Use of SP-Sephadexto fractionate and obtain semi-pure preparation from whole pituitaries of Indian water Buffaloes (Bubalus bubalis)

Abstract

Different charge isoforms of native Luteinizing hormone (LH) (dimeric form) V_e/V_o = 1.49 can be fractionated on SP-Sephadex into four different charge isoforms (LHUB, LH25, LH50, and LH100) by stepwise elution using different molarities of Na₂HPO₄. LHUB was found to be difficult to purify, whereas LH50 and LH100 were found to be pure and highly immunoreactive against anti-bLHβ serum as indicated by the results obtained from direct binding ELISA and Western blot analysis. SDS-PAGE of LH50 and LH100 showed two bands corresponding to the two subunits of LH. LH25 can be purified to homogeneity by rechromatography on S-300. Purification of buLH as a highly immunoreactive preparation has also been described using SP-Sephadex. This LH preparation (SP25B), which was obtained after slight modification in the pre-existing protocol, has been found to be highly immunoreactive against anti-bLHβ serum in direct binding ELISA. Being a very simple and reproducible method, it can be used to obtain pure LH preparation, as a reference material, in a short period of time for various immunoassays and bioassays.