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Biochemical Markers in Ophthalmology
Published in Ching-Yu Cheng, Tien Yin Wong, Ophthalmic Epidemiology, 2022
Abdus Samad Ansari, Pirro G. Hysi
Several conventional low-throughput methods can be utilized in proteomics. Antibody-based methods such as enzyme-linked immunosorbent assay (ELISA) and western blotting depend on the availability of antibodies targeted toward specific proteins or epitopes [142]. Gel-based methods include two-dimensional gel electrophoresis and differential gel electrophoresis. These utilize electric current to separate proteins, before further analysis is completed using alternate testing such as MS [143]. Chromatography-based methods can separate proteins from biological mixtures through various modalities such as ion exchange, size exclusion, and affinity chromatography. Other gel-free chromatographic techniques include gas chromatography and liquid chromatography [144].
Methods of Evaluation in Orthopaedic Animal Research
Published in Yuehuei H. An, Richard J. Friedman, Animal Models in Orthopaedic Research, 2020
Hybridization is the process of matching complementary strands of DNA or RNA or both to form a double stranded molecule. In situ hybridization (ISH) is the hybridization of a DNA or RNA probe to a target molecule that has not been extracted from its original cellular location, within a chromosome or in a fixed tissue section.253 Immunohistochemistry is analogous to ISH for nucleic acids and is used to detect the distribution of a specific protein within a cell or tissue. In immunohistochemistry, a specific antibody serves as the probe to detect the protein of interest. Gel electrophoresis is a method of separating DNA, RNA, or protein molecules based on their size and electrical charge. This technique makes use of the fact that, under an electrical field small molecules migrate through a gel matrix (agarose or acrylamide) faster than larger molecules. Southern blotting or transfer is a technique which is used to transfer DNA that has been electrophoresed through an agarose gel onto a solid support for hybridization. Northern blotting or transfer is the process of transferring RNA onto a solid filter support for hybridization. Western blotting or transfer is the process of transferring proteins that have been electrophoresed through an acrylamide gel onto a solid filter support for detection of a specific protein by antibody labeling.
Breathomics and its Application for Disease Diagnosis: A Review of Analytical Techniques and Approaches
Published in Raquel Cumeras, Xavier Correig, Volatile organic compound analysis in biomedical diagnosis applications, 2018
David J. Beale, Oliver A. H. Jones, Avinash V. Karpe, Ding Y. Oh, Iain R. White, Konstantinos A. Kouremenos, Enzo A. Palombo
Electrophoresis is defined as the migration of ions under the influence of an electric field, and gel electrophoresis is a commonly used method to separate proteins by size and charge (Kubáň et al., 2012). The development of capillary electrophoresis (CE) was enabled by advances in GC column technology, but instead of using pressurized gas or liquid as the mobile phase, CE uses high voltages to generate an electrophoretic flow of ionic species within a narrow-bore (20–200 µm i.d.) capillary. CE can be linked to either an ultraviolet–visible spectroscopy (UV-vis) or MS detector, and the resulting data look very similar to LC chromatograms; although in CE the data are referred to as an electropherogram rather than a chromatogram and retention time is referred to as migration time.
Coordinated interaction between Lon protease and catalase-peroxidase regulates virulence and oxidative stress management during Salmonellosis
Published in Gut Microbes, 2022
Perumalraja Kirthika, Vijayakumar Jawalagatti, Amal Senevirathne, John Hwa Lee
Proteome isolation was conducted in Salmonella wild-type strain JOL401 and Lon protease mutant strain JOL909. Overnight cultures of both strains were freshly inoculated into LB broth (2% inoculum) and allowed to grow until the late log phase. When the OD600 reached 0.8–1.0, cells were harvested by centrifugation at 13000 × g for 30 min. Cells were washed with phosphate-buffered saline (PBS) and subjected to osmotic lysis using the hypotonic solution with 1% lysozyme. Purified proteins were quantified using the Bradford method. 2-D gel electrophoresis was conducted as described in a previous report.54 Resolved gels were stained with Coomassie blue. Proteins of interest were further characterized using amino-acid N-terminus sequencing after blotting onto a polyvinylidene difluoride membrane. The obtained sequences were used as a query in a BLAST analysis of the ST genome through the National Center for Biotechnology Information.55
The tumour/normal tissue ratio of Keap1 protein is a predictor for lymphovascular invasion in colorectal cancer: a correlation study between the Nrf2 and KRas pathways
Published in Biomarkers, 2022
Liang-Che Chang, Chung-Wei Fan, Wen-Ko Tseng, Jim-Ray Chen, Chung-Ching Hua
The primary antibodies used were anti-Nrf2 (sc-722; Santa Cruz Biotech, CA, USA; 1:900 dilution), anti-Keap1 (sc-33569; Santa Cruz; 1:600), anti-Bach1 (NBP1-88722; Novus Biologicals, Littleton, CO, USA; 1:250), anti-p62 (ab-109012; Abcam, Cambridge, MA, USA; 1:1000), anti-HO1 (ab-13243; Abcam; 1:600), anti-KRas (SC-30; Santa Cruz; 1:1000), anti-Erk1/2 (SC-514302; Santa Cruz; 1:1000), anti-PI3K (SC-1637; Santa Cruz; 1:600), anti-Raf1 (SC-7267; Santa Cruz; 1:1000) and anti-β-actin (sc-47778; Santa Cruz; 1:600). The secondary antibodies were obtained from Santa Cruz (sc-2005) and Abcam (ab-6721). Gel electrophoresis was done as previously described (Chang et al.2018). In brief, the proteins were separated on a 12% Tris-glycine PAGE gel and then transferred to a PVDF membrane which was subsequently incubated first with primary antibodies at 4 °C overnight and then with secondary ones for 1 hour at room temperature. The protein levels were detected by chemiluminescence and visualised. The level of each protein was normalised to that of β-actin. An example of protein electrophoresis is illustrated in Figure 2.
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.