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Immunolocalization of the microtubule cytoskeleton
Pages 5 (1255 words)
Proteins are probably the most diverse substances found in the cells found in both the single and multicellular organisms. Estimated around 10 000 different proteins, each protein has its specific function. Cellular functions are often localized in specific compartments of the cell, and being able to localize the unknown proteins may provide important information in determining the function of the proteins…
The most prominent methods are: Western blot, spectrophotometry, enzyme assay, immunoprecipitation and immunostaining. In immunostaining, the method used during this procedure, an antibody is used to detect a specific protein epitope. These antibodies can be monoclonal or polyclonal. Then enzymes such as horseradish peroxidase or alkaline phosphatase are commonly used to catalyse reactions that give a coloured or chemiluminescent product. Fluorescent molecules can be visualised using fluoresence microscopy.
During this experiment, the distribution of the protein tubulin in normal rat kidney (NRK) cells is explored. A monoclonal antibody that is specific for the -subunit of tubulin is used. Tubulin polymerizes into long, 25-nm wide microtubules that we will visualize with tubulin antibodies. The formation and maintenance of microtubules is cold sensitive. At 4C, microtubules are destabilized and they depolymerize. At 37C, human body temperature, they remain polymerized. Photograph 1 shows cells incubated at 4 0C, while photograph 2 shows cells incubated at 37 0C. At this stage of the procedure, microtubules cannot be detected in either of the photographs.
3 separate plates are used to create the NRK cells culture. ...
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