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Understanding Chondrex Inc.'s "Grades" of Purified Collagen

The 28 known types of collagen comprise around 30% of total body protein content in animals, making it the most abundant protein in the Animalia kingdom. Collagen is a major structural protein that can be found primarily in the extracellular matrix (ECM) of connective tissues (tendons, bones, ligaments) as well as the basement layers of skin and internal organs. The different types of collagen can be categorized into five groups based on their quaternary protein structures:

  1. Fibril-forming collagen - Types I, II, III, V, VII XI, XXIV, XXVII; tropocollagen molecules self-assemble into fiber bundles. Most abundant subclass of collagen.
  2. Network-forming collagens – Types IV, VI, VIII, X; tropocollagen molecules self-assemble into mesh like sheets.
  3. FACITs – Types IX, XII, XIV, XVI, XIX-XXII, XXVI; fibril-associated collagens with interrupted triple helices
  4. MACITs – Types XIII, XVII, XXIII, XXV; membrane-associated collagens with interrupted triple helices
  5. MULTIPLEXINs – Types XV, XVIII; multiple triple-helix domains and interruptions

Despite the differences in their molecular assemblies, each type shares a characteristic protein motif: the collagen triple helix (Fig. 1). A single collagen triple helix molecule is termed tropocollagen. These tropocollagen molecules can self-assemble to create the larger protein structures described above.

Tropocollagen Molecule

Figure 1: The collagen triple helix motif. Source: Protein Data Bank.

Tropocollagen is composed of three collagen alpha chains that contain a repetitive amino acid sequence characteristic of collagen proteins: Gly-X-Y. The “X” and “Y” residues can be any amino acid, but they are most commonly proline or hydroxyproline*. When the amino acid sequence does not follow this repeated pattern, the 3D shape of the protein changes from the triple helix conformation. These sections are called Non-Collagenous (NC) Domains as they have conformations not classically associated with collagen proteins.
*Hydroxyproline is mainly found in collagen, therefore our Hydroxyproline Assay Kit can be used to quantify total collagen levels in samples. A full list of our Collagen Detection Kits can be found here.

The amino acid sequence of each specific alpha chain composing a triple helix varies between the collagen stypes. For instance, type II tropocollagen consists of three alpha 1 chains of type II collagen (denoted as ?1[II]3), whereas type V tropocollagen can be composed of three alpha 1 chains (?1[V]3), two alpha 1 chains and one alpha 2 chain (?1[V]2?2[V]), or one alpha 1, one alpha 2, and one alpha 3 chain (?1[V]?2[V]?3[V]). A different gene encodes each unique alpha chain (i.e. gene COL2A1 encodes the alpha 1 chain of type II collagen).

Chondrex, Inc.’s collagen proteins are isolated from tissues using pepsin-digestion. Pepsin digestion solubilizes collagen in the native/undenatured form, but it does cleave the NC domains of collagen. Collagen that has had the NC domain(s) removed this way are termed atelocollagen. The NC domains are more antigenic than the triple helix domains, therefore atelocollagen is a widely used biomaterial for implantable medical devices. 

For over 20 years, Chondrex, Inc. has been providing type II collagen for developing Collagen-Induced Arthritis (CIA) in animals as a model of rheumatoid arthritis. During that time, we have also become familiar with many other types of collagen associated with autoimmune diseases. Figure 2 below provides some key background information about the different types of purified collagen that Chondrex, Inc. offers. 

Cell Culture Grade Collagen

Native form type I collagen derived from skin tissue. To better mimic the natural skin ECM and promote cell adhesion and growth, the Cell Culture Grade collagen also contains minor skim ECM components. Therefore, this grade of collagen is slightly less pure than our other grades, but it is ideal as a scaffold for 2D and 2D cell cultures. Contains both monomeric (tropocollagen) and polymeric (fibrillar) collagen.
Purity: >95% by SDS-PAGE
Application: 2D or 3D cell culture scaffold
Types Offered: I
Species Offered: Bovine, Porcine

Immunization Grade Collagen

Highly purified, pepsin-solubilized, native form collagen purified by salt precipitation and DEAE-cellulose chromatography. Mostly monomeric collagen but may contain polymeric collagen. Endotoxin level ?1 EU/mL. Please contact us for specific lot information.
Purity: >90-99% by SDS-PAGE
Application: In vivo (arthritis induction, antibody production), Tissue engineering, Cell Culture
Types Offered: I, II, III, IV, V, IX, XI
Species Offered: Bovine, Canine, Chicken, Goat, Human, Mouse, Porcine, Rabbit, Rat, Sheep

ELISA Grade Collagen

Native form, pepsin-solubilized collagen derived from various tissues. This grade of collagen has been filtered to only include monomeric collagen since polymeric collagen could provide multiple binding sites for antibodies recognizing the same epitope, leading to an overestimation of results. 
Purity: >99% by SDS-PAGE
Application: ELISA antigen
Types Offered: I, II, III
Species Offered: Bovine, Chicken, Human, Monkey, Mouse, Porcine, Rat

T-cell Grade Collagen

Heat-denatured collagen from various tissues. Monomeric collagen only. T-cells cannot recognize native forms of proteins, therefore this grade of collagen has been heat denatured. Pepsin, commonly used in collagen solubilization, is highly antigenic and contamination of collagen with pepsin may result in an overestimate of T-cell responses to collagen. Therefore, this grade of collagen has received ion-exchange chromatography to remove pepsin completely. 
Purity: >90-99% by SDS-PAGE
Application: T-cell assays, in vivo antigen
Types Offered: I, II
Species Offered: Bovine, Chicken, Human, Monkey, Mouse, Porcine, Rat

Cyanogen Bromide (CB) Collagen Peptide Fragments

Peptide fragments from type II collagen that has been cleaved by CB. Monomeric collagen only. The fragments have been re-natured into the triple-helical conformation via step-wise cooling and are ready for use an antigen in antibody assays. If using for T-cell assay, collagens should be heat-denatured since T-cells only recognize denatured peptides. 
Purity: >95% pure by SDS-PAGE
Application: T-cell assays, ELISA antigen
Types Offered: II
Species Offered: Bovine, Chicken, Mouse

If you have any questions remaining about the different types or grades of collagen that Chondrex, Inc. offers, please contact us.

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