Preparations of full-length RAG1 and RAG2 complex, and HMGB1 protein were described previously (Shimazaki et al., 2012 (link)).Burst kinetic assays to determine the active fraction of the RAG complex and coupled cleavage assay with both 12- and 23RSS were done as previously described (Shimazaki et al., 2009 (link)).To determine the oligomerization status of RAG proteins, we have done gel filtration analysis on different versions of RAG complexes, including the core/core RAG complexes (core RAG1 plus core RAG2), as well as combinations of core and full-length versions of RAG1 and RAG2. The preparations of RAG complexes analyzed by gel filtration thus far are all consistent with tetrameric RAG complexes.
Briefly, the cleavage reaction contains 5 nM of 5′ end labeled 12RSS substrate, 5 nM unlabeled 23RSS substrate, 20 nM of the tetrameric RAG complex and 1 μM HMGB1(high-mobility group protein B1), 25 mM K-morphorinepropanesulfonic acid (MOPS)-KOH, pH 7.0, 30 mM potassium glutamate, 30 mM potassium chloride (KCl), 5 mM MgCl2, 1 mM DTT, and 0.05 mg/ml BSA and is incubated at 37°C for 60 min. The reaction is stopped by adding 0.1% SDS and 20 mM EDTA, and then heat denatured. Products are separated on denaturing gels and visualized using a Molecular Image FX (Bio-Rad).The intensity of autoradiography is quantified using Quantity One (Bio-Rad).
Briefly, the cleavage reaction contains 5 nM of 5′ end labeled 12RSS substrate, 5 nM unlabeled 23RSS substrate, 20 nM of the tetrameric RAG complex and 1 μM HMGB1(high-mobility group protein B1), 25 mM K-morphorinepropanesulfonic acid (MOPS)-KOH, pH 7.0, 30 mM potassium glutamate, 30 mM potassium chloride (KCl), 5 mM MgCl2, 1 mM DTT, and 0.05 mg/ml BSA and is incubated at 37°C for 60 min. The reaction is stopped by adding 0.1% SDS and 20 mM EDTA, and then heat denatured. Products are separated on denaturing gels and visualized using a Molecular Image FX (Bio-Rad).The intensity of autoradiography is quantified using Quantity One (Bio-Rad).