated next-generation bio-inspired materials.
Topics: Molecular Weight; Nanostructures; Peptides; Protein Conformation
PubMed: 29697126
DOI: 10.1039/c8cs00115d -
Contributions To Nephrology 2017Hemodialysis is a process of mass separation by a semipermeable membrane, utilized to cleanse blood from waste products retained in case of kidney failure.... (Review)
Review
Hemodialysis is a process of mass separation by a semipermeable membrane, utilized to cleanse blood from waste products retained in case of kidney failure. Traditionally, dialysis membranes have been classified based on composition and hydraulic conductance, creating the net differentiation between cellulosic versus non-cellulosic on one hand and low-flux versus high-flux on the other. With the evolution of biomaterials and improved spinning technology, new membranes have been introduced in the market with specific characteristics and refined individual properties. Therefore, we should consider new parameters to classify dialysis membranes including polymer blending, surface functionalization, molecular weight cut-off (MWCO), hydrophilic/hydrophobic properties, thickness and architecture, adsorption capacity, and electric potential. All these parameters may be utilized to characterize a membrane alone or in combination. Recently, a new parameter has been identified as an important element to characterize a new class of membranes. Beyond the classic MWCO describing the molecular weight at which the sieving value in pure convection = 0.1, the molecular weight retention onset (MWRO) is a new parameter that defines membrane sieving properties. The retention onset value is the molecular weight at which the sieving value = 0.9. The relationship between MWCO and MWRO describes the steepness of the sieving curve and the membrane pore size distribution with important consequences on the final mass separation process and solute removal.
Topics: Biocompatible Materials; Classification; Humans; Membranes, Artificial; Molecular Weight; Renal Dialysis
PubMed: 28910795
DOI: 10.1159/000479260 -
The Journal of Physical Chemistry. B Jul 2022Shear-thickening fluids that absorb the impact energy of high-velocity projectiles are of great interest for aerospace and body-armor applications. In such a frame, we...
Shear-thickening fluids that absorb the impact energy of high-velocity projectiles are of great interest for aerospace and body-armor applications. In such a frame, we investigate transient states of neat and aqueous polyelectrolytes (PE) having low molecular weights and containing poly([2-(methacryloyloxy)ethyl]trimethylammonium) as polycations and poly(acrylamide--acrylic acid) as polyanions. We compare results with those of bulk water. We employ nonequilibrium molecular dynamics to simulate oscillatory shear, mainly in the linear viscoelastic regime. We find that neat PE exhibits properties of a viscoelastic solid, whereas water and the aqueous mixture of PE conform to viscoelastic liquids with Maxwellian behavior at low angular frequencies. Terminal relaxation times are ∼0.499 and ∼1.385 ps for water and the aqueous mixture of PE, respectively. At high angular frequencies, storage moduli show anomalous behaviors that correspond to transitions between shear thinning and shear thickening in complex shear viscosities. The change in potential energy with the increase of the angular frequency is mainly driven by intramolecular interactions for neat PE, whereas short-range Coulomb interactions are the major contributions for water and the aqueous mixture of PE. Upon observation of the molecular configurations, only the local polyionic structure in the aqueous mixture of PE shows improvement when increasing the angular frequency, whereas the rest remains barely affected. Thus, the water structure in the aqueous mixture of PE allows the storage of energy elastically through the hydrogen-bond network at large angular frequencies, whereas the mechanical contribution of polyions weakens and fully vanishes at the beginning of shear thinning, explaining the superimposed data with data of bulk water. Our method and findings set the path for future molecular simulations in the nonlinear viscoelastic regime with more complex underlying molecular mechanisms.
Topics: Molecular Conformation; Molecular Weight; Polyelectrolytes; Viscosity; Water
PubMed: 35732066
DOI: 10.1021/acs.jpcb.2c01448 -
Frontiers in Immunology 2022
Topics: Humans; Immune Checkpoint Inhibitors; Immunotherapy; Molecular Weight; Neoplasms
PubMed: 35663988
DOI: 10.3389/fimmu.2022.920442 -
Carbohydrate Research Nov 2019Fucoidan is a unique polysaccharide that has various biological activities partly owing to its capability to act as mimetics of natural ligands of protein receptors.... (Review)
Review
Fucoidan is a unique polysaccharide that has various biological activities partly owing to its capability to act as mimetics of natural ligands of protein receptors. However, its use is limited due to a number of reasons including those associated with molecular weight and composition variation in relation to an algae type and habitat, raw material collection time, extraction method and duration. The main problem which limits its application in therapy is high molecular weight and seasonal composition. To expand the scope of its application, it is necessary to develop a validated procedure of high-molecular-weight fucoidan depolymerization or synthesis of its oligomeric elements . Therefore, there is a need for the synthesis of polysaccharideoligomeric components and/or polymer mimetics which allow for the creation of chains with a certain degree of sulfation, molecular weight and yield. This paper presents the most commonly used methods of fucoidan homopolysaccharide and heteropolysaccharide fragments synthesis as well as problems associated with their synthesis, including fucoidan analogues available in the form of glycopolymers that are obtained by using different methods of radical polymerization. These fucoidan mimetic glycopolymers have a biological activity similar to that of native fucoidans with high yield, which allows for their use as potential agents in the pharmaceutical industry.
Topics: Chemistry Techniques, Synthetic; Molecular Weight; Polymerization; Polysaccharides
PubMed: 31526929
DOI: 10.1016/j.carres.2019.107806