Chromogenic Polymer Hydrogel-Based Enzyme Screening Assay: A High Throughput Method to Screen the Carbohydrate-Active Enzymes Using Synthetic Substrates

To begin, activate the 96-well filter assay kit plate by adding 200 microliters of activation solution to each well. Incubate at room temperature without agitation for 10 minutes.

Use a centrifuge and any standard 96-well plate for collection to spin down the extant activation solution. Add 100 microliters of sterile water to the Chromogenic Polymer Hydrogel, or CPH substrates, and apply a vacuum or spin to remove the stabilizer. Repeat the wash two more times.

To prepare the enzyme reaction, add 150 microliters of 100-millimolar sodium acetate buffer pH 4.5, and 5 microliters of endo-cellulase solution with three different concentrations, to each well of the assay kit plate.

Place the product plate underneath the assay kit plate to collect any potential leakage from the reaction plate during shaking. Then, incubate the assay kit plate at room temperature in a horizontal shaker at 100 RPM for 30 minutes.

For receiving reliable data, it's necessary to agitate the reaction mixture during the incubation.

Place the assay kit plate with the product plate underneath it in the centrifuge, and spin down at 2,700 x g for 10 minutes, to transfer the reaction product into the wells of the product plate.

Following the spin, visually inspect the product plate to check that the volume of liquid in each well is approximately the same. Using a plate reader, read the absorbance of the collection plate at 630 nanometers for the different green CPH substrates.