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Molecular Analysis in Mechanobiology
Published in Jiro Nagatomi, Eno Essien Ebong, Mechanobiology Handbook, 2018
After electrophoresis, proteins are transferred to a hydrophobic membrane (PVDF or nitrocellulose) and the membrane is incubated with a blocking buffer. In the Western blotting step, antibodies are used to probe the membrane; most commonly using an indirect method with an enzyme-conjugated secondary antibody. Antibody incubation steps are usually performed for 1 h; however, incubation with the primary antibody overnight at 4°C at a relatively lower dilution can sometimes increase sensitivity and reduce background. After antibody binding and washing, a developing solution is added containing either a colorimetric or chemiluminescent substrate. Enzymatic substrate cleavage is a kinetic process and care must be taken to stop the development process before plateau signal levels are reached. Development of colorimetric assays can be visually monitored. Chemiluminescence is detected using photographic film in a closed cartridge and may require more optimization of development time. However, chemiluminescence-based assays provide much greater sensitivity. Western blots can be “stripped” and re-probed several times to detect assay multiple proteins from a single blot.
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).
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.