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| | In Vitro Csn5 Activity Assay |
| Description: | Csn5 activity was measured using purified CSN complex and the fluorescent substrate OGNedd8–SCFSkp2, in which a unique cysteine engineered into Nedd8 was labeled with Oregon Green 488, after which the product was conjugated to SCFSkp2 as previously described [Duda et al., Cell, 134:995-1006]. Csn5-dependent cleavage of OGNedd8 from SCFSkp2 resulted in a decrease of fluorescence polarization. The assay was performed in buffer containing 40 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 0.01% Triton X-100, 1% glycerol, 25 mM trehalose and 15 μg/ml ovalbumin. Polarization data was collected with a PHERAstar plate reader using black low-volume 384-well plates. |
| Affinity data for this assay | |
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