Pre equilibrated anti flag m2 affinity gel

20 ml liquid cultures of Sf9 cells at 1.5 × 10⁶ cells/ml, supplemented with 200 μl of 0.1 M sodium ascorbate and 20 μl of ⁵⁵FeCl₃ (1 mCi/ml, PerkinElmer), were infected with baculoviruses at an MOI of 1 and incubated at 25 °C for 48 h. Cells were then centrifuged at 500×g for 10 min at 4 °C. The cell pellets were first washed with 5 ml of citrate buffer (50 mM citrate and 1 mM EDTA in PBS, pH 7.0) and then with 10 ml PBS. The pellets were resuspended in 1 ml of lysis buffer (as above, for DNA2). The lysates were incubated on ice for 30 min and then centrifuged for 30 min at 17,200×g and 4 °C. Next, the lysates were incubated with 20 μl of pre-equilibrated anti-FLAG M2 affinity gel (Sigma Aldrich) for 1 h at 4 °C rotating. The beads were then washed three times with 1 ml of wash buffer A (as above) and three times with wash buffer B (as above). The beads were then incubated with 100 μl of elution buffer (as above) for 1 h at 4 °C. 1 ml of Ultima Gold liquid scintillation liquid (PerkinElmer) and 90 μl of the eluted proteins were added to scintillation counting tubes, which were then vortexed for mixing. Counts per minute (cpm) were measured with a Tri-Carb scintillation counter (Packard) using standard ³H settings. The remaining 10 μl elutions were analysed by SDS–PAGE and InstantBlue staining. The counts were normalised based on protein levels.