Identification and purification of proteins from germ cell-conditioned medium (GCCM)

Germ cells are known to regulate Sertoli cell and testicular function possi bly through released factor(s) or via cell-cell contact. However, the ident ities of many of these putative biological factors are not known. The aim o f this study is to present a strategy to identify and purify germ cell-deri ved proteins found in germ cell-conditioned medium (GCCM) at a quantity suf ficient to permit protein microsequencing. The purification scheme of a nov el germ cell-derived protein from GCCM designated GC-26 is presented along with several germ cell proteins using a combination of high pressure liquid chromatography (HPLC) columns. The purity of GC-26 and other germ cell pro teins were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel ele ctrophoresis (PAGE) and silver staining. The identities of GC-26, a 26-kDa polypeptide, and other proteins were determined by direct protein microsequ encing. These partial NH2-terminal amino acid sequences were compared with the existing databases at Protein Identification Resource (PIR), GenBank, a nd BLAST. These analyses revealed that these proteins are unique. This stra tegy should be useful for the micropurification of proteins from other biol ogical samples and/or fluids.