Monoclonal Antibodies Against Murine Ia Antigens: Studies On Structure, Function, Epitopes, And Idiotypes
Soldano Ferrone, Chella S. David in Ia Antigens, 2019
Ia antigens were labeled with 35S for 30 to 60 min by addition of 200 µCi 35S-methionine (in 200 µl) to 107 spleen cells (200 µl) or day 3 LPS blasts. NP-40 extracts with labeled la were purified on lentil lectin Sepharose and in some instances preabsorbed with rabbit anti-IgM for removal of immunoglobulin. After precipitation with Protein A sepharose, about 500,000 cpm were applied to 10% polyocrylamid slab gels.15 In a special form of two-dimensional gel electrophoresis performed according to Koch and Haustein,16 proteins were separated in the first dimension under nonreducing conditions, and in the second dimension under reducing conditions. This technique allows the identification of unreduced complexes and their corresponding subunits.
Interferons and their Mechanisms of Action
Velibor Krsmanović, James F. Whitfield in Malignant Cell Secretion, 2019
IFN primarily act as regulators of gene expression. Two-dimensional gel electrophoresis has revealed an increased expression of several proteins in various systems.32,65,66 These responses vary widely with cell type, IFN species, and for each of them with the gene under consideration (see Section III.B for a detailed coverage of molecular mechanisms of gene activation). One of the greatest challenges in the field has been, and still is, to identify most of these modulated gene products and to delineate their precise role in the overall response to IFNs or, conversely, to appreciate to which extent their IFN-regulated expression influenees cell physiology. Some of these genes, for instance, Class I and Class II antigens of the major histocompatibility complex, have been extensively studied, and the prime importance and consequences (eventually detrimental as well) of their increased expression through IFN treatment have been briefly described (see Section II.D). Table 1) summarizes our present knowledge of IFN-induced proteins. We will focus on two genes for which reasonable experimental evidence has now accumulated for an involvement in, at least, part of the cellular responses to IFN: the Mx gene and the components of the 2,5A system.
Proteomics Approaches to Uncover the Drug Resistance Mechanisms of Microbial Biofilms
Chaminda Jayampath Seneviratne in Microbial Biofilms, 2017
Two-dimensional gel electrophoresis (2-DE) was first developed by O’Farell in the 1970s and has been the cornerstone of proteomics until the advent of gel-free proteomics [96,97]. Traditionally, in gel-based approaches, proteins are first separated using a gel and then the extracted spots are digested with a protease, most commonly trypsin [98]. Following 2-DE, various staining methods such as Silver stain, Coomassie Blue and fluorescent dyes are used to visualise the gel (Figure 6.1a). In fact, one of the advantages of gel-based proteomics techniques is that they provide a visual representation in the gel of the proteome under investigation, containing valuable information about the relative size, isoelectric point and abundance of the proteins. Gel-based techniques can separate thousands of proteins and the currently used protocols can produce 2,000–3,000 detectable proteins spots. The differential expression profile of the proteins can be analysed using appropriate software such as ImageMaster (Amersham Biosciences), Progenesis (Nonlinear Dynamics), Decyder (GE Healthcare Life Sciences), PDQuest (BioRad), Melanie (Geneva Bioinformatics), ELISE and HERMes. It should be noted that the analytical methods and algorithms used in different software tools vary considerably [99].
Nanoparticle-protein corona complex: understanding multiple interactions between environmental factors, corona formation, and biological activity
Published in Nanotoxicology, 2021
Aysel Tomak, Selin Cesmeli, Bercem D. Hanoglu, David Winkler, Ceyda Oksel Karakus
Gel electrophoresis is a widely used technique that separates and identifies bound proteins based on molecular weight (one-dimensional gel electrophoresis) or based on both isoelectric point and molecular weight (two-dimensional gel electrophoresis, 2-DE). In particular, the ability of one-dimensional SDS-PAGE to validate protein corona formation and identify its components has been widely studied in nanoscience. For example, it was used to elucidate the molecular composition of protein coronas associated with PLGA NPs (Partikel, Korte, Mulac, et al. 2019), magnetic NPs (Martens et al. 2019), AuNPs (Chandran, Riviere, and Monteiro-Riviere 2017), and silica NPs (Mirshafiee, Kim, Park, et al. 2016). Furthermore, SDS-PAGE analysis has been successful in separating bound proteins from NP surfaces and in identifying isolated individual proteins. However, it suffers from labor-intensive sample preparation, poor band resolution, and low separation of proteins with similar molecular weights (Monopoli et al. 2011). 2-DE combines two different electrophoretic procedures (isoelectric focusing and SDS-PAGE) to achieve a greater separation of complex protein mixtures. It has, for example, successfully characterized the composition of proteins adsorbed onto polystyrene NPs (Gessner, Paulke, and Müller 2000a; Grassi et al. 2019). After separating proteins by SDS-PAGE or 2-DE, visualization of separated proteins is achieved by staining gels with Coomassie blue, silver staining, or fluorescent staining.
Proteomic profiling of giant skeletal muscle proteins
Published in Expert Review of Proteomics, 2019
Sandra Murphy, Paul Dowling, Margit Zweyer, Dieter Swandulla, Kay Ohlendieck
Mass spectrometry-based proteomic cataloguing studies of the skeletal muscle proteome from various species have used both gel electrophoretic and liquid chromatographic techniques. The initial proteomic mapping of skeletal muscle tissues heavily depended on high-resolution two-dimensional gel electrophoretic methods for large-scale protein separation [22]. More recent investigations on the near-to-complete skeletal muscle proteome employed liquid chromatography combined with advanced mass spectrometry [23]. However, two-dimensional gel electrophoresis is still extensively used for comparative proteomic surveys and the biochemical analysis of post-translational modifications [5,24]. The technical limitations of two-dimensional gel electrophoresis are especially relevant in relation to the systematic identification of low-abundance proteins, highly hydrophobic proteins and high-molecular-mass proteins. Many extremely large muscle-associated proteins do not properly transfer from the first-dimension isoelectric focusing gel to the second-dimension slab gel system [22]. Alternative approaches with one-dimensional gels and GeLC-MS/MS methodology can overcome some of these technical issues, but are usually less discriminatory in their separation power [25].
Recent insights into Mycobacterium tuberculosis through proteomics and implications for the clinic
Published in Expert Review of Proteomics, 2019
Deepa Bisht, Devesh Sharma, Divakar Sharma, Rananjay Singh, Vivek Kumar Gupta
Aslam and coworkers [5] recently described various proteomics approaches and their applications. Proteomics techniques can be broadly classified under five headings: Conventional, advanced, quantitative, high-throughput, and bioinformatics analysis. Conventional techniques are chromatography based (gel filtration, ion-exchange and affinity) for purification and analysis is usually done by enzyme-linked immunosorbent assay (ELISA) or western blotting. They are restricted to analysis of only few proteins and also incapable of defining protein expression level [6,7]. Gel based techniques like sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2DGE), and two-dimensional differential gel electrophoresis (2D-DIGE) were used for separation of complex protein samples. 2D-DIGE utilizes CyDye labeled proteins that can be visualized by exciting the dye at a specific wavelength [8]. Compared to others, it is a more recently developed method that uses proteins covalently tagged with spectrally different fluorescent dyes to quantify gel spots. However, they have significant drawbacks associated with the separation method and staining procedures. Proteome microarrays or chips (composed of thousands of proteins from one species, affinity purified and functionally active), which are high-throughput platforms for global profiling of molecular interactions in a single experiment were then established. The main challenge with protein microarray was to explore the function of the complete genome [9].
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