![]() ![]() Although Western blotting is very useful for protein identification and quantification, the whole procedure is laborious and time-consuming. After blocking for non-specific adsorption, specific protein recognition is achieved by interaction with an appropriate primary antibody and secondary antibody, which is often conjugated to a fluorophore or an enzyme such as horseradish peroxidase ( Mishra et al., 2019 Han et al., 2020). The common procedure of Western blotting analysis starts with electrophoresis separation of proteins followed by transfer to a nitrocellulose membrane. Western blotting is a standard molecular biology technique for sensitive and selective protein detection ( Nybo, 2012 Kim, 2017). This work provided aptamers as useful molecular tools for selective protein recognition in Western blotting analysis. Lastly, the identified aptamers were also effective in recognition of different fusion proteins with the same tag, thus greatly expanding the scope of the potential applications of these aptamers. Compared with conventional antibody-based immunoblotting, such aptamer-based procedure gave a cleaner background and was able to selectively label target protein in a complex mixture. The generated aptamers G1, M1, and H1 specifically bound to their cognate target proteins with nanomolar affinities, respectively. In this work, multiple fluorescent DNA aptamers were isolated by in vitro selection to selectively label commonly used tag proteins including GST, MBP, and His-tag. Aptamers represent an alternative class of simple and affordable affinity reagents for protein recognition, and replacing antibodies with aptamers in Western blotting would potentially be more time- and cost-effective. Successful detection of the target proteins in these methods relies on the specific interaction of the antibodies, which often bring a high production cost and require a long incubation time. Selective protein recognition is critical in molecular biology techniques such as Western blotting and ELISA. 4State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing, China.3Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, China.2State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing, China.1Department of Biomedical Engineering, College of Engineering and Applied Sciences, Jiangsu Key Laboratory of Artificial Functional Materials, Nanjing University, Nanjing, China.Novex AP Chemiluminescent Substrate should be combined with Nitro Block II for use with nitrocellulose membranes.Yao Wang 1,2,3 Zhe Li 1,4 Hanyang Yu 1,2,3 * The emitted light is stable up to 24-96 hours allowing for multiple exposures.Ĭhoose from a standalone substrate, Novex AP Chemiluminescent Substrate or the WesternBreeze Chemiluminescent Immunodetection kits which contains all solutions necessary for your application including blocking solutions, primary antibody diluent, ready-to-use secondary antibody solution, ready-to-use chemiluminescent substrate, wash solutions, incubation trays, pre-cut filter papers, polyester sheet for even substrate development on the membrane. Light emission occurs only during the enzyme-substrate reaction therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases. CDP-Star is dephosphorylated by AP to yield meta-stable dioxetane phenolate anion intermediate that decomposes and emits light with a maximum intensity at a wavelength of 475 nm. For western blot detection based on alkaline phosphatase (AP), 86 kDa, we offer our CDP-Star substrate that is designed to deliver picogram level sensitivity and is compatible with both traditional x-ray film and CCD-based imaging. ![]()
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