Large-Scale Fractionation of Molluscan Shell Matrix

• Published in: Protein expression and purification 2001-10, Vol.23 (1), p.175-179
• Main Authors: Marin, Frédéric, Pereira, Lucilia, Westbroek, Peter
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Blotting, Western; Chemical Fractionation; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix - chemistry; Extracellular Matrix - immunology; Mollusca - anatomy & histology; Mollusca - chemistry; Proteins - chemistry; Proteins - immunology; Proteins - isolation & purification
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.2001.1487

The numerous proteins occluded within the molluscan shell play a key role in the control of the mineralization process. Although extensively studied, these proteins are still poorly known, mainly because they are difficult to fractionate. In the present paper, we present, for the first time, a simple combined strategy for separating successfully large amounts of molluscan shell proteins. Since shell proteins do not absorb at 280 nm, our approach is based on the “blind” separation of these proteins by a preparative denaturing electrophoresis. They are subsequently detected on dot-blot with polyclonal antibodies raised against the unfractionated soluble matrix. In the present case, this approach allows one to collect enough purified proteins to obtain amino-acid composition as well as N-terminal sequences, and to perform in vitro tests and glycosylation studies. Furthermore, this method permits one to raise polyclonal antibodies against the isolated proteins.