In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Microcontact Printing:

1. Cut out field of micropattern of the PDMS stamp using a scalpel
2. Wash the field containing the micropattern by flushing it with ethanol (100%) and distilled water.
3. Dry the PDMS stamp with nitrogen or argon gas.
4. Pipette 50 μl BSA-Cy5 work solution (0.67 mg/ml) onto the PDMS stamp so that the whole micropattern field is covered with solution. Incubate for 30 min at room temperature.
5. Wash the micropattern field by flushing it with Phosphate Buffered Saline (PBS) and distilled water.
6. Dry the PDMS with nitrogen or argon gas.
7. Place the PDMS stamp under its own weight onto the middle of one epoxy-coated coverslip and incubate for 30 min at room temperature in a petri dish.
8. Label the position of the PDMS stamp on the backside of the coverslip and strip the stamp from the slide with a forceps.
9. Stick a Secure Seal Hybridization chamber over the microcontact-printed field on the coverslip. The label helps to localize the center of the field.
10. Pipette 60 μl streptavidin work solution (50 μg/ml) into the reaction chamber and incubate the sample for 60 min at room temperature.
11. Wash the sample with 1 ml PBS by adding the buffer into one port of the chamber and removing it simultaneously at the second port with a pump.
12. Pipette 60 μl biotinylated antibody work solution (10 μg/ml in PBS supplemented with 0.1% Tween 20; “PBST”) into the reaction chamber and incubate for 60 min at room temperature.
13. Wash the sample with 1 ml PBST and subsequently 1 ml PBS by adding the buffer into one port of the chamber and removing it again at the second port with a pump.
14. Store the micropatterned surfaces in PBS in the dark at room temperature until cells are ready for seeding.

Incubation of cells onto the micropatterned surface:

1. Grow adherent cells to 50% confluence in a 3 cm tissue culture plate.
2. Detach cells expressing bait and prey proteins of interest with Ethylenediaminetetraacetic acid (EDTA) solution and centrifuge 5 min at 1,000 rpm.
3. Discard the supernatant and dissolve the cell pellet in the according growth medium.
4. Centrifuge 5 min at 1,000 rpm.
5. Discard the supernatant and dissolve the cell pellet in appropriate growth medium.
6. Exchange the PBS from the reaction chamber on the micropatterned coverslip by 60 μl of the cell suspension.
7. Create a humidified chamber by soaking a sterile pad in distilled water and putting it into the petri dish to prevent the sample from running dry.
8. Incubate the cells over-night at 37 °C in a 5% CO₂ atmosphere on the micropatterned surfaces.
9. Before analyzing the cells on the microscope, exchange the medium by Hank s buffered salt solution including Ca²⁺ and Mg²⁺.

Microscopy:

1. The coverslip is placed on a suitable mount and the cell morphology is checked under white-light.
2. The quality of the BSA-Cy5 patterns is checked by excitation at 647 nm.
3. Micropatterns of fluorescent prey protein (GFP or YFP) are recorded at 488 or 514 nm under Total Internal Reflection (TIR) excitation.

Representative results:

If there is interaction between the target proteins in cells grown on a micropatterned surface, the prey will follow the bait redistribution. The resulting micropattern can be visualized by the fluorescence label of the prey protein. Importantly the contrast obtained provides a direct measure of the interaction strength (see Figure 1). Therefore a simple evaluation of the interaction of two proteins becomes possible – without the necessity to further process the measured primary data.

Figure 1. Rearrangement of the bait in the live cell plasma membrane at different interaction strengths. TIR images of T24 cells transfected with (a) CD71-GFP on CD71-antibody, (b) CD4 and cytosolic YFP on CD4-antibody and (c) GPI-GFP-DAF on CD59-antibody microbiochips are shown. The data are characteristic for strong (a), no (b) or weak interaction (c). (a) is reproduced from (Weghuber et al., in press), (b) from (Schwarzenbacher et al., 2008).