Immunolocalization of the microtubule cytoskeleton - Essay Example

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. . The coverslip is then immersed in 3.7% formaldehyde (in PBS) at room temperature. Immersion in 0.2% Triton-X 100 (in PBS) detergent at room temperature follows. To prevent the subsequent antibody from sticking non-specifically to the cells on the coverslip, the coverslips should be incubated with cell-side-up with a 100-l droplet of 3% (w/v) bovine serum albumin (BSA).

The incubation is done on a piece of Parafilm in a humid Petri dish for 15 minutes. An anti-tubulin dilution is prepared with 1 part antibody + 75 parts 1% (w/v) BSA in PBS. A 200 l total of antibody is necessary for the three coverslips. This tubulin antibody is a monoclonal antibody (12G10) and recognizes the -subunit of tubulin. Then each coverslip is overlayed each with a 100-l droplet of the diluted tubulin antibody. After that, a secondary antibody with 1 + 200 in 1% (w/v) BSA in PBS is used.

The coverslips are placed back into the Petri dish chamber and overlay with a 100-l droplet of secondary antibody and they are incubate for 30 minutes at 37C. Therefore, we have 3 coverslips, all treated with the secondary antibody, 2 of them are treated with the primary antibody (one of them is incubated at 37C, and the other incubated at 4C) and 1 that is not treated with the primary antibody (incubated at 37C). The coverslips then are placed back into the Petri dish chamber and are overlaid for 30 minutes at 37C with a100-l droplet of Vectastain ABC reagent.

This reagent contains a complex of avidin and the enzyme horseradish peroxidase (HRP). They are washed again with PBS. The drops of DAB (diaminobenzidine; a carcinogenic compound) are applied to the coverslips and incubate at room temperature for 5 minutes. After DAB incubation,