Quick Menu

Collagen Detection

The extracellular matrix (ECM) is a highly dynamic and complex three-dimensional structure composed of a macromolecule network including collagen, glycosaminoglycans (GAGs), elastin, fibronectin, and several other non-cellular structures. The purpose of the ECM is to provide a homeostatic environment for cells to reside in, and although the demands for cell homeostasis differ between tissues and organs, the major constituent of ECMs is collagen (1). The collagen family of proteins is the most abundant protein family in mammals, comprising approximately 30% of total protein mass (2,3). It is the main component of connective tissues and plays a critical role in the inflammatory process of rheumatic diseases (3-5). Factors that may modulate collagen production include cancer, hypoxia, genetic disorders, inflammation, and aging (6-12). If you would like to learn more about the characteristics of collagen proteins, as well as the different “grades” of purified collagen Chondrex Inc. offers, please see our Understanding Chondrex, Inc.'s "Grades" of Purified Collagen blog.

In order to study the physiological and pathological turnover of connective tissues and its components, Chondrex Inc. provides Collagen Detection ELISA Kits for quantifying various species of type I and type II collagen (below), as well as total collagen detection (below), glycosaminoglycans (GAGs) detection, DNA Assay Kit, and metabolic quantification assays for tissue and cell culture samples. 

To learn more about our type I collagen, type II collagen, and total collagen detection kits, please continue reading below. We provide information about the specificity of our collagen detection kits, as well as notes about preparing samples for use in our various collagen detection assays.

If you have any other questions about which collagen detection kit is right for you, please contact us.

Collagen Type I Detection Kit

Product Catalog # Price (USD)
Bovine Type I Collagen Detection Kit 6014 450.00
Canine Type I Collagen Detection Kit 6019 450.00
Human Type I Collagen Detection Kit 6021 485.00
Mouse Type I Collagen Detection Kit 6012 450.00
Porcine Type I Collagen Detection Kit 6015 450.00
Rabbit Type I Collagen Detection Kit 6016 450.00
Rat Type I Collagen Detection Kit 6013 450.00
Type I Collagen C-Telopeptide (CTX-I) Detection Kit 6033 365.00
Type I Collagen N-Telopeptide (NTX-I) Detection Kit 6040 365.00

Collagen Type II Detection Kit

Product Catalog # Price (USD)
Type II Collagen Detection Kit, Multi-Species 6018 450.00

Hydroxyproline Assay Kit

Product Catalog # Price (USD)
Hydroxyproline Assay Kit 6017 399.00

Reagents For Collagen Solubilization

Product Catalog # Price (USD)
Buffered Normal Goat Serum 9066 85.00
Elastase from Porcine Pancreas 30047 36.00
ELISA Blocking Agent 9073 35.00
ELISA Buffered Blocking Agent 9072 79.00
Normal Goat Serum 9067 38.00
Pepsin 6011 10.00

Semi-Quantitative Tissue Total Collagen Detection Kit

Product Catalog # Price (USD)
Sirius Red/Fast Green Collagen Staining Kit 9046 65.00

Sirius Red Total Collagen Detection Kit

Product Catalog # Price (USD)
Sirius Red Total Collagen Detection Assay PLATE Kit 9062P 163.00
Sirius Red Total Collagen Detection Kit 9062 120.00

Type I Collagen Detection ELISA Kits
Type l collagen is the most abundant collagen type and is found in most connective tissues such as skin, bone, tendon, ligament, heart, and lungs (2). A well-studied type I collagen disorder is fibrosis and the marker of lung fibrosis is an increase in collagen deposition (12). Chondrex, Inc. offers Type I Collagen Detection ELISA Kits for nine different species, including kits for C- and N-Telopeptides of type I collagen. The total assay working time is less than six hours and 40 samples can be measured in duplicate.

Species Specific Type I Collagen Detection ELISA Kits: Designed to quantify the amount of type I collagen from cell culture, tissue culture, or tissue specimens. These kits are highly specific for native form (undenatured) type I collagen, while having low reactivity to denatured collagen. Therefore, special collagen solubilization protocols must be used to prevent denaturing collagen proteins in samples. Chondrex, Inc. provides Tips for Collagen Solubilization for customers who have purchased our Type I Collagen Detection ELISA Kits. Please contact us to receive the sample preparation protocol.

Mouse and Human C-Telopeptide (CTX-I) and N-Telopeptide (NTX-I) Detection ELISA Kits: The CTX-I (competitive ELISA) and NTX-I (sandwich ELISA) Detection ELISA Kits are designed to quantify degraded type l collagen peptide fragments. Proteinases mediate resorption of type I collagen from bone and generate specific peptide fragments of degraded collagen. For example, matrix metalloproteinases (MMPs) exclusively produce C-terminal degraded fragments (ICTP) from type I collagen, while cathepsin K produces CTX-I fragments from the C-terminus and NTX-I fragments from the N-terminus of type I collagen (13). These degraded collagen fragments reflect the metabolism of type I collagen and are indicative of proteinase activities in various disease states. Therefore, immunoassays have been developed to monitor the levels of these degraded fragments in biological fluids. Chondrex, Inc. has developed CTX-I and NTX-l Detection ELISA kits for mouse and human samples using monoclonal antibodies which recognize conserved peptide sequences between the species. 

Select Citations for Type I Collagen Detection Kits

Gay MH et al., Nose to back: compatibility of nasal chondrocytes with environmental conditions mimicking a degenerated intervertebral disc. Eur Cell Mater 37:214-232, (2019). PMID: 30900738

Goel PN et al., Notch signaling inhibition protects against LPS mediated osteolysis. Biochem Biophys Res Commun 515(4):538-543, (2019). PMID: 31176486

Kumar D et al., Peptide hydrogels—a tissue engineering strategy for the prevention of oesophageal strictures. Advanced Functional Materials 27:1702424, (2017).

Leipner C et al., Imatinib mesylate attenuates fibrosis in coxsackievirus b3-induced chronic myocarditis. Cardiovasc Res 79:118-126 (2008). PMID: 18326555

Lorenz J et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses. Osteoarthritis and Cartilage 24(2):325-34, (2016). PMID: 26327449

Shimizu Y et al., Angiotensin II subtype 1a receptor signaling in resident hepatic macrophages induces liver metastasis formation. Cancer Science 108(9):1757-1768, (2017). PMID: 28660748

Type II Collagen Detection ELISA Kit
Type II collagen is unique among the collagen family, and its tissue distribution is limited to avascular tissues such as cartilage and the vitreous body of the eyes (12,13). Because type II collagen can induce arthritis in experimental animals, autoimmunity to type II collagen is suspected in the pathogenesis of certain autoimmune diseases in humans such as rheumatoid arthritis (RA), eye diseases associated with RA, and relapsing polychondritis, which affects specific tissues containing type II collagen (13). The Type II Collagen Detection ELISA Kit is designed to quantify the amount of native type II collagen in cell/tissue cultures and tissue samples from multiple species (human, monkey, porcine, bovine, rat, mouse, rabbit, equine, and chick). The total assay working time is less than four hours and 40 samples can be measured in duplicate.

The kit is highly specific for native form (undenatured) collagen detection while having low reactivity to denatured collagen. Therefore, special collagen solubilization protocols must be used to prevent denaturing collagen proteins when preparing tissue samples for use with the assay. Chondrex, Inc. provides Tips for Collagen Solubilization for customers who have purchased our Type II Collagen Detection ELISA Kit. Please contact us to receive the sample preparation protocol.

Selected Citations for Type II Collagen Detection ELISA Kit

Piltti J, Fernandez-Echevarría C, Marcellino D, Lammi MJ, Rho-kinase inhibitor Y-27632 and hypoxia synergistically enhance chondrocytic phenotype and modify S100 protein profiles in human chondrosarcoma cells. Scientific Reports 7(1):3708, (2017). PMID: 28623370

Khader A, Arinzeh TL, Biodegradable zinc oxide composite scaffolds promote osteochondral differentiation of mesenchymal stem cells. Biotechnology and Bioengineering 117(1):194-209, (2020). PMID: 31544962

de Paz-Lugo P, Lupiáñez JA, Meléndez-Hevia E, High glycine concentration increases collagen synthesis by articular chondrocytes in vitro: acute glycine deficiency could be an important cause of osteoarthritis. Amino Acids 50(10):1357-1365, (2018). PMID: 30006659

Hydroxyproline Assay Kit
The Hydroxyproline Assay Kit quantifies the total collagen content (any type and species) from tissue homogenates, cell cultures, and tissue culture. Hydroxyproline, a major component of collagen, makes up about 13.5% of its amino acid composition. Due to the highly restricted distribution of hydroxyproline in collagen and elastin, the hydroxyproline content generally reflects the amount of collagen in samples. Therefore, quantitating hydroxyproline has been utilized for evaluating tissue fibrosis or collagen deposition (14,15). 

Conventional hydroxyproline assays require cumbersome procedures and special tools. In contrast, Chondrex, Inc.’s Hydroxyproline Assay Kit employs an improved assay system that can be operated with ease and precision using 96-well plates. The hydroxyproline assay works for determining total collagen content for both native and denatured collagen. Collagen solubilization of samples is not required, but the samples must be hydrolyzed before the assay is performed. By hydrolyzing the sample, a single hydroxyproline is formed from the total collagen sample. Please see our kit protocol to learn more about the hydrolyzation process and special considerations that need to be made. The total working time for the Hydroxyproline Assay Kit is less than one hour and 40 samples can be measured in duplicate.

Selected Citations for Hydroxyproline Assay Kit

Park JS et al., Targeting of dermal myofibroblasts through death receptor 5 arrests fibrosis in mouse models of scleroderma. Nat Commun 10(1):1128, (2019). PMID: 30850660

Pattappa G et al., Physioxia has a beneficial effect on cartilage matrix production in interleukin-1 beta-inhibited mesenchymal stem cell chondrogenesis. Cells 8:936,(2019). PMID: 31434236

Ramani K et al., IL-17 receptor signaling negatively regulates the development of tubulointerstitial fibrosis in the kidney. Mediators of Inflammation 2018:5103672, (2018). PMID: 30405320

Sirius Red Total Collagen Detection Kit (Quantitative Assay)
Sirius Red Total Collagen Detection Kit utilizes Sirius red dye as a detection reagent to determine solubilized collagen concentration in samples. Sirius red is a unique dye which specifically binds to the [Gly-X-Y]n helical structure on fibrillar collagen (type I - V) (16,17). This kit does not discriminate between collagen species and types and is therefore suitable for detecting total collagen content in samples. Due to the low level of collagen in cell culture media, additional concentration steps may be necessary if assaying cell culture media samples.
The Sirius Red Total Collagen Detection Kit will only detect solubilized native (undenatured) collagen from samples. If your study requires distinguishing between native and denatured collagen, the hydroxyproline assay can be used to detect total collagen. The difference between native (Sirius red assay) and total collagen (hydroxyproline assay) will equal denatured collagen levels. While the Sirius Red Total Collagen Detection Kit is designed to be used with a spectrophotometer, our Sirius Red Collagen Detection Plate Kit is designed for use with a 96-well ELISA plate. The plate kit is convenient for assaying various collagen-containing samples (tissue specimens, cell culture media, cultured cells) at one time. The total assay working time ranges from 30-60 minutes depending on the quantity of samples and is measured at an absorption of 540nm. 

Selected Citation for Sirius Red Total Collagen Detection Kit

Schafer S et al., IL-11 is a crucial determinant of cardiovascular fibrosis. Nature 552:110-115, (2017). PMID: 29160304

Taokaew S, Alghunaia A, Newby BZ, Zosteric acid, a bioactive component in eelgrass Zostera marina, reduced collagen I expression in a repaired mouse fibroblast scratch. Natural Product Communications 2019:1–6, (2019).

Zhao L et al., Ocular surface repair using decellularized porcine conjunctiva. Acta Biomaterialia 101:344-356, (2020). PMID: 31706041

Sirius Red/Fast Green Collagen Staining Kit- (Semi-Quantitative Assay)
Sirius Red and Fast Green is a dye combination used to distinguish collagen from surrounding materials. Sirius Red specifically binds the [Gly-X-Y]n helical structure of fibrillar collagens, whereas Fast Green binds to non-collagenous proteins (16,17). As Sirius Red and Fast Green have absorptions at 540 nm and 605 nm respectively, the OD values of the extracted dyes can be used to calculate the collagen and non-collagenous protein content of samples. By exploiting the unique features of these two dyes, Chondrex, Inc. provides a simple semi-quantitative assay kit to determine the amounts of collagen and non-collagenous proteins in cultured cell layers and tissue sections. 

Because this assay does not require collagen solubilization, it can be used to measure total collagen content in various tissues. The assay results can be normalized by results of non-collagenous proteins with Fast Green staining OD values. For general histological studies in which tissue sections are 10-20 μm thick, the assay sensitivity for collagen and non-collagenous proteins is greater than 3 μg/section and 50 μg/section, respectively. This kit contains enough reagents to stain 30-50 samples.

Selected Citations for Sirius Red/Fast Green Collagen Staining Kit

Andricovich J et al., Loss of KDM6A activates super-enhancers to induce gender-specific squamous-like pancreatic cancer and confers sensitivity to BET inhibitor. Cancer Cell 33(3):512-526.e8, (2018). PMID: 29533787

Chen H et al., WWP2 regulates pathological cardiac fibrosis by modulating SMAD2 signaling. Nature Communications 10(1):3616, (2019). PMID: 31399586

Won JE et al., Hierarchical microchanneled scaffolds modulate multiple tissue-regenerative processes of immune-responses, angiogenesis, and stem cell homing. Biomaterials 227:119548, (2020). PMID: 31670033

Extracellular matrix (ECM) assay for Glycosaminoglycans (GAGs), DNA, and MTT Assays
Besides collagen detection assays, glycosaminoglycans (GAGs) and cell proliferation assays can be used to quantify connective tissue components.   

Glycosaminoglycans (GAGs) are negatively charged polysaccharides located in most connective tissues and ECM, as well as on the surfaces of many types of cell. GAGs have a widespread function and are known to play critical roles in health, hydration, and regeneration (18). Recent findings in idiopathic pulmonary fibrosis patients show an upregulation of GAGs, which possibly leads to deteriorating pro-fibrotic environment (19). In addition to type II collagen, GAGs are implicated as an autoantigen of rheumatoid arthritis (RA), as anti-GAGs antibodies associated with ECM degradation exist in serum from RA patients. In fact, immunization with GAGs can induce arthritis in mice; however, the implications of GAGs in RA pathogenesis are still under research (20,21). 

Chondrex, Inc. provides a sulfated GAGs Assay Kit (cat# 6022) using the cationic dye, 1,9 dimethylmethylene blue (DMB) which binds to highly charged sulfated GAGs, except hyaluronan (21). This kit utilizes an improved DMB solution tjhat minimizes interference with negatively charged contaminants, such as DNA and RNA. The kit also employs chondroitin sulfate as an appropriate standard for the analysis of ECM in cartilage. The total assay working time less than one hour and 40 samples can be measured in duplicate.

Deoxyribonucleic acid (DNA) and MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) assays are routinely used to assess cell proliferation and viability. These assays can be used to correlate cell health and environmental factors in culture, such as drug treatments and tissue engineering (scaffolds) (22).  For example, in cartilage tissue engineering, artificial cartilage quality is evaluated by DNA amounts translated as chondrocyte numbers, as well as amounts of collagen and GAGs in the ECM (23, 24). 

Chondrex Inc.  provides a DNA Assay Kit (cat# 6023) employing the Hoechst 33258 fluorescent dye which specifically binds to Adenine-Thymine base pairs, resulting in fluorescence at excitation 360 nm/emission 460 nm. As the dye-DNA binding and the fluorescence intensity are unaffected by contaminating proteins and other substances in an optimized assay condition, this DNA assay kit works accurately with samples containing other analytes (tissue samples and cell culture samples). The total assay working time is less than one hour and 40 samples can be measured in duplicate.

MTT is a positively charged compound that can enter viable eukaryotic cells and reflect cytosolic metabolic behavior under different cell culture conditions. MTT assays are routinely carried out to deduce toxicity of developed scaffolds and determine biocompatibility between collagen producing cells and scaffold materials (25). Soluble MTT is taken into cells where it is reduced, and ultimately insoluble formazan is produced and accumulates both inside and on the surface of metabolically active cells (26). The resulting formazan can be solubilized and quantified to reflect cell proliferation and viability.

Chondrex, Inc. provides an MTT Cell Proliferation Assay Kit which employs a simple method to assay cell viability and cell density using an absorbance microplate reader at 570nm. This kit can be used for up to 250 samples in duplicate. 

or more details download our ECM Analysis Flow Chart Flyer and read our MTT Blog.

Selected Citations for EMC Detection Kits

Maier F, Lewis CG, Pierce DM, The evolving large-strain shear responses of progressively osteoarthritic human cartilage. Osteoarthritis Cartilage 27(5):810-822, (2019). PMID: 30660720

Sears V, Gosh G, Harnessing mesenchymal stem cell secretome: Effect of extracellular matrices on proangiogenic signaling. Biotechnology and Bioengineering 117(4):1159-1171, (2020). PMID: 31956977
 

Reagents for Collagen Solubilization
Both the Type I Collagen Detection Kits (species specific) and the Type II Collagen Detection Kit (multi-species) are specific for native collagen and have low reactivity with denatured collagen. Therefore, sspecial collagen solubilization protocols must be used to prevent denaturing collagen proteins in samples. Additional supplies, such as guanidine hydrochloride, pepsin (catalog # 6011, 6020), and elastase (catalog # 30047), may be necessary to process samples for use with these kits. Please contact Chondrex, Inc. to receive our Tips for Collagen Solubilization.

Buffered Normal Goat Serum (cat# 9066): Chondrex, Inc recommends pre-treatment of plastic surfaces with buffered normal goat serum to minimize non-specific binding of collagen that can lead to underestimated results. In addition, the use of blocking agent in the buffer can interfere with the results for some assays. Pre-treatment with buffered normal goat serum can minimize this risk. 

Normal Goat Serum (cat# 9067): Goat serum from healthy donors used for buffer preparation. 

Elastase from Porcine Pancreas (cat# 30047): Pancreatic elastase is a serine protease which will hydrolyze native elastin abundant in connective tissue. Elastase is a dissociating enzyme used for collagen solubilization from tissue samples at neutral pH. Elastase will digest cross-linkages of insoluble polymeric collagen in neutral pH tissue samples leaving a solubilized monomeric collagen as the byproduct of the reaction. Because of its effective enzymatic properties, elastase is the enzyme of choice for the isolation of Type II cells from lung tissue (27,28).

Pepsin from Porcine Gastric Mucosa (cat# 6011, 6020):  The enzymatic characteristics of pepsin have been studied since 1820 and the enzyme has become widely used for countless solubilization protocols (29). Pepsin is an endopeptidase produced in the digestive system and exhibits maximum activity at pH 2.0 and is inactive at pH 6.5 and above. It can be as a dissociating enzyme for solubilizing collagen from tissue samples at an acidic pH. Pepsin cannot digest native collagen but cleaves collagen into telopeptides and atelocollagen which forms soluble collagen. However, prolonged pepsin digestion can denature collagen. Similarly, heating collagen to 40⁰C or above can also denature collagen. Therefore, sample preparation for collagen analysis should be kept at 4⁰C to avoid denaturation (30-32).

Selected Citation for Collagen Solubilization Reagents

Seidl CI et al., CRISPR-Cas9 targeting of MMP13 in human chondrocytes leads to significantly reduced levels of the metalloproteinase and enhanced type II collagen accumulation. Osteoarthritis Cartilage 27(1):140-147, (2019). PMID: 30223022

 

References

  1. Theocharis AD, Skandalis SS, Gialeli C, Karamanos NK, Extracellular matrix structure. Adv Drug Deliv Rev 97:4‐27, (2015). PubMed PMID: 26562801
  2. Ricard-Blum S, The collagen family. Cold Spring Harb Perspect Biol 3(1):a004978, (2011). PubMed PMID: 21421911
  3. Deshmukh SN, Dive AM, Moharil R, Munde P, Enigmatic insight into collagen. J Oral Maxillofac Pathol. 20(2):276-83, (2016). PubMed PMID: 27601823
  4. Rheumatoid arthritis. Nat Rev Dis Primers 4:18002, (2008). PubMed PMID: 29417950
  5. Fang Q, Zhou C, Nandakumar KS, Molecular and cellular pathways contributing to joint damage in rheumatoid arthritis. Mediators Inflamm 2020:3830212, (2020). PubMed PMID: 32256192
  6. Xu S et al., The role of collagen in cancer: from bench to bedside. J Transl Med 17(1):309, (2019). PubMed PMID: 31521169
  7. Ohlund D et al., Type IV collagen is a tumour stroma-derived biomarker for pancreas cancer. Br J Cancer 101(1):91-7, (2019). PubMed PMID: 19491897
  8. McKay TB, Hjortdal J, Priyadarsini S, Karamichos D, Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro. PLoS One 12(4):e0176017, (2017). PubMed PMID: 28426715
  9. Petrova V et al., The hypoxic tumor microenvironment. Oncogenesis 7:10, (2018).
  10. Kehlet S et al., Excessive collagen turnover products are released during colorectal cancer progression and elevated in serum from metastatic colorectal cancer patients. Sci Rep 6:30599, (2016). PubMed PMID: 27465284
  11. Birch HL, Extracellular matrix and ageing. Subcell Biochem 90:169-190, (2019). PubMed PMID: 30779010
  12. Goldstein RH, Control of type I collagen formation in the lung. Am J Physiol 261(2 Pt 1):L29-40, (1991). PubMed PMID: 1872414
  13. Marshall GE, Konstas AG, Lee WR, Collagens in ocular tissues. Br J Ophthalmol 77(8):515–524, (1993). PubMed PMID: 8025051
  14. Cremer MA, Pitcock JA, Stuart JM, Kang AH, Townes AS, Auricular chondritis in rats. An experimental model of relapsing polychondritis induced with type II collagen. J Exp Med 154(2):535-40, (1981). PubMed PMID: 7021752
  15. Wheater G et al., The clinical utility of bone marker measurements in osteoporosis. J Transl Med 11:201, (2013). PubMed PMID: 23984630
  16. Cundy T, Reid IR, Grey A, CHAPTER 31 - Metabolic bone disease. Clinical Biochemistry: Metabolic and Clinical Aspects (Churchill Livingstone, Third Edition, 2014) Pages 604-635, ISBN 9780702051401.
  17. Segnani C et al., Histochemical detection of collagen fibers by sirius red/fast green is more sensitive than van Gieson or sirius red alone in normal and inflamed rat colon. PLoS One 10(12):e0144630, (2015). PubMed PMID: 26673752
  18. Casale J, Crane JS, Biochemistry, glycosaminoglycans. Treasure Island (FL): StatPearls Publishing. (2020).
  19. Westergren-Thorsson G et al., Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis. The international journal of biochemistry & cell biology 83:27-38, (2017). PubMed PMID: 27974233
  20. Wang JY, Roehrl MH, Glycosaminoglycans are a potential cause of rheumatoid arthritis. Proc Natl Acad Sci USA. 99(22):14362-7, (2002). PubMed PMID: 12391302
  21. Barbosa I et al., Improved and simple micro assay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies. Glycobiology 13:647-653, (2003). PubMed PMID: 12773478
  22. Pabbruwe MB et al., A comparison of colorimetric and DNA quantification assays for the assessment of meniscal fibrochondrocyte proliferation in microcarrier culture. Biotechnology letters 27(19):1451-5, (2005).  PubMed PMID: 16231215
  23. Yoon JH, Halper J, Tendon proteoglycans: biochemistry and function. J Musculoskelet Neuronal Interact 5:22-34 (2005). PubMed PMID: 15788868
  24. Buckwalter JA, Mankin HJ, Articular cartilage: tissue design and chondrocyte-matrix interactions. Instr Course Lect 47:477-486, (1998). PubMed PMID: 9571449
  25. Varkey A et al., Impact of silk fibroin-based scaffold structures on human osteoblast MG63 cell attachment and proliferation. Int J Nanomedicine 10 Suppl 1(Suppl 1):43‐51, (2015). PubMed PMID: 26491306
  26. Twentyman PR, Luscombe M, A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br J Cancer 56:279–285 (1987). PubMed PMID: 3663476
  27. Worthington Tissue Dissociation Guide by Worthington Biochemical Corporation http://www.worthington-biochem.com/tissuedissociation/elastase.html
  28. Fields GB, Interstitial collagen catabolism. J Biol Chem 288(13):8785-93, (2013). PubMed PMID: 23430258
  29. Last JA, Reiser KM, Collagen biosynthesis. Environ Health Perspect 55:169-77, (1984). Review. PubMed PMID: 6428877
  30. Fruton JS, A history of pepsin and related enzymes. Q Rev Biol 77(2):127-47, (2002). Review. PubMed PMID: 12089768
  31. Johnston N, Dettmar PW, Bishwokarma B, Lively MO, Koufman JA, Activity/stability of human pepsin: implications for reflux attributed laryngeal disease. Laryngoscope 117(6):1036–1039, (2007). PubMed PMID: 17417109
  32. Dunn BM, Overview of pepsin-like aspartic peptidases. Current Protocols in Protein Science Chapter 21 (1): 21.3.1–21.3.6, (2001)  PubMed PMID: 18429164