Immunoblotting (Western blot)
By means of immunoblotting, we determine the presence of proteins in cells, or study the mechanism of action of therapeutic antibodies or compounds in cell lines of interest. We can determine whether total protein levels or phosphorylation status of proteins change over time upon treatment. Changed phosphorylation status can also be determined after addition of increasing concentrations of ligand.
Cells are seeded in plates and allowed for adherence/growth overnight.
They are treated with increasing amounts of ligand, therapeutic antibodies, or compound for a certain period, followed by lysis of the cells.
By immunoblotting, a primary and secondary antibody are used to detect protein bands. An increased band intensity compared to other bands indicates a higher expression in a certain sample. Absence of a band indicates that the target is not expressed. β-Actin is taken along as control.
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