The preparation and use of biotinylated trypsin in western blotting for the detection of trypsin inhibitory proteins

Abstract

Methods were developed for the preparation of biotinylated trypsin of high specific activity. This was used as a probe for the detection of serine proteinase inhibitory proteins which had been separated by sodium dodecyl sulfate-polyacrylamide gradient slab gel electrophoresis and electroblotted to nitrocellulose. The method was extremely sensitive and specific and could detect 0.15 ng of the serine proteinase inhibitor, trasylol (0.023 pmol active inhibitor). The method was also widely applicable and could be used to detect a range of serine proteinase inhibitors in human serum with molecular weights of 50 to ˜ 180 kDa, soybean trypsin inhibitor variants, and secretory leukocyte proteinase inhibitor extracted from human intervertebral disc and articular cartilage. The identity of several of the serine proteinase inhibitors detected using biotinylated trypsin was verified by Western blotting using specific antibodies. Under the conditions used, electrotransfer of trasylol was quantitative; densitometric examination of blots indicated that there was a linear relationship between the amount of active trasylol electrophoresed (1-50 ng) and the intensity of the blot obtained with biotinylated trypsin as probe, indicating that serine proteinase inhibitory proteins may also be quantified by this technique using densitometric scanning. © 1994 Academic Press, Inc.