LUMIER-Assays Dr. Manfred Koegl Protein Interaction Unit

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LUMIER-Assays Dr. Manfred Koegl Protein Interaction Unit Genomics And Proteomics Core Facility

LUMIER assays are a reliable, cheap, high-throughput method to measure protein-protein interactions in mammalian cells. The assay tests the interaction of two proteins which are transiently expressed in mammalian cells. Like classical co-immuneprecipitations, the method is based on a co-purification protocol. Idea by Barrios-Rodiles et al., (2005) Science 307:1621-5.

LUMIER-Assays Two tagged proteins (here labelled X and Y), are transiently expressed in mammalian cells. Cells are lysed, and the first tag is used for affinity purification of protein X on magnetic beads. The second tag is a luciferase and used for detection of protein Y. IgG-coated magnetic beads Y Luci affinity tag X *”LUMIER”´, Idea by Barrios-Rodiles et al., (2005) Science 307:1621-5.

LUMIER workflow Expression constructs for 1. Transiently transfect into HEK293 cells Expression constructs for protein Y (luciferase-tagged) + / - protein X (protein A-tagged) 2. Prepare cell lysate, split in 2 aliquots 3. Allow binding to magnetic beads 4. Wash 5. Measure luciferase activity Calculate bound luciferase relative to total luciferase

Controls sample Y negative control X Y (hexuplicates +/- s.d.)