It is of particular importance to unravel the interaction network of molecules in living cells to understand the mechanisms that regulate cell metabolism and function. An increasing number of methodologies which provide qualitative and quantitative information are the driving force on our way to obtaining holistic pictures of cell function. Co ip, TAP tag or photo cross linking essays allow for purification of pre molecules in vitro.
In addition, several approaches were designed for the analysis of protein interactions in the living cell. These include two hybrid screens or complementation essays of fluorescent proteins. Furthermore, some spectroscopic approaches like fret or FCS and also non spectroscopic methods have been established to address this question.
The aforementioned techniques have been developed for recording large populations of cells yet suffer from being rather indirect and therefore hardly quantitative. In contrast, a few high-end quantitative approaches were introduced. However, extending high throughput is difficult to combine high throughput capabilities with the possibility of extracting quantitative information.
We recently developed a new concept for identifying protein protein interactions. The micro pattering technique here is a schematic illustration of the micro pattering SA grids of BSA Sci five are printed on functionalized glass cover slips and interspaces are filled with streptavidin, followed by biotinylated ligands. In most cases, antibodies against the membrane.
Protein bait in cells grown on such microbiome chips. The bait will be arranged in the plasma membrane according to the antibody micro pattern. Interactions with a second fluorescently labeled protein prey are probed by measuring the degree of co pattering Using A silicon master PD MS stamp with an array of squares with feature size and depth.
The three micrometer are produced from a gel and cut out. The stamps are rinsed with 100%ethanol and double de distilled water and dried with nitrogen before. They are incubated with 0.67 milligrams per milliliter of sci-fi.
Labeled BSA solution for 30 minutes At room temperature during incubation, Epoxy modified glass cover slips are cleaned in the same way. Same way after inking, the stamps are washed with PBS and water. For BSA sci five Transfer.
A stamp is dried by nitrogen and immediately afterwards placed under its own weight under a glass cover slip for 30 Minutes. The location of the micro pattern field is marked on the backside of the cover slip. Upon removing the stamps, cover slips are sealed with adhesive silicone masks.
Chambers are loaded with a 50 microgram per milliliter Streptavidin solution incubated for one hour and rinsed with one milliliter PBS afterwards. Subsequently, the streptavidin patterns are incubated with 10 micrograms per milliliter biotinylated antibody for one hour and after washing with PBS cells are loaded into the chamber. When the cells have attached to the surface, the sample is ready for measurements.
The Only Requirement for the readout is a fluorescence microscope capable of illuminating the sample in total internal reflection configuration. Such systems are commercially available from different suppliers and became easy to handle for the user over the last years. Our detection system is set up on an epi fluorescence microscope.
Argon and krypton ion lasers are used for selective fluorescence excitation of the used fluoro fours samples are illuminated in an objective-based total internal reflection configuration using a 100 fold magnification high NA oil immersion objective. After appropriate filtering using standard filter sets, fluorescence is imaged onto a back illuminated CCD camera Readout is performed in time delay and integration mode, which allows for scanning of large sample areas. For this samples are shifted using a motorized XY stage synchronized to the line shift of the camera.
The back reflected laser beam is imaged on a two segment photo diode and the differential signal is used to control a Z pizo for fast refocusing. The reader is equipped with an automated focus hold system operating during the scanning process with one scan at 488 nanometer for selective excitation of GFP, the second scan at 647 nanometer for selective excitation of PS Five. For data analysis, we use a contrast calculation program written in MATLAB based on the fluorescence intensity within and outside the captured antibody region.
The contrast level and thereby the degree of protein interaction is analyzed for fast analysis of a great number of cells. Also commercially available automated software can be used. For instance, the scan R imaging software offered by Olympus has been successfully tested for the contrast evaluation of micro pattering experiments.
We Analyze the well-known interaction of CD four and LCK to proteins in early T-cell signaling as shown here. Fluorescent LCK clearly redistributes on CD four antibody coated regions. Disruption of the zinc clasp structure, which is a main interaction site of these two proteins by addition of a zinc cell later reduce the contrast of the LCK micro patterns.
In summary, the main advantages of the micro patterning technique are as follows, quantification of local spatially resolved protein interactions. Furthermore, the detection of weak interactions also in the plasma membrane of living cells, the detection of modulated bait prey interactions, analysis of the resting state, and the applicability of the approach for high throughput.
