After measuring the protein of interest (POI) with a specific antibody (marked by a chemiluminescent reaction), a second set of antibodies is used to quantify the protein defined as the LC. However, gels relying on these tests may still be subject to differential protein transfer or human loading error, and thus journal reviewers usually require a second control. Protein levels are first measured with colorimetric assays, such as the Bicinchoninic Acid (BCA) assay. In these cases each sample must contain the same amount of total protein. Increasingly, investigators are utilizing measurements of antibody binding such as fluorescence intensity to quantify differences between samples of interest. Antibodies against β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), along with other high-abundance housekeeping proteins, are used most often because they bind to proteins in nearly any sample. A control antibody, or loading control (LC) often serves this purpose. Demonstrating absence requires proof that protein is in each lane of the gel. The Western, or immunoblot, is widely used for determining the presence or absence of a protein within a cellular homogenate, limited only by the availability of a specific antibody.
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