| Assay Method Information | |
| | YKL-40: HS Assay |
| Description: | The standard experimental protocol for YKL-40 indirect binding AlphaScreen assay comprised of 5 step additions to wells of a 96 well plate: (i) in the 1 step, 8 uL of biotinylated heparan sulphate was added at 5×60 nM concentration in MST buffer containing 1% DMSO; (ii) in the 2nd step, 8 uL of a small molecule compound inhibitor was added at 5×1 uM or 5×100 uM concentration in MST buffer containing 2% DMSO; (iii) in the 3rd step, 8 uL of YKL-40-histag was added at concentration 5×7.5 nM in MST buffer containing 0% DMSO, after which the plate was spun down for 2 minutes at 1500 g and incubated 1 h at 37° C.; (iv) in the 4th step, 8 uL of Nickel Chelate Acceptor beads were added at concentration 5×10 ug/mL in MST buffer containing 1% DMSO, after which the plate was spun down for 1 minute at 250 g and incubated in dark for 1 hour at RT; (v) in the 5th step, 8 uL of Streptavidin Donor beads were added at concentration 5×10 ug/mL in MST buffer containing 1% DMSO, after which the plate was spun down for 1 minute at 250 g and incubated in dark for 1 hour at RT. Positive and negative control wells received just 8 uL of MST buffer containing 2% DMSO without inhibitor in the 2nd step (ii). In addition, negative control received just 8 uL of MST buffer containing 0% DMSO without YKL-40-histag in the 3nd step (iii). Finally, the plate luminescence was excited at 680 nm and the emission read at 520-620 nm using the AlphaScreen module in the The Spark 10 M multimode microplate reader (Tecan Trading AG, M nnedorf, Switzerland). Inhibition percentage and IC50 values were determined using GraphPad Prism 7.0 software (GraphPad Software, San Diego, Calif., USA). |
| Affinity data for this assay | |
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