Blue sepharose cl 6b beads

NADP⁺ binding assay was performed as previously described (33 (link)). Briefly, 200 ng of 6xHis-FLAG-IDH1 and variant proteins were incubated with 30 μl Blue Sepharose CL-6B beads (Amersham Biosciences) which mimics NADP⁺ at 4°C for 2 hours, then washed with 20 mM Tris-HCL (pH 8.6) for 3 times. The beads were then subjected to SDS-PAGE, followed by Western blotting analysis. For NADP+ binding assay with IDH1 dimer or monomer, 1 mg IDH1 WT protein was used for sucrose gradient centrifugation to separate monomer or dimer, then 1 ug monomer or dimer protein was used for in vitro kinase assay with or without FGFR1. After kinase assay, 200 ng protein were incubated with 30 μl Blue Sepharose CL-6B beads (Amersham Biosciences) at 4°C for 2 hours, then washed with 20 mM Tris-HCL (pH 8.6) 3 times. The beads were subjected to SDS-PAGE, followed by Western blotting. The same amount of protein was loaded in parallel as loading control.

1 μg of 6xHis-FLAG-IDH1 protein was incubated with 100 μl Blue Sepharose CL-6B beads (Amersham Biosciences) at 4°C for 2 hours, then washed with 20 mM Tris-HCL (pH 8.6) for 3 times and separated to 3 tubes equally. The beads were incubated with indicated concentration of NADP⁺ at 4°C for 1 hours. Then the samples were centrifuged to separate the supernatant and beads. The supernatant or beads were subjected to SDS-PAGE, followed by Western blotting analysis.