please provide a standardized protocol for in vitro collagen determination.
thanks in advance.
What is being quantified?
Collagen content in cells?
There are ELISA kits easily available for the particular type of collagen
Hello Navdeep Singh
You may use the Hydroxyproline assay as hydroxyproline content is highly correlated to the collagen content, and this assay has been frequently used to quantify collagen. This method has been cited as the ‘gold standard’ collagen quantification assay. Although this assay is relatively simple and low cost, it does not discriminate between collagen types or other non-collagen molecules that contain hydroxyproline.
For the protocol for Hydroxyproline assay, please refer to the article attached below.
Article A Modified Hydroxyproline Assay Based on Hydrochloric Acid i...
The other methods that are available are:
SDS-PAGE densitometry
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separates molecules by molecular weight and charge. Because the molecular weights of many collagen alpha-chains are documented, they can be identified using a prepared ladder of proteins. Nevertheless, separating the alpha-chains is challenging and requires strong denaturants. Semi-quantification of protein in gel bands from SDS-PAGE is possible via densitometric analysis. Although SDS-PAGE alone does not confirm the identity of proteins, they can be identified through antibody binding in a Western Blot.
HPLC
High-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-FLD) has been used for the identification and quantification of trivalent crosslinks hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP). This method uses reverse-phase chromatography with ion-pairing agents. While HPLC-FLD has been a gold standard assay for collagen crosslink quantifications, its lengthy chromatography and usage of harsh ion-pairing agents are significant disadvantages.
ELISA
ELISA is a plate-based assay to quantify target molecules based on antigen recognition and thus can be used to measure individual collagen subtypes or crosslinks. Commercial ELISA kits exist for several collagen subtypes. ELISA’s drawbacks include its high cost and requirement for separate runs for different molecules (for example, different collagen types, different collagen crosslinks, or collagen from different biological species). Antibodies specific to minor collagen types for many species are not readily available. Identifying or quantifying different collagen types in the same sample is not possible, and because different collagen types have a high degree of sequence homology, careful attention is needed when choosing custom epitopes for collagen subtypes.
If you are interested in some more methods of quantification, you may refer to the article attached below.
Article Collagen: quantification, biomechanics and role of minor sub...
Best.
- Badges
- Science method