This method can help answer key questions in the field of protein protein interactions by enabling the detection of novel binding partners for extracellular proteins, both in high throughput and with high sensitivity. The main advantage of this method is that, with just a few micrograms of protein, you can generate hundreds of slides using the micro array printer. Use a standard micro arrayer for slide printing.
This instrument has a capacity of 57 slides per run, uses a print head holding 48 spotting pins, and can accommodate up to 8, 000 spots per slide. Generate working plates using 384 well plates at 10 microliters of sample per well from the stock plates. Perform this step manually, prior to slide printing using the micro arrayer.
Then spot proteins with quill type spotting pins onto epoxy silane slides at 60%humidity to prevent dehydration of the protein spots. Cy3 labeled bovine serum albumin can be spotted in duplicate between each protein sample to visualize the array for mask fitting. Subsequent to printing, remove the protein micro array slides from the humidified environment.
Next, use an ultrasonic fogger to generate a fine mist of blocking solution that settles onto the slide surface. Then, block the slides overnight with 5%milk in PBST to enactivate the surface. Store slides at minus 20 degrees Celsius in 50%glycerol to prevent freezing.
Interactions between extracellular proteins are often characterized by their low affinities. To enable the detection of these interactions, by increasing the binding avidity, we developed a multivalent approach based on the capture of the queried protein expressed as FC tagged extracellular domains on protein A coated micro beads. Spin the carrier IgG, that has been labeled with Cy5 mono reactive dye as described in the text protocol.
To calculate the molar ratio of the query protein and Cy5 IgG, divide the molecular weight of the query protein by the molecular weight of the IgG and multiply by 40 micrograms per milliliter to determine the concentration of Cy5 IgG needed. Once all the ratios have been determined form the micro bead protein complex by combining the FC tagged query and the Cy5 IgG with the protein A micro beads. Mix the suspension in PBS on a tube rotator for 30 minutes at room temperature, protected from light.
To begin the screening, warm the prepared slides at room temperature before loading onto the hybridization station. To perform the screening, first wash the micro array with PBST for one minute to remove residual glycerol. Load 200 microliters of one milligram per milliliter protein A in PBS 5%milk and incubate for 30 minutes to prevent uncomplexed protein A micro beads from binding to FC tagged proteins that may be present in the micro array.
After the hybridization station washes the slides with PBST, load 200 microliters of the query micro bead complex, in the presence of one milligram per milliliter protein A and incubate for 30 minutes. Following hybridization and washing of the slides by the hybridization station, rinse slides with water and place them in individual 50 milliliter conical tubes. Dry the tubes by spinning at 900 times G for five minutes.
Finally, scan the slides with a micro array scanner appropriate to detect Cy3 and Cy5 fluorescence by exciting at 532 and 635 nanometers, respectively. Shown here is a representative image of a printed slide. The Cy3 labeled bovine serum albumin spots are in green.
The spots were printed in duplicate as a quality control of the printing process. Representative results of an orphan protein, screened for identification of binding partners using the extracellular protein micro array technology, is shown here. The duplicate red spots are identified as a hit for the screened query protein.
This method that is described is highly versatile and can be used for the printing of a gamut of protein libraries, be they specific protein families or large compilations of proteins such as ours. Any protein of interest can be evaluated. While attempting this procedure it is important to handle the slides with care and to ensure that they do not dry out so as to prevent the loss of activity of the proteins printed.
Following the screen of a protein of interest, it is highly advised to further validate any hits observed by using orthogonal technologies, such as surface plasmon resonance. After its development, this technique has paved the way for researchers to study extracellular protein interactions in humans.
