Collagen can be difficult to purify and analyze, even though collagens are abundant and constitute about 30% of the body’s total protein. This is due to the extensive network that is formed by collagen molecules via different types of crosslinking which makes the collagen molecules insoluble and challenging to extract.
Currently, various types of assays exist to analyze collagen:
– ELISA for specific pro-domains of collagen
– ELISA for specific types of collagen
– Western blotting using specific collagen antibodies
– Sirius Red based staining of tissue sections
– Sirius Red based assays for soluble collagen
– Determination of Hydroxyproline residues (either by a colorimetric kit or by HPLC) after hydrolysis of the sample.
QuickZyme Biosciences has developed a set of assays for the rapid and easy analysis of soluble or insoluble collagens from any tissue and any species.
Collagen assays available from QuickZyme Biosciences:
- QuickZyme Soluble Collagen assay
- QuickZyme Total Collagen assay
- QuickZyme Sensitive Tissue Collagen Assay
- QuickZyme Hydroxyproline assay
- QuickZyme Sensitive Tissue Hydroxyproline Assay
QuickZyme Soluble Collagen assay
This assay recognizes soluble or (acid/pepsin) solubilized fibrillar collagen. The assay is colorimetric, has a 96-well plate format, and is based on precipitation of collagen with Sirius-Red, an anionic dye with sulphonic acid groups. The dye is released from the precipitated complex at high pH, followed by colorimetric detection. The assay is optimized such that other proteins (such as albumin) do not interfere. Gelatin (denatured collagen) is not recognized by this assay. The assay requires a soluble or solubilized form of fibrillar collagen.
Application: The assay is used for the measurement of soluble collagen in e.g. cell culture media, and (acid or acid/pepsin) solubilized collagens e.g. from cellular extracts. The assay is not recommended for tissues since most of the tissue collagen is crosslinked and insoluble.
QuickZyme Total Collagen assay
This assay recognizes all types of collagen (mature, immature, procollagen, degraded collagen, crosslinked collagen, collagen from various sources and species). The assay is colorimetric, has a 96-well plate format, and is based on the quantification of hydroxyproline, a modified version of the amino acid proline that occurs almost exclusively in collagen. Hydroxyproline is released from collagen upon acid hydrolysis of the collagen containing sample. Hydrolysis is carried out at 950 C and the hydrolysate can directly be used for hydroxyproline analysis without washing or drying steps.
Application: The assay is used for the quantification of total collagen. This includes all procollagen, unfolded collagen, mature collagen as well as collagen degradation products and gelatin of all collagen types present in the sample. Since the first step is complete hydrolysis of the sample, there are no difficult collagen extraction steps. The assay is applicable for all types of samples including tissue from any species. This assay is influenced by matrix effects due to unidentified substances in tissue samples. Matrix effects can be prevented in many samples through dilution of the hydrolysate. If sufficient dilution of the hydrolysate is not desirable due to low collagen concentration, such as in liver tissue, we recommend the QuickZyme Sensitive Tissue Collagen assay.
QuickZyme Hydroxyproline assay
This assay is similar to the QuickZyme Total Collagen assay, with the difference that protocols and tubes for collagen hydrolysis are not included.
Application: This assay has the same application area as the Total Collagen assay but is intended for customers who have their own method of hydrolysis, or have a collection of hydrolyzed samples to be analyzed.
QuickZyme Sensitive Tissue Collagen assay
This assay is very similar to the QuickZyme Total Collagen assay, but is not influenced by matrix effects, making it the assay of choice for tissues with low collagen concentrations and substantial matrix effects, such as liver tissue.
Application: The assay is used for the measurement of total collagen in (tissue) samples with low concentration of collagen and matrix effects that would require dilution in the QuickZyme Total Collagen assay.
QuickZyme Sensitive Tissue Hydroxyproline assay
This assay is similar to the QuickZyme Sensitive Tissue Collagen assay, with the difference that protocols and tubes for collagen hydrolysis are not included.
Application: This assay has the same application area as the QuickZyme Sensitive Tissue Collagen assay, but is intended for customers who have their own method of acid hydrolysis, or have a collection of hydrolyzed samples to be analyzed.