Type I Collagen Purification from Rat Tail Tendons

Type I collagen, or collagen I, is the most abundant protein in the human body and provides strength and resiliency to tissues such as bone, tendons, ligaments, and skin. Collagen I forms macromolecular networks in which resident mesenchymal cells are embedded. Cell-extracellular matrix interactions are critical not only for maintenance of tissue properties but also for guiding and orienting the phenotype of resident cells. Cues from the extracellular matrix have been shown to be critical in pathophysiologies such as fibrosis, aging, and cancer. Hence, the details of these interactions are being scrutinized to better understand the mechanisms of such diseases and conditions. Many in vitro assays, such as cell-embedded collagen lattices, preparation of hydrogels, adhesion assays, etc., have been developed to study various aspects of cell-extracellular matrix interactions. All these in vitro models rely on utilizing high-quality purified collagen I. Here, we provide state-of-the-art collagen I extraction protocol and useful tips to produce high-quality purified collagen I solutions. We also provide a detailed protocol for pepsin digestion of collagen I, for a highly reliable collagen concentration assay, and guidelines for conducting quality controls to validate purified collagen solutions. Collagen I prepared with these procedures is highly suitable for many in vitro applications.