Rapid high-resolution electrophoresis of multimeric von Willebrand Factor using a thermopiloted gel apparatus.
Rapid and highly reproducible nonreducing agarose gel electrophoresis (NRAGE) of von Willebrand Factor (vWF) multimers was performed using a thermostated minigel apparatus that monitors and precisely controls internal gel temperature. The substitution of lithium dodecyl sulfate (LiDS) for sodium dodecyl sulfate (SDS) allowed electrophoresis to be performed below the 16 degrees C Krafft point of SDS and facilitated NRAGE of vWF over the entire range of 0-35 degrees C. Internal gel temperature was regulated by a thermocouple probe inserted directly into the gel during electrophoresis which interfaced with a thermopilot that continually measures and adjusts temperature to within +/- 0.5 degrees C. At 10 degrees C operative temperature, NRAGE at 1.5% agarose concentration was completed in 20 min at 250 V. Electrophoresis could be performed in only 10 min at 500 V, but at such high voltages, localized temperature fluctuations as much as 6 degrees C resulted in perturbation of banding patterns in those vicinities. In the optimized method, both high molecular weight multimers and proteolytic fragments of vWF were separable suggesting clinical applicability of this system for the diagnosis of von Willebrand Disease and thrombotic thrombocytopenic purpura.
Smejkal
Gary B
GB
Cleveland Clinic Foundation, Department of Clinical Pathology, Cleveland, OH, USA. gary.smejkal@proteomesystems.com
Shainoff
John R
JR
Kottke-Marchant
Kandice M
KM
D004587:Electrophoresis, Agar Gel / Q000295:instrumentation*; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation*; D014841:von Willebrand Factor / Q000302:isolation & purification*
D014841:von Willebrand Factor