Tips for Collagen Proteins

Collagens represent a large family of proteins that are the essential components of the extracellular matrix (ECM), the chief structural component of skin, and the most abundant protein in the human body representing approximately 30% of the total dry weight. Collagen is an attractive substance for various medical applications, such as implants, transplants, organ replacement, tissue equivalents, vitreous replacement, plastic and cosmetic surgery, surgical suture, and dressings for wounds and burns among others.

Based on their supramolecular structures, the collagens are divided into two main classes, fibril-forming collagens (type I, II, III, and V) and non-fibril-forming collagens (type IV and VI). Different collagen types are necessary to confer distinct biological features to the various types of tissues in the body.

• Collagen Standards

  Collagen proteins exhibit an amino acid composition rich in glycine, proline, and hydroxyproline residues. Because of the relatively high sequence similarity between different types of collagens, it is necessary to choose collagen standards with native 3D structure instead of denatured structure. Under physiological conditions, only the native collagen networks can self-assemble in a highly type-specific, three-dimensional architecture to entrap other ingredients, and are expected to function as cellular scaffolds for tissue engineering. The use of properly folded type-specific collagens, exhibiting native tertiary structure is recommended as a control for functional assays and testing specificity of anti-collagen antibodies.

• Collagen Extraction

  Collagen is insoluble in organic solvents. Water-soluble collagen represents only a small fraction of total collagen and it depends on the age and type of tissue extracted. Limited digestion using proteolytic enzymes such as pepsin is a convenient method to extract collagen, although the resulting collagen could be partially digested. Continuous refrigeration throughout collagen extraction is paramount to avoid degradation and denaturation.

• Collagen-Based Assays

  In general collagen-based assays require special attention to buffer pH, temperature, and concentration. Acidic pH is critical to maintaining collagens' stability and solubility. Since collagens are usually more unstable than other proteins, assays should be designed to perform at temperatures <10°C. Optimal concentrations of collagen are needed to obtain reliable measurements but if too high then it can result in spontaneous polymerization (gelatin formation) commonly occurring with collagens. An example of an ELISA assay for collagen can be found here.

• Testing Collagens by SDS-PAGE and Western Blot

  The best separation of collagens by electrophoresis requires the addition of detergents as well as reducing and chaotropic agents at optimal concentration. This ensures maximal resolution of the characteristic chains allowing for unambiguous identification of specific collagen types. Due to the variable size of different collagens, 6% acrylamide gel can be used for collagens I, II, and III, while 10% acrylamide gel is more suitable for collagens IV, V, and VI.

  When performing western blot for collagens it is highly recommended to use standards as positive and negative controls for the collagen type to be measured along with an antibody validated for this application. The quality of the antigen is important for good results and therefore attention to collagen extraction, sample preparation, and denaturing SDS-PAGE as described above is critical.

• Collagen Synthesis and Dynamics

  Collagen is first synthesized as a larger precursor molecule called procollagen which contains additional peptides at both ends. An important, early post-translational modification of collagens is the hydroxylation of selected proline and lysine amino acids in the procollagen protein. Other modifications include glycosylation by the addition of galactose and glucose. As the procollagen is translocated to the cell surface, specific enzymes called procollagen proteinases remove both of the extension peptides from each end of the molecule.

• Collagen Crosslinking

  In the extracellular spaces, crosslinking in collagens take place as the triple-helical collagen molecules line up and begin to form fibrils and then fibers, which is thought to affect the mechanical properties of collagen fibers-composed tissue such as muscle or bone. The degree of cross-linking can affect the yield of collagen during extraction and also affect the exposure of some epitopes thus requiring further optimization of antigen retrieval methods when performing immunohistochemistry. A useful method to determine the extent of collagen cross-linking in tissue is using specific dyes like picrosirius red.