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  • Title: Isolation of EGF-dependent transforming growth factor (TGF beta-like) activity from culture medium conditioned by fetal rat calvariae.
    Author: Centrella M, Canalis E.
    Journal: J Bone Miner Res; 1987 Feb; 2(1):29-36. PubMed ID: 3502665.
    Abstract:
    A transforming growth factor of the beta class (TGF-beta), defined by its ability to induce normal rat kidney cells (NRK, clone 49F) to form anchorage-independent large colonies in soft agar in the obligate presence of epidermal growth factor, has been prepared from culture medium conditioned by fetal rat calvariae. This activity was purified by acetic acid extraction, gel permeation chromatography, and two reversed-phase HPLC (rpHPLC) steps. Bone culture derived-TGF beta-like activity was soluble in 1.0 M acetic acid, eluted from Sephadex G-75 at relative molecular mass (Mr) 25,000, from mu Bondapak C18 rpHPLC at 63 +/- 5% methanol in 0.1 M acetic acid, and from mu Bondapak CN rpHPLC at 36 +/- 2% n-propanol in 0.1% trifluoroacetic acid. Based on specific activity estimations at each stage of purification, TGF beta-like activity was purified 2500-fold with a 14% recovery, and 1 l of conditioned medium yielded 1-2 micrograms of factor. Silver-stained polyacrylamide gels of this material after CN mu Bondapak rpHPLC revealed a predominant band of (Mr) 24,000.
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