Figure 4

In vitro interaction assay between resin-bound His ₆ -Wzd _C41 (34 kDa) and native Wze (27 kDa) proteins. Coomassie-stained gel A and B: (A) Lane 1 contains the BenchMark Prestained Protein Ladder (Invitrogen). Lane 2: The two proteins were incubated together in a 1:1 ratio with the Ni²⁺ charged resin for 4 h at 16°C in 2.5 ml buffer B1 containing 10 mM Tris–HCl pH 7.5, 100 mM NaH₂PO₄, 50 mM imidazole, 1% Triton, 5 mM MgCl₂. Lanes 3 to 6: 5 ml wash fractions with buffer B1 in panels A and B (in panel B, 200 μM ATP was added to the wash in lanes 4 to 6). Lane 7: protein elution using 1 ml buffer containing 10 mM Tris–HCl pH 7.5, 100 mM NaH₂PO₄, 1 M imidazole, 1% Triton, 5 mM MgCl₂, 2% sarcosyl. (C) Western blot of gel in panel B, detection was carried out using anti-phosphotyrosine antibody.