Summary
In plasma, protein S is found in its free form and as a complex with C4b-binding protein.
After 125I-protein S was added tonormal human plasma and applied to SDS-8% polyacrylamide gel
electrophoresis, the autoradiogram of the gel showed only one single band at free
protein S position. Applying this evidence, we have developed a peroxidase staining
Western Blotting method to quantitate total protein S in human plasma which consists
of sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting
to nitrocellulose membrane and a sensitive avidin-biotinylated peroxidase staining
method (ABC technique). The measurement of protein S by the immunoblotting was reproducible
and the coefficient of variation was 7%. As little as 1 ng of protein S could be detected.
C4b-binding protein did not affect the measurement of protein S. Compared to other
immunoassays, this peroxidase staining immunoblotting method has the advantage of
directly estimating the apparent molecular weight of protein of interest, eliminating
nonspecific stain and having high sensitivity without using radioisotope.
