High throughput colorimetric atpase assays kit

Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin/streptomycin (P/S) for cell culture, blocking solution for Western blot, MANT-ADP for ATPase binding assay, Opti-MEM medium, siRNA (HSP90β, scramble), lipofectamin 2000, lipofectamine RNAi Max for transfection, qRT-PCR kit were obtained from Life Technology. RIPA lysis buffer, ECL were purchased from Thermo; protease inhibitors and phosphor-stop were acquired from Roche. Strep-tactin superflow plus were purchased from Qiagen. Bradford protein assay kit was purchased from Bio-rad. High Throughput Colorimetric ATPase Assays kit were from Innova Biosciences. Caspase-3 activity assay kit was purchased from Cell Signaling. Apoptosis detection kit was purchased from BD. For antibodies, Raf-1, IKK-1/2, HSP90β, p-HSP90β, HSP70, CDC37, PPIA, p-MEK, MEK, p-ERK, ERK, GAPDH, PARP and Ub antibodies were purchased from Santa Cruz; Anti-flag antibodies was from Sigma; CDK4, p-Rb, Caspase-9, eEF2 and Strep-HRP antibodies were from Cell Signaling. All chemicals not listed above were purchased from Sigma-Aldrich.

The ATPase activity was measured using the High Throughput Colorimetric ATPase Assays kit (Innova Biosciences). Free phosphate from the AaFtsH dodecamers and hexamers was eliminated by incubation with 100 μl of PiBind resin (Innova Bioscience) for 30 min. Also mix A (50 mM Tris-HCl pH 8.0; 150 mM NaCl; 10 mM MgCl₂), was previously incubated with PiBind resin (Innova Bioscience). Reactions were started by mixing AaFtsH (0.25 μM final concentration) in mix A with 50, 100, 250, 500, 1000, or 1800 μM ATP (final concentrations). Reaction rates were measured every 2 min for a total of 10 min, in triplicates. ATP concentrations were chosen such that the maximum concentrations were more than 10× the previously reported K_M values (30 (link), 31 (link)). Twenty-five microliters of P_iColorLock mix was added to stop the reaction, and after 5 min, 10 μl of stabilizer reagent was added. OD₆₅₀ was measured after 30 min using a CLARIOstar (BMG-Labtech) microplate reader; every replica was measured three times and an average of these readings was calculated for each replica (Fig. S4). Released Pi concentrations were calculated from a calibration curve of standard Pi concentrations measured. K_M and K_cat were calculated for both hexamers and dodecamers. Michaelis–Menten constants were obtained, assuming the steady state of the reaction, with GraphPad Prism software with a 95% CI.