Transcript LUMIER-Assays

LUMIER-Assays
Dr. Manfred Koegl
Protein Interaction Unit
Genomics And Proteomics Core Facility
Genomics & Proteomics
Core Facilities
Page 2
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.
Dr. Manfred Koegl
Genomics & Proteomics
Core Facilities
Page 3
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
Luci
X
Y
affinity tag
Dr. Manfred Koegl
*”LUMIER”´, Idea by Barrios-Rodiles et al., (2005) Science 307:1621-5.
Genomics & Proteomics
Core Facilities
Page 4
LUMIER workflow
Expression constructs for
protein Y (luciferase-tagged)
+ / - protein X (protein A-tagged)
1. Transiently transfect into HEK293 cells
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
Dr. Manfred Koegl
Genomics & Proteomics
Core Facilities
Page 5
Controls
sample
X
negative control
Y
Y
AP1-Complex: JUN binding to FOS
luciferase units (abritrary)
20000
10000
(hexuplicates +/- s.d.)
0
Dr. Manfred Koegl
ProteinA-JUN
ProteinA
Luci-FOS
Luci-FOS