Collagens, Collagen Antibodies and the Extracellular Matrix


The extracellular matrix (ECM) often refers to the space between cells. This space is typically populated by various cell types originating from different lineages. Therefore the ECM is a multicellular environment that varies greatly in tissue types and structure but often is charged with the functions of cell adhesion, cellular communication and differentiation. In animals we can think of the ECM as both the interstitial matrix as described above and the basement membrane onto which many epithelial cells are bound.

The ECM is composed of various secreted molecules and proteins. ECM proteins are often fibers or fibrillar proteins such as collagens, elastin, fibronectin and laminins. Collagens are the most abundant proteins in the ECM. In fact collagens are the most abundant proteins in the human body. For instance in the bone matrix collagens account for up to 90% of the protein present. Collagens are secreted by exocytosis in a precursor form as procollagens and are then processed by cleavage by procollagen specific proteases to be assembled into their final form. 



Featured Collagens, Collagen Antibodies and the Extracellular Matrix Products


Immunohistochemistry of Anti-Collagen I Antibody
Immunohistochemistry of Anti-Collagen II Antibody

Anti-Collagen I











Purified Collagen Proteins



Rockland provides highly purified type specific collagen proteins derived from several sources for your research needs. Rockland’s type specific purified collagens are chromatographically and immunologically pure. These proteins are free from other collagens, serum proteins and non-collagen extracellular matrix proteins. Reaction with other type anti-collagen antibodies is minimal (i.e. Collagen Type I react strongly with anti-Collagen Type I antibody but will show minimal reactivity with anti-Collagen Type III antibody by way of example).

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Anti-Collagen Antibodies

At least eleven (11) genetically distinct gene products are collectively referred to as 'collagen types' or other proteins and proteoglycans of the extracellular matrix. In humans, collagens are composed of about twenty (20) unique protein chains that undergo various types of post-translational modifications and are ultimately assembled into a triple helix (see Figure 2 below). This results in great diversity between collagen types. Collagens are highly conserved throughout evolution and are characterized by an uninterrupted "Glycine-X-Y" triplet repeat that is a necessary part of the triple helical structure.

For these reasons, generating type specific antibodies to collagens presents unique challenges. It is an absolute requirement to use non-denatured three-dimensional epitopes to develop these type specific antibodies. Rockland extensively purifies collagens for immunization from human and bovine sources by limited pepsin digestion and selective salt precipitation. This maintains the three-dimensional structure of the collagen.



Figure 2 - The structure of a typical collagen molecule. The top panel represents a single collagen molecule, which always consists of three protein chains. Each chain is an alpha helix, and in type I collagen molecules there are two alpha 1 chains and one alpha 2 chain, which is abbreviated as [α1(I)]2 [α2(I)]. The three protein chains (wound into a triple helix) make up one collagen molecule. The red line shown in one of the strands of the triple helix represents the alpha helical winding of the peptide backbone of that peptide chain. As shown in the bottom panel, each triple-helical collagen molecule is 300nm long and forms a rod. Collagen molecules spontaneously assemble in a head-to-tail alignment with a small gap that separates the 'head' of one molecule with the 'tail' of the next molecule, and in a staggered side-by-side arrangement. Adjacent collagen molecules are displaced about 67nm, or slightly less than one-fourth of the length of a single molecule. The side-by-side interactions are stabilized by covalent bonds (red bars) between the N-terminus of one molecule and the C-terminus of an adjacent molecule. [Adapted from Mathews CK, Van Holde KE, Ahern KG. Biochemistry. San Francisco, CA: Benjamin Cummings, 1999.] 



After isolation from rabbit antiserum, these antibodies are repeatedly and exhaustively cross-adsorbed by immunoaffinity purification to produce 'type' specific antibodies that are well suited to detect extracellular matrix proteins in normal and disease state tissues. For instance, anti-Collagen Type I is first purified against immobilized type I collagen and then is passed over immobilized type II collagen repeatedly until no additional antibody is bound. This process is applied successively for all other collagen types rendering the Collagen Type I antibody specific for collagen I with minimal cross reactivity to all other collagen types.




Extracellular Matrix Antibodies




Many cells bind to components of the extracellular matrix, with cell adhesion occurring in two ways; 1) focal adhesions, connecting the ECM to actin filaments of the cell, and 2) hemidesmosomes, connecting the ECM to intermediate filaments such as ke ratin. This cell-to-ECM adhesion is regulated by specific cell surface cellular adhesion molecules (CAM) known as integrins. Fibronectins bind to ECM macromolecules and facilitate their binding to transmembrane integrins, initiating intracellular signaling pathways as well as association with the cellular cytoskeleton via a set of adaptor molecules.











The Rockland Advantage: Collagen Proteins and Antibodies 


Critical Thinking. Because Rockland collagen antibodies are designed with specificity to recognize only non-denatured three-dimensional epitopes, assays that maintain native collagen structure work best. Rockland produces, tests and validates collagen proteins and collagen antibodies in our own laboratories and is considered a critical resource for highly specific anti-collagen antibodies. Trust Rockland for your critical requirements for collagen proteins and antibodies and other reagents needed for your work to be successful.



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