Western blotting

nl-10-2-2014-3Information on altered expression of toxicologically relevant genes is important when analyzing the underlying mechanism of toxic responses in cells. Western blotting is a common method to identify and quantify level-changes of proteins in tissues and cells exposed to possible toxic compounds.

Proteins in a sample are separated based on their size by gel electrophoresis. After this separation, the proteins are transferred to a solid membrane. The membrane is incubated with different antibodies that specifically bind proteins of interest. Fluorochromes attached to the antibodies make it possible to quantify bound antibody based on fluorescence intensity. The amount of bound antibody depends on the amount of the specific protein in each sample, and comparing fluorescence intensity from the samples yields relative protein levels.

At NIOM, we use the Odeyssey CLx Infrared Image System to develop a digital image and quantify fluorescence from each sample. This technique gives us a fast and easy answer to identify the result from the western blotting.

nl-10-2-2014-2When cells are exposed to different substances, the intracellular environment can change. This may lead to altered expression of different proteins. An example is shown in Figure 2. Samples of BEAS-2B cells is exposed to a methacrylate commonly used in resin-based dental materials and analyzed by western blotting. The target protein in Figure 2 has a molecular weight of 53 kDa (p53) and can be seen as the marked bands. Knowledge of the investigated protein and comparison to the migration in the gel yield a control for specific antibody binding.