Sondagem de alta densidade microarrays proteína funcional para detectar interacções proteína-proteína

The overarching goal of this procedure is to identify protein, protein interactions within a given proteome in a parallel unbiased manner, which is not possible with other technologies. This is accomplished by first hybridizing and epitope tagged protein of interest with a highly purified, full length functional protein library deposited in a coordinate plane on the surface of a microscope slide. Once the protein has reached equilibrium with each of its respective target proteins on the array, the unbound fraction is washed away and the protein interactions are detected with a monoclonal antibody to the epitope conjugated to a fluorescent moiety using a standard microarray scanner.

The resulting images are interlaced with information regarding the intensity of each feature with respect to bound protein and fluorescent signal. This information is extrapolated with the aid of the microarray analysis software to generate a list of intensity values for each feature on the array. The information regarding the protein-protein interacting pair can be exported to downstream bioinformatic analysis software to score the feature specific data and identify protein protein interactions between the protein of interest and the proteins on the microarray.

The raw signal intensity data is compiled over two or more protein microarrays to account for inter and intra assay variation, and the data is normalized to score each of the interactions. The resulting output generates a target list of interacting proteins, which can be used to guide further interrogation of protein protein interactions. In vivo Functional protein microarray was developed in Michael Snyder’s lab over 15 years ago.

Since then, there have been many successful applications of this technology due to its versatility and biochemical proteomic assays. This technique facilitates the study of protein interactions and modular signaling networks, as well as alternative connectivity and previously known signaling cascades To culture and purify the V five fusion kinase probes use freshly streaked yeast strain Y 2 58 containing V five fusion protein plate, the yeast on synthetic complete uracil, 2%dextrose agar, and grow at 30 degrees Celsius for three days from frozen culture. Inoculate starter cultures from a single colony and grow overnight in synthetic complete uracil.

2%dextrose on a shaking platform or wheel at 30 degrees Celsius. The following morning, inoculate 400 milliliters of a synthetic complete uracil. 2%raffinose culture with sufficient starter culture to achieve a final optical density at 600 nanometers or a OD 600 of 0.1.

Grow the inoculums to an OD 600 of 0.6, then induce V five kinase fusion construct expression by adding enough three x yeast extract. Pep tone supplemented with 6%galactose to dilute the induction media by a factor of three or a final galactose concentration of 2%Induce cells at 30 degrees Celsius for six hours on a shaking platform. Use a two liter erlenmeyer flask to ensure appropriate aeration.

Harvest the cells by spinning 400 milliliters of the cell suspension at 1000 times G for five minutes at four degrees Celsius. Wash the cells once with 50 milliliters of ice cold PBS buffer and transfer to a 50 milliliter conical tube. Wash the pellet again in ice cold phosphate buffered saline or A PBS buffer and transfer to two milliliter snap cap tubes for lysis.

After spinning the cells at 20, 000 times G for one minute at four degrees Celsius, remove the buffer with a pipette and place the tubes on ice. Lyce the cells with 0.5 millimeters zirconia beads in a one to one-to-one volume of cell pellet to beads to PBS lysis, buffer. Then vortex the mixture three times using an agitation platform for two minute intervals at four degrees Celsius.

Centrifuge the lysate at 20, 000 times, G in a tabletop micro fuge for 10 minutes at four degrees Celsius. Transfer the clarified lysate to a tube containing approximately 100 microliters of pre-washed nickel affinity resin and incubate on a nutate for two hours at four degrees Celsius. To capture the histamine six x tagged V five fusion protein pellet the resin using a tabletop centrifuge for five minutes at 1000 times G and four degrees Celsius following removal of the supernatant.

Wash the resin with wash buffer for 10 minutes at four degrees Celsius on the mutator. Repeat the wash twice more with fresh wash buffer. Elute the proteins with two 100 microliter volumes of 200 millimolar ole.

Add glycerol to a final concentration of 30%of the samples and store at minus 80 degrees Celsius until used in the assay. To detect interactions. Dilute the V five fluoro four conjugated antibody to 260 nanograms per milliliter in probe, buffer and mix thoroughly by shaking.

Then dilute the V five fusion protein Probe over a concentration range of five to 500 micrograms per milliliter. Next, remove the protein microarrays from the freezer and bring to four degrees Celsius in the refrigerator just prior to use. Add the blocking buffer directly to the slide holder containing the protein microarrays and cover the top with paraform to prevent leakage.

Block the arrays in blocking buffer for one hour by shaking at 50 RP on a stage at four degrees Celsius. After blocking, transfer the arrays to a humidified chamber, tiled to four degrees Celsius and add 90 microliters of diluted probe directly to the array surface. Overlay the arrays with a raised lifter slip and incubate without shaking in the humidified chamber at four degrees Celsius for one and a half hours Following incubation, add the slide to a 50 milliliter conical tube containing enough pre chilled probe buffer to completely envelop the slide, allowing the lifter slip to gently slide off of the protein microarray.

Wash the arrays three times for one minute each in three conical tubes. Apply the antibody solution directly to the array immediately after completing the wash and overlay with erased lifter slip. As before, incubate the arrays for 30 minutes at four degrees Celsius in the humidified chamber.

Perform the same wash step as before, and spin in a 15 milliliter conical tube at 800 times G in a tabletop centrifuge for five minutes. At room temperature, air dry the arrays in a slide holder in the dark for 30 minutes prior to scanning the array at 647 nanometers. In this experiment, the binding activity of TDA one V five was investigated.

Yeast protein microarrays spotted in duplicate with approximately 4, 200 full length GST fusion proteins were incubated with the empty vector control or with TDA one V five probes. The inset panels compare the identical region in both arrays. Of particular note is RIM 11, which interestingly has also been identified as a phosphorylated target of TDA one in a previously published study.

Once targets have been identified, they can be tested for biochemical and functional activity in vivo. If you’re watching this video, you should have a better understanding of how to use functional protein microarrays as a means to identify candidate protein protein interactions for further in vivo validation and interrogation.