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. 2016 Oct 11;11(10):e0164662. doi: 10.1371/journal.pone.0164662

Fig 3. ATP hydrolysis and oligomerization assay of NLRP1(227–990).

Fig 3

(A) HPLC chromatogram showing the profile of a solution of ADP and ATP standards run on an ion exchange Partisil SAX column (grey solid line), retention times for ADP 2.81 minutes and ATP 6.12 minutes. The sample prepared by unfolding of NLRP1(227–990) contained mainly ATP (>85%) (black solid line). The peak with a retention time of about 2 minutes is background from the buffer. (B) Malachite green assay performed on a solution of NLRP1(227–990) in the presence of ATP 1mM (black squares) and in the presence of ATP 1mM and MDP 0.1mg/mL (black triangles). In both cases the concentration of protein was 0.1 mg/mL and the experiment was performed at room temperature for 1 hour. A blank experiment, without NLRP1 and MDP (black diamonds) and a positive control with DnaK (black stars) were also performed. (C) Size exclusion chromatography (Superose 6 10/300 column) of NLRP1(227–990) in absence (black solid line), in presence of MDP and ATP (grey dotted line) and in presence of MDP and a non-hydrolysable ATP analogue (grey solid line). Black arrow heads indicate the retention volumes of molecular weight standards.