proximity ligation and rca
TRANSCRIPT
Direct observation of individual
endogenous protein complexes
in situ by proximity ligation
SÖderberg et al., Nature Methods, 2006, 3, 995.
Science, 1994, 265, 2085.
Rolling Circle Replication
Nucleic Acids Research, 1998, 26, 5073.
Rolling Circle Replication
Nature Biotechnology, 2002, 20, 448.
Proximity ligation
Nature Methods, 2006, 3, 995.
Rolling Circle Replication/Proximity ligation
Nature Methods, 2006, 3, 995.
U2OS Skov-3
CHO-K1 HFB
TIME CELLS
Mitotic Cells
Nature Methods, 2006, 3, 995.
Rat1 TGR-1 fibroblast
Rat1 TGR-1 fibroblastc-myc knockout
C-Myc / EGFR proximity probes Her-2 / EGFR proximity probes
Nature Methods, 2006, 3, 995.
U2OS stained with 2 different hybridization probes
SHSY5Y cells
Identification of interaction of c-Myc/Max with RNA Pol II
Nature Methods, 2006, 3, 995.
Normal colon tissue
Normal colon tissue(Enzyme based immunostaining)
Human tonsil tissues
Burkitt Lymphoma sample
Nature Methods, 2006, 3, 995.
U-937 cells transformed with v-myc
U-937 cells transformed with v-myc + PMA + IFN-
Nature Methods, 2006, 3, 995.
4-OH-Tamoxifen: Antagonist to ER
Small molecule inhibitors of c-Myc/Max interaction
Conclusions
-P-LISA can detect protein-protein interaction at the single molecule level.-RCA increases the number of fluorophores/detected interaction.-Multi protein complexes can be studied.-Doesn’t need expression of fusion proteins.-Disruption of protein-protein interaction can be studied.
-P-LISA can be performed only on fixed cells-Real time tracking cannot be possible-Selection of antibodies for each pair
-Validate results from Yeast Two Hybrid or other assays-Can be used as molecular ruler