| Assay Method Information | |
| | Inhibition of the activity of Gtfs determined by Zymographic Assay |
| Description: | A previously reported zymographic assay was utilized for the investigation of Gtf enzymatic activity, as described in Example 1. A 1:100 in fresh 5 mL THB with 50 μL of selective compounds at a series of concentrations was used to dilute overnight S. mutans UA159 cultures and grown to OD470 of 1.0. The final concentration of DMSO used in the assays was 1%. After the centrifugation at 4° C., the supernatants were isolated and filtered through a 0.22-μm-pore-size filter membrane and dialyzed at 4° C. against 0.02 M sodium phosphate buffer (pH 6.8), with 10 μM phenylmethylsulfonyl fluoride (PMSF), followed by a second dialysis against 0.2 mM sodium phosphate containing 10 μM PMSF. 4 mL of samples were concentrated to 40 μL by 100K Amicon Ultra-4 centrifugal filter (Merck Millipore Ltd.). Next, 10 μL of each concentrated culture supernatant was applied to 8% SDS-PAGE in duplicate. One gel was subjected to Coomassie blue dye for protein detection, while the other one was subjected to zymographic assay. The resultant white opaque glucan bands were visualized against a black background. |
| Affinity data for this assay | |
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