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.