Fast Magnetic Bead Purification Of Interacting Cellular Proteins

Biomedical research, and in particular cancer research, is progressing from focusing on small numbers of molecules or cellular events to global functional analysis. Therefore, methods that allow one to look at a broader angle at cellular processes, such as mRNA expression levels or protein interaction patterns, are needed more and more often to study fundamental processes.

Co-affinity purification of two proteins from a complex mixture is one of the standard methods for the detection of protein-protein interactions.

To circumvent the need for specific antibodies in affinity puri-fication and subsequent detection, proteins can be expressed in fusion with a “tag”, i.e. an extension that has a high-affinity binding site for a generic antibody. When two differently tagged proteins are used, the first tag can be used for the specific purification of the complex, and the second tag for the detection of the co-purifying protein.

In one variation of this protocol, the tag for detection is not an antibody tag, but an enzyme that can be detected via its cata-lytic activity, such as luciferase [1] highly amplifying the read out signal. This avoids the need of gels and blots for detection of the tag, and allows microplate based miniaturization and automation of the protocol affinity tag is Staphylococcus aureus protein A, which can be purified on immunoglobulins as the affinity matrix.The use of magnetic beads for immobilization of immuno-globulins is advantageous for miniaturization and automation of the assay.