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Proteins and proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
The western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3D structure of the protein (native/non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or INDF), where they are probed (detected) using antibodies specific to the target protein. Now many reagent companies specialize in providing antibodies (both monoclonal and polyclonal antibodies) against tens of thousands of different proteins. Conunercial antibodies can be expensive, although the unbound antibody can be reused between experiments. This method is used in the fields of molecular biology, biochemistry, immunogenetics, and other molecular biology disciplines. The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern (Figure 3.15).
Experimental Protocols for Generation and Evaluation of Articular Cartilage
Published in Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi, Articular Cartilage, 2017
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi
Protein extracted for Western blot use may also be used for ELISA; however, detection of proteins poorly solubilized by detergents, such as collagen, may be poor. Ideally, ELISA should be used where quantification of proteins, for example, quantification of signaling components, or phosphorylation events is wanted. Protein preparations for Western blot make use of a gentle detergent lysis buffer and exclude enzymatic digest. Western blot allows for separation of proteins based on molecular weight through a polyacrylamide gel and is a qualitative to semiquantitative procedure. However, the gel-based separation by size allows for detection of components with similar antigenic sites that may vary in size, for example, determining if a specific protein is cleaved by enzymatic activity, or where the specificity of the antibody is poor.
Cytotoxicity activity, in silico molecular docking, protein- and DNA-binding study of a new Ni(II) Schiff base complex
Published in Journal of Coordination Chemistry, 2018
Niladri Biswas, Sumit Khanra, Arnab Sarkar, Shamee Bhattacharjee, Deba Prasad Mandal, Ankur Chaudhuri, Sibani Chakraborty, Chirantan Roy Choudhury
Western blot (also called protein immunoblotting, because an antibody is used to specifically detect its antigen) is a widely accepted analytical technique used to detect specific proteins in the given sample. By analyzing location and intensity of the band formed, expression details of the target proteins in the given cells could be obtained. AGS and A549 cell lysates were obtained and equal amounts of protein from each sample were diluted with loading buffer, denatured, and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by protein transfer to nitrocellulose membrane. The effect of complex 1 on PCNA expression was checked on AGS and A549 cancer cells. The blot was incubated with anti-PCNA antibody, followed by blotting with HRP-conjugated secondary antibody. The blots were then detected by using a chemiluminescent kit from Thermofisher. This analysis was performed two times.
Histological and immunohistochemical study of the effect of liraglutide in experimental model of non-alcoholic fatty liver disease
Published in Egyptian Journal of Basic and Applied Sciences, 2023
Mai Salah Nour, Zeinab Abd El-Hay Sakara, Nawal Awad Hasanin, Shereen Mohamed Hamed
In Western blot technique, proteins are separated based on their molecular weight via gel electrophoresis before being transported to a membrane, where each protein forms a band. The amount of protein is shown by the band’s thickness. The method was explained by Mahmood and Yang [21]. The PVDF membranes are produced by (Millipore, Bedford, MA). Primary antibodies: anti-beclin 1 (mouse monoclonal antibody, cat. no. sc -48,341, from Santa Cruz Biotechnology) and anti-alpha-smooth muscle actin (mouse monoclonal antibody cat. no. A2547, from Sigma-Aldrich). Bound antibodies were found using the chemiluminescent substrate (NEN Life Science Products, Boston, MA).