| Assay Method Information | |
| | Binding of the M6P Analogs of the Invention with CI-M6PR |
| Description: | 96-Well plates (Maxisorp Nunc) are incubated overnight at 4° C. with 200 μl of PMP (pentamannose 6-phosphate) at the concentration of 200 μg ml−1, in carbonate buffer (NaHCO3/Na2CO3 at 0.1M, pH 9.6). The following day, the solution containing the residual PMP is discarded and the wells are saturated, for 1 h at ambient temperature, with 360 μl of 1% gelatin (Type A from Porcine Skin) diluted in PBS (1.9 mM NaH2PO4, 8.1 mM Na2PO4 and 154 mM NaCl, pH 7.4). The wells are then rinsed five times with PBS to which 0.2% gelatin has been added. All the washes and also the dilutions are carried out in the solution of PBS to which 0.2% gelatin has been added. The M6P analogs to be tested at the various concentrations (from 10−2 to 10−7 M) are pre-incubated in the presence of pre-biotinylated CI-M6PR (M6PRb) (2.5 μg mol−1) for 20 min, then 200 μl of the mixture are incubated in the wells for 2 h, at ambient temperature. After three washes, the wells are incubated for 1 h with a streptavidin-peroxidase solution (250 μl per well; 3.10−8 M). After three more washes, 200 μl of a solution of OPD (o-phenylenediamine, 1 mg ml−1 in citrate buffer, pH 5.0, and 1 μl 30% H2O2.ml−1; Sigma Aldrich) are added. After incubation for 20 min in the dark at ambient temperature, the optical densities are measured at the wavelength of 450 nm.The affinities of the phosphonate disaccharide 18 and carboxylate disaccharide 24 of formula (I) of the invention were measured and compared with those of their respective monosaccharide homologs.The affinity denotes the binding capacity (in the case in point by means of a covalent bond) of the M6P analogs for CI-M6PR. |
| Affinity data for this assay | |
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