Biomedical research and, in particular, cancer research is progressing from focusing on small numbers of molecules or cellular events to global functional analysis, feeding these results into new approaches for the prevention, diagnosis, and treatment of cancer. Methods that allow researchers to look across a broader angle at cellular processes such as mRNA expression levels or protein interaction patterns are needed more often to study fundamental processes.
Coaffinity 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 purification 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 copurifying 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 catalytic activity such as luciferase, highly amplifying the read-out signal. This avoids the need for gels and blots for detection of the tag and allows microplate-based miniaturization and automation of the protocol (Figure 1).
In this article, we describe the use of Tecan’s HydroFlex™ washer equipped with a magnetic bead plate carrier for fast purification of a large number of samples using magnetic beads. For the detection of the luminescence signal Tecan’s Infinite® F200 multimode reader was used. In the protocol in our lab at the German Cancer Research Center (DKFZ), the 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 immunoglobulins is advantageous for miniaturization and automation of the assay.
Figure 2 depicts the workflow of the experiment. After transient expression of the protein pair in tissue culture cells, a lysate is prepared and allowed to bind to immunoglobulin-coated magnetic beads. Nonbinding proteins are removed from the beads in a washing step. Luciferase activity is determined in the starting material as well as in the purified beads. The fraction of bound luciferase activity in the positive versus the negative control is taken as a readout for binding.
An important step in such an automated protocol is the washing procedure used for purification of the protein complexes. The options available in the HydroFlex protocol allow fine-tuning of the washing procedure. The relative speed of the washing steps ensures a minimal loss of protein complexes during the wash, while maintaining a high enrichment factor, as can be measured via the activity of the luciferase tag.