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Characterization Techniques for Bio-Nanocomposites
Published in Shrikaant Kulkarni, Neha Kanwar Rawat, A. K. Haghi, Green Chemistry and Green Engineering, 2020
Electrophoresis is also a useful tool in colloidal science and can be applied: To separate complex mixtures with very high resolution. Two-dimensional gel electrophoresis (2-DE) is used to analyze proteins.To separate molecules linearly based on their isoelectric point in the first dimension and molecular mass in the second one [31].To isolate small inorganic clusters like gold [32] or semiconductors and magnetic particle clusters [33]).To separate the nanoparticles with a discrete number of functional groups [34], e.g., gold nanoparticles conjugated to nucleic acids [35, 37], or peptide conjugated semiconductors [38].
Protein Engineering and Bionanotechnology
Published in Anil Kumar Anal, Bionanotechnology, 2018
In case of complex proteins, two-dimensional gel electrophoresis (2D-PAGE) is used where proteins are separated based on two properties, that is, net charge and molecular mass. With the progress achieved in this method, it is suitable for separating up to 10,000 proteins in a single gel analysis. One of the major advantages of 2D-PAGE is the ability to resolve protein that has undergone PTMs. This method serves in protein expression profiling where expression of two samples can be compared both qualitatively and quantitatively. It is also used in cell map proteomics for microorganisms, organelles, and protein complex (Graves and Haystead 2002).
Proteins and proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Two-dimensional gel electrophoresis or 2D electrophoresis is a form of gel electrophoresis commonly used to separate a large number of proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2D electrophoresis begins with ID electrophoresis but then separates the molecules by a second property in the direction 90° from the first. In ID electrophoresis, proteins are separated in one dimension, so that all the proteins lie along a lane, but separated from each other by an isoelectric point. The result is that the molecules are spread out across a 2D gel. Because it is unlikely that two molecules will be similar in two distinct properties, molecules are more effectively separated by 2D electrophoresis than in ID electrophoresis. Separating proteins by isoelectric point is called isoelectric focusing. Thereby, a gradient of pH is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pHs other than their isoelectric point, proteins will be charged. If they are positively charged, they will be pulled toward the more negative end of the gel and if they are negatively charged, they will be pulled to the more positive end of the gel. The proteins applied in the first dimension will move along the gel and will accumulate at their isoelectric point. That is, the point at which the overall charge of the protein is 0 (a neutral charge). The result of this is a gel with proteins spread out on its surface. These proteins can then be detected by a variety of means, but the most commonly used are silver and coomassie staining. In this case, a silver colloid is applied to the gel. The silver binds to cysteine groups within the protein. The silver is darkened by exposure to ultraviolet light. The darkness of the silver can be related to the amount of silver and, therefore, the amount of protein at a given location on the gel. This measurement can only give approximate amounts but is adequate for most purposes (Figure 3.17).
Omics to address the opportunities and challenges of nanotechnology in agriculture
Published in Critical Reviews in Environmental Science and Technology, 2021
Sanghamitra Majumdar, Arturo A. Keller
Two-dimensional gel electrophoresis (2DE) separates protein mixtures based on charge (isoelectric point) in the first dimension and by mass in the second dimension on 2D-gels, which in combination with MS have been used for decades for plant proteome profiling. However, it has its limitations owing to poor sensitivity, low reproducibility, and low throughput. Especially for plant whole lysates that contain an array of secondary metabolites like pigments and phenolics, these compounds can introduce streaking in a gel (Vannini et al., 2014). With rapid development in MS techniques, quantitative proteomics have evolved from gel-based to gel-free approaches. Like metabolomics, proteomics can also be categorized into untargeted and targeted method. Due to the exploratory nature of untargeted proteomics, it is the preferred approach to screen for candidate protein markers to elucidate plant responses to ENM exposure. This approach can facilitate high-throughput analysis of protein abundance across different ENM exposures, tissue types, stages of growth, physiological conditions and stress conditions (Hart-Smith et al., 2017). In a gel-based approach, proteins are separated by 2DE and the spots showing comparable differences are excised from the gel. The proteins in the gel fractions are digested into peptides, which are then characterized by LC-MS. A typical gel- and label-free quantitative proteomic analysis used for discovery studies employ a bottom-up approach, where the proteins in a sample are cleaved into peptides, which are then separated, identified and quantified using LC-MS/MS (Figure 3) (Hu et al., 2015). The steps and key challenges in proteomic analysis in ENM-plant interaction study are summarized below.