Abstract:

Pressure Cycling Technology (PCT) is an emerging method for extracting molecules of a wide range of molecular weights for biochemical and molecular biological experiments. PCT is accomplished with an instrument, the Barocycler™, which utilizes changes in hydraulic pressure to manipulate the biological sample within a closed system (i.e., the Pulse Tube™). These tubes allow the volume of extracting medium to be altered during PCT by movement of a small piston in each tube, which reacts to the pressure of hydraulic fluid (30% ethylene glycol) surrounding the Pulse Tube™. The current study was conducted to evaluate performance of PCT as a means of extracting estrogen (ER) and progestin receptor (PR) proteins from lyophilized reference specimens used in proficiency testing and evaluation of suspected environmental estrogen mimics. Various quantities of reference specimens were subjected to PCT at different pressures and cycle counts using 40 mM Tris-HCl buffer, for different times. As a control, identical quantities of reference specimens were homogenized with the same buffer using conventional methods (i.e., with a Polytron PT 10–35). Extracts from the two procedures were centrifuged at 105,000 x g for 30 min, 4°C. ER were measured by titrating extracts with increasing concentrations of [³H]estradiol-17 in the presence and absence of a 200-fold excess of unlabeled diethylstilbestrol. PR were measured similarly with [³H]R5020 in the presence and absence of unlabeled R5020. Receptor protein yield and integrity were assessed from specific binding capacities (fmol/mg protein) and apparent dissociation constants (Kd values, M), respectively. Preliminary results illustrate the PCT method and conditions may be altered to optimize extraction of a particular labile protein analyte.

Keywords: PRESSURE CYCLING; ESTROGEN RECEPTOR; PROGESTIN RECEPTOR; PROTEIN EXTRACTION; LIGAND TITRATION; LYOPHILIZED TISSUE

Document Type: Research article