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Analysis of Single Cells Using Lab-on-a-Chip Systems
Published in Frances S. Ligler, Jason S. Kim, The Microflow Cytometer, 2019
Typical applications that benefit from the LOC cytometry approach are the monitoring of protein expression or apoptotic processes in eukaryotic cells. Monitoring cellular protein expression is a critical step for characterization of cell populations or assay optimization and can be achieved by staining the protein of interest with specific antibodies. Fluorescently labeled antibodies are used to detect cells bearing specific antigens (lipids, proteins or carbohydrates). The antibody may be directly conjugated to a fluorescent probe or a fluorescent secondary antibody may be used. The detection of proteins generally depends on the availability of a suitable, specific antibody. When using fluorescently labeled antibodies, cells can often be stained directly on the chip, eliminating time consuming washing steps.7
Nanodevices for Early Diagnosis of Cardiovascular Disease: Advances, Challenges, and Way Ahead
Published in Alok Dhawan, Sanjay Singh, Ashutosh Kumar, Rishi Shanker, Nanobiotechnology, 2018
Alok Pandya, Madhuri Bollapalli
The other format, called indirect immunoassay, has the ability to improve the sensitivity of the detection. In this method, the analyte of interest is bound to a specific antibody after immobilization onto the surface. A labeled secondary antibody against this primary antibody is then incubated for detection purposes. It is important that the secondary antibody be raised in another species than the primary antibody to avoid nonspecific binding. By far the most common type of immunoassay detection is achieved by applying two specific antibodies for the analyte in a sandwich format immunoassay. Antibody-sandwich immunoassays may be the most useful of the immunosorbent assays for detecting antigens because they are frequently between two and five times more sensitive than those in which the antigen is directly bound to the solid phase (Allinson, 2011). The antigen being investigated is actually sandwiched between two antibodies, which are the coated antibody immobilized on a surface and the detection antibody, which is usually conjugated to a marker that can cause a quantifiable response proportional to its concentration. Autoanalyzers that enable automated routine biochemical tests in hospital laboratories for biomarker diagnosis are used today to perform immunoassays (Chan, 1994).
Protein Expression Methods
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Western blotting is used for immunohistochemical visualization of proteins separated by a polyacrylamide gel. The Western blot is a close relative to Southern and Northern blots, described in Section 2.10, “Southern and Northern Blots” section. The first step in Western blotting is transferring the protein from the gel to a polymer membrane, typically either polyvinylidene fluoride (PVDF) or nitrocellulose. This is accomplished by “sandwich” electrophoresis, in which a sandwich is created containing filter paper, the polyacrylamide gel, the polymer membrane, and more filter paper. Electrophoresis is then used to transfer the proteins from the gel to the polymer membrane, using either a semidry blotting apparatus or a wet blotting apparatus (Figure 5.6). After the transfer is complete, the membrane is blocked with a solution of bovine serum albumin (BSA) or powered nonfat milk reconstituted in buffer. The purpose of the blocking step is to coat the entire membrane with protein, thus avoiding nonspecific absorption of the antibodies used for protein visualization. The blocked membrane is then probed with a primary antibody specific for the protein being expressed. In many cases, the antibody is specific for an epitope tag, which was appended to the protein during subcloning. The primary antibody is then probed with a labeled secondary antibody, specific for the primary antibody. For maximum sensitivity, the secondary antibody is typically labeled with either alkaline phosphatase (AP) or horseradish peroxidase (HRP), both of which catalyze a chemiluminescent reaction that can be visualized using a high-sensitivity camera or black-and-white film.
Generation and evaluation of anti-mouse IgG IgY as secondary antibody
Published in Preparative Biochemistry & Biotechnology, 2020
Qi Zhang, Dongyang He, Long Xu, Shikun Ge, Jinquan Wang, Xiaoying Zhang
Secondary antibody is widely applied in immunoassay, such as Western blot, ELISA, Immunohistochemistry, Immunocytochemistry, Flow cytometry and Immunoprecipitation.[5] Secondary antibody is prepared in other hosts (e.g.,: goat anti-rabbit, rabbit anti-mouse) that bind to a primary antibody or antibody fragment, usually labeled with enzyme or luciferin.[6] The secondary antibody can have different specificity to the primary antibody, including the entire antibody molecule (H + L), fab fragment, fc fragment or heavy chain, or light chain (κ, λ).[7]