A Method For Rapid Visualization Of The Size- Distribution Of Factor VIII-Related Protein In Plasma

Factor VUI-related protein circulates in normal human plasma as a series of multimeric forms with apparent molecular weights ranging from 1 to 20×10⁶. So far, combined electrophoretic and immunologic methods permitted demonstration of variable concentration and size- distribution of factor VIII-related protein in von Willebrand’s disease as compared to normal. We now have devised a one-step method for determining the size pattern of this plasma protein. Fresh plasma, containing 1% SDS and 0.8M urea, was layered on top of 2.5% polyacrylamide gels with 2.75% by weight of methylene bisacrylamide/acrylamide. Following extended electrophoresis in 0.2% SDS-0.1M Tris/HCl (pH 7.4), the gels were soaked in 5% formaldehyde and then extensively washed with 10% ethanol. Proteins were visualized employing an ultrasensitive ammoniacal silver stain. This staining revealed a multimeric protein pattern in the upper part of the gel the distribution of which was recorded by densitometry. The protein was identified as factor VIII by two-dimensional immunoelectrophoresis. The method was reproducible and allowed densitometric evaluation within 24 hr.