This kit is designed for two purposes: 1) identification of proteins that specially bind linear polyUb chains in whole cell or tissue lysates; 2) validation of direct interactions between linear polyUb chains and their binding proteins. The non-cleavable 6xHis-tagged linear Ub6 is resistant of deubiquitination when incubating with whole cell or tissue lysates, thus allowing maximally capture proteins that specifically bind linear polyUb chains. After binding, 6XHis-tagged non-cleavable linear Ub6 and the binding proteins can be enriched with Nickel XPure Agarose Resin (included). The bound proteins and the non-cleavable polyUb chains can be eluted from Ni resin using a buffer containing 250 mM imidazole (included) or by incubating with TEV protease (not included). Either of the elution method can significantly reduce the amounts of non-specific binding proteins when compared with elution using SDS sample buffer.
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