Proximity Ligation And RCA

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