Differential Radial Capillary Action of Ligand Assay: A High-Throughput Technique to Identify Bacterial Nucleotide Second Messenger-Binding Intracellular Proteins

Bacteria produce nucleotide second messengers, NSMs - intracellular signaling molecules - in response to appropriate stimuli. These molecules bind to target proteins and regulate bacterial functions.

To identify these target proteins using differential radial capillary action of ligand assay, take a multi-well plate with bacterial cell lysate containing soluble proteins. These proteins are derived from different bacterial clones expressing individual ORFs - open reading frames - in the genome, ensuring each well has a distinct protein.

Add a mix of guanosine tetra- and penta-phosphates - NSMs labeled with radioactive phosphorus. These molecules bind to target proteins in the lysate. With a pin tool, collect an equal amount of liquid from each well. Place the tool on a nitrocellulose membrane to deposit the liquid as spots.

Target proteins bind to the membrane through non-covalent interactions preventing their diffusion. This sequesters the bound radiolabeled NSMs at the central point of application. Free NSMs radially diffuse with the liquid phase via capillary action.

Use phosphor imaging to detect the radioactive signals and obtain a digital image of the spots. Define the spots using two circles. The outer one depicts the periphery of the diffused NSMs. The inner circle represents the sequestered NSMs.

Calculate the binding fraction - the intensity of radioactivity detected from the inner circle over the total radioactivity of the diffused spot. Locate spots with high binding fractions to identify the wells with NSM-bound target proteins.

For DRaCALA screening of the target proteins, add 20 microliters of the thawed whole-cell lysates to individual wells of a 96-well V-bottom microtiter plate, and add 2.5 units of Serratia marcescens endonuclease to each well. After 15 minutes at 37 degrees Celsius, place the lysates on ice for 20 minutes.

Next, mix equal volumes of phosphorus 32-labeled guanosine pentaphosphate and guanosine tetraphosphate, and add 1x lysis buffer #1 to the mixture to obtain a four-nanomolar guanosine pentaphosphate solution.

Using a multichannel pipette and filtered pipette tips, mix 10 microliters of the guanosine pentaphosphate mixture with the cell lysate for a five-minute incubation at room temperature.

At the end of the incubation, wash a 96X pin tool three times in a 0.01% solution of non-ionic detergent for 30 seconds, followed by 30 seconds of drying on a paper towel per wash, before placing the pin tool in the 96-well sample plate. After 30 seconds, lift the pin tool straight up and place it straight down on a nitrocellulose membrane for 30 seconds.

After five minutes of drying, place the nitrocellulose membrane in the transparent plastic folder for storage phosphor screen exposure and visualization by phosphor imaging, as demonstrated.

To quantify and identify potential target proteins in the analysis software associated with the phosphor imager, open the .gel file of the visualized plates.

To define the spots to be analyzed, use the "Array analysis" function to set up a 12-column by 8-row grid. To circumscribe the outer edge of the whole spots, define big circles. Export the "Volumn+Background" and "Area" of the defined big circles to a spreadsheet, to circumscribe the small inner dots. Size down the defined circles.

Export the "Volumn+Background" and "Area" of the defined small circles and save all the data in the spreadsheet. Position circles to overlap with spots as necessary, resizing to slightly bigger than the actual spots.

Use the equation to calculate the binding fractions in the spreadsheet, and plot the data. Then, identify the potential binding proteins in the wells that show high binding fractions compared to the majority of other wells.