The ATPase cycle of Hsp70 chaperones controls their transient association with substrate and, thus, governs their function in protein folding. Nucleotide exchange factors (NEFs) accelerate ADP release from Hsp70 which results in rebinding of ATP and release of the substrate. This chapter describes several methods suitable to study NEFs of Hsp70 chaperones. On the one hand, steady-state ATPase assays provide information on how the NEF influences progression of the Hsp70 through the entire ATPase cycle. On the other hand, nucleotide release can be measured directly using labeled nucleotides, which enables identification and further characterization of NEFs.
