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