Quality Control of Bupleurum Species by Newly Established Eastern Blotting

Abstract

In the investigation of staining technique for several compounds, a polyvinyliden difluoride (PVDF) membrane was found as transfer membrane (Towbin et al., 1979), and applied to western blotting technique that utilizes antigen-antibody binding property for the specific and sensitive detection of peptides and proteins by immunostaining (Granger, 1988; Reig & Klein, 1988). In the case of low molecule compounds, thin-layer chromatography (TLC) immunoblotting of glycosphingolipids was reported using a nitrocellulose membrane (Towbin et al., 1984), but the transfer efficiency from TLC plate to the membrane was poor and not constant. Recently, the direct TLC immunostaining of glycolipids and glycosphingolipids was demonstrated without the blotting step from TLC plate to the membrane (Meisen, 2004; Suetake & Yu, 2003). Furthermore, TLC immunostaining of gangliosides (Miyamoto et al., 2006) and dot blot analysis of gangliosides on PVDF membrane (Chabraoui et al., 1993) were reported as the direct immunostaining. These immunostaining techniques were limitedly applied for glycolipids, glycosphingolipids and gangliosides because the other low molecule compounds were easily washed out into the buffer solution without fixing on the TLC plate or PVDF membrane. Since small molecular compound such as saponin cannot fix on the PVDF membrane or TLC plate, no success of immunostaining for saponin has been reported. We reached to a new idea that a saponin was divided into two functional parts, sugar and aglycone moieties. Sugar parts in saponin could be oxidized to give aldehyde groups which were conjugated with carrier protein like bovine serum albumin (BSA) and then fixed onto the PVDF membrane as saponin-BSA conjugate because BSA was strongly binded on the membrane. In contrast, the aglycone part was recognized as epitope by monoclonal antibody (MAb) against saponin and then we succeeded to immunostaining of saponin on the PVDF membrane. The first success was reported on the immunostaining of solasodine glycosides by our group (Tanaka et al., 1997) followed ginsenosides (Fukuda, 1999, 2000, 2001). In 2001 we suggested a new name, [eastern blotting] for glycyrrhizin as a new immunostaining technique (Shan et al., 2001). In eastern blotting the developed TLC plate was covered with PVDF membrane and all contents were blotted from TLC plate to the membrane by press and heating. After the