Assessing the ATP Binding Ability of NLRP3 from Cell Lysates by a Pull-down Assay

Abstract

NACHT-, LRR-, and PYD-containing protein 3 (NLRP3) is a member of AAA+ ATPase family that upon activation forms inflammasomes. Several studies demonstrated that ATP binding and hydrolysis are important for NLRP3 function as an inflammasome sensor. Furthermore, compounds targeting ATP binding motifs and interfering with ATPase activity of NLRP3 inhibit NLRP3 inflammasome formation. Measuring ATPase activity of proteins and binding of radiolabeled ATP to specified proteins are well-established methods that require purified protein. Here, we describe a method for assessing NLRP3 binding to ATP using ATP-conjugated beads and lysates of cells that either express endogenous NLRP3 or are transfected with plasmids encoding NLRP3. Efficiency of binding is followed after elution from the beads and detection with Western blot and immunolabelling. The method can be used to evaluate the functionality of NLRP3 variants or to check whether compounds or NLRP3 binding partners interfere with binding of ATP.

Keywords: ATP agarose beads; ATP binding; Lysate; NLRP3; Pull-down assay.