Sorry - Because of organizational restructuring of the unit the LUMIER service is temporarily discontinued (not affecting already on-going projects).
LUMIER-assays (LUminescence-based Mammalian IntERactome mapping), originally published by Barrios-Rodiles et al., provide a novel method to test protein-protein interactions. It is based on the co-purification of two tagged proteins from transiently transfected mammalian cells. See here for a powerpoint explanation of the basic method.
Advantages of LUMIER assays include
- a quantitative read-out
- co-precipitation as simple assay principle
- the possibility of testing the effect of external triggers such as hormones, growth factors or drugs on the interaction
LUMIER Assay In Brief
The assay tests for the interaction of two proteins, X and Y, which are transiently co-expressed in mammalian cells. Protein X is expressed in fusion with a tag which allows efficient purification on affinity beads. Protein Y is expressed in fusion with luciferase, which allows sensitive detection. After cell lysis, protein extracts are purified on magnetic affinity beads, which results in an enrichment of protein X. Co-purification of Y is detectable as luciferase activity in the enriched material. A significant luminiscence signal indicates that the two protens XY are interacting.
The assay works with small numbers of cells in a microtiterplate format and can be automated for increased throughput. One advantage of LUMIER is that testing the effect of various exogenous stimuli on an interaction is straightforward. For example, cells can be treated by addition of small molecules (hormones, drugs) or stimulated by growth factors before lysis to see if a particular interaction is responsive to such stimuli.
Finding novel interactions: Array LUMIER
The assay can also be used to find novel protein-protein interactions for a given protein. In this case, arrayed collections of proteins are tested one by one for binding to the protein of interest. The speed of the assay allows screening of complete signalling pathways, or cellular compartments. More...
What we need from you
To do a LUMIER assay, we need expression constructs for the two proteins. Each protein should be available in two forms: Protein A-tagged, and luciferase tagged. On request we can provide aliquots of GATEWAY-compatible versions of these vectors in several variations. (for technical reasons we have only GATEWAY-compatible versions and no vectors with multiple cloning sites).
What we do
LUMIER assays are done as follows:
- Expression constructs are transiently transfected into HEK293 cells. Two days after transfection, cells are lysed in a buffer detergent-containing buffer, and protein A-tagged proteins are purified on immunoglobulin-coated magnetic beads. Luciferase activity is measured in the lysate before and after washing.
- As a negative control, the expression construct for the protein A-tagged protein will be replaced by a construct of protein A only.
- As a positive control, a known protein-protein interaction, such as JUN binding to FOS, is done in parallel with the customer's samples. A positive result for the positive control indicates that the assay has worked as expected. The service does not include any test verifying the expression of the transiently expressed proteins by Western blotting or any other method.
What we return
Customers will receive a table containing all relevant data from the LUMIER assays. Note that expression constructs will not be provided to our customers. The DKFZ will not claim any rights to the results of this service, other than the right to make that data publicly available after a lock-up period of two years.
For more details, see our FAQs, or contact Frank Schwarz. Information on pricing you'll find at the GPCF pricelist (only available to internal customers).
If you are more interested in a high-throughput screening for protein-protein interactions, please have a look at our Y2H-Screening Service .
