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Approaches for Identification and Validation of Antimicrobial Compounds of Plant Origin: A Long Way from the Field to the Market
Published in Mahendra Rai, Chistiane M. Feitosa, Eco-Friendly Biobased Products Used in Microbial Diseases, 2022
Lívia Maria Batista Vilela, Carlos André dos Santos-Silva, Ricardo Salas Roldan-Filho, Pollyanna Michelle da Silva, Marx de Oliveira Lima, José Rafael da Silva Araújo, Wilson Dias de Oliveira, Suyane de Deus e Melo, Madson Allan de Luna Aragão, Thiago Henrique Napoleão, Patrícia Maria Guedes Paiva, Ana Christina Brasileiro-Vidal, Ana Maria Benko-Iseppon
An excellent criterion for selecting the solution for the extraction of globular proteins is the relationship between protein quantity and its biological activity. Thus, the protein purifier must avoid producing an inactive crude extract even if it contains a high protein concentration or an active extract, but with a minimum amount of the targeted protein. Protein detection can be done through absorbance at 280 nm or by colorimetric methods as Bradford, Lowry, Biuret, and bicinchoninic acid (Zheng et al. 2017; Silva et al. 2019; Licini et al. 2020).
Immunological Approaches
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
Deborah E. Dixon, Susan J. Steiner, Stanley E. Katz
To perform the direct competition method, the solid phase (usually a plastic microtiter plate or tube) is coated with antibody specific for the test antigen. Appropriate amounts of both the test sample containing the antigen and the enzyme-labeled antigen are added. There is competition for the antibody between the unlabeled and labeled antigen. Substrate is added, and the color produced by the enzyme action is inversely proportional to the concentration of antigen in the sample. This method is satisfactory for protein detection. However, sensitivity is variable and dependent upon the purity and stability of the antigen conjugate.
Proteome Changes Associated with the VEGFR Pathway and Immune System in Diabetic Macular Edema Patients at Different Diabetic Retinopathy Stages
Published in Current Eye Research, 2022
Ruyi Han, Ruowen Gong, Wei Liu, Gezhi Xu
Many protein detection methods9–18 have been used to reveal possible molecular biomarkers in different DR stages, including western blot (WB),13,15,17,18 enzyme-linked immunosorbent assay (ELISA),14 sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with liquid chromatography mass spectrometry,9 two-dimensional fluorescence difference gel electrophoresis coupled with tandem mass spectrometry (2 D-DIGE/MS/MS),10,13,15–17 and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).11,12,15 The number of proteins identified is largely limited in WB and ELISA due to the necessity for protein antibodies, while the development of 2 D-DIGE/MS/MS and LC-MS/MS greatly increases the number of detected proteins. However, traditional 2 D-DIGE/MS/MS and LC-MS/MS relying on the data-dependent acquisition mode or selected reaction monitoring19–22 still remain challenging regarding the detection sensitivity and in quantifying large fractions of proteomes across multiple samples. Recently, a data-independent acquisition (DIA)-based LC-MS/MS strategy was established,19–22 which is independent of the detection or knowledge of the precursor ions to trigger acquisition of the fragment ion spectra. Therefore, DIA-based LC-MS/MS is well-suited for applications in simultaneously detecting hundreds of proteins.
From proteomic landscape to single-cell oncoproteomics
Published in Expert Review of Proteomics, 2021
Vivian Weiwen Xue, Sze Chuen Cesar Wong, William Chi Cho
With the advancement of qualitative and quantitative oncoproteomics, the understanding of functional pathways in cancer mechanisms shows the process from macro to micro perspective. The global proteomic profiling comprehensively displays the changes of protein expression along the carcinogenesis. Abnormal proteome in cancers can be used for cancer diagnosis, monitoring, and discovering novel therapeutic targets. MS-based protein detection is widely used in global oncoproteomic analysis, whereas it requires starting amount of protein samples from hundreds of cells to process routine detection. This poses a challenge for studying cancer cell heterogeneity, interactions and the detailed signal transduction in trace samples or at the single-cell level. Nowadays, cancer genome and transcriptome research can be achieved accurately and rapidly in a high-throughput manner by next-generation sequencing. However, proteomic analysis is still mainly based on MS detection. Unlike DNA and RNA, protein molecules cannot be amplified, which limits the sensitivity of protein detection.
Analytical techniques for multiplex analysis of protein biomarkers
Published in Expert Review of Proteomics, 2020
Alain Van Gool, Fernado Corrales, Mirjana Čolović, Danijela Krstić, Begona Oliver-Martos, Eva Martínez-Cáceres, Ivone Jakasa, Goran Gajski, Virginie Brun, Kyriacos Kyriacou, Izabela Burzynska-Pedziwiatr, Lucyna Alicja Wozniak, Stephan Nierkens, César Pascual García, Jaroslav Katrlik, Zanka Bojic-Trbojevic, Jan Vacek, Alicia Llorente, Felicia Antohe, Viorel Suica, Guillaume Suarez, Ruben t’Kindt, Petra Martin, Deborah Penque, Ines Lanca Martins, Ede Bodoki, Bogdan-Cezar Iacob, Eda Celikbas, Suna Timur, John Allinson, Christopher Sutton, Theo Luider, Saara Wittfooth, Marei Sammar
The ELISA principle can also be applied for single-cell protein secretome analyses using single-cell barcode chips capturing a panel of 32 pre-specified proteins from a single cell in each microchamber. The cells of interest may be stimulated and sorted before loading the cells on a chip. Chips are analyzed using Isoplex machinery, which contains automated cellular imaging and includes a complete ELISA workflow. The software provides information on the secreted proteins across different categories: homeostatic, proliferative, inflammatory, chemotactic, regulatory, and immune effector functions and provides a polyfunctionality strength index [16] (Table 1). Other commercially available methods for multiplex protein detection are reviewed below and summarized in Table 1.