Collagen-IV Bovine
Description
Purification and Properties
Collagen-IV Bovine is purified using proprietary chromatographic techniques, yielding high-purity protein (>90%) .
Mechanisms:
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Basement Membrane Assembly: Forms covalently linked networks critical for filtration and barrier functions .
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Regulation of Angiogenesis: NC1 domains suppress tumor growth and vascularization .
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Pathological Implications: Dysregulated hydrolysis by MMP-2/MMP-9 links to retained fetal membranes (RFM) in dairy cows .
Medical and Research Applications
Collagen-IV Bovine is utilized in both clinical and laboratory settings.
Applications:
Notably, bovine collagen’s similarity to human collagen minimizes immune responses, though antibodies to bovine type IV collagen have been observed in some autoimmune conditions .
Research Findings and Mechanisms
Recent studies highlight Collagen-IV Bovine’s role in placental expulsion and disease pathogenesis.
Retained Fetal Membranes (RFM) in Cows:
In vitro studies confirm that FAK signaling modulates COL-IV degradation via Src, MMP-2, and MMP-9 pathways .
Product Specs
Collagen is a fibrous protein that forms the structural basis of various tissues, offering tensile strength and maintaining structural integrity. It represents a major constituent of the extracellular matrix. Collagen and its derivative, gelatin, have found widespread use in medical, pharmaceutical, and consumer products for over a century. These materials, derived from animal sources, are readily available and cost-effective. However, many collagen formulations lack high purity levels and may trigger inflammatory reactions in certain individuals. Moreover, concerns regarding potential contamination of bovine-derived products with prions, such as the agent responsible for mad cow disease and its human counterpart, Creutzfeldt-Jakob disease, have emerged in recent years. Animal-derived collagens undergo extensive post-translational modifications throughout their lifespan in the extracellular matrix. These modifications affect both the extractability of collagen from tissues and their biophysical properties. Consequently, commercially available collagens extracted from animal tissues often exhibit significant batch-to-batch variability. Furthermore, these bulk materials can be challenging to analyze. Products containing animal-derived collagen carry the risk of eliciting adverse inflammatory or immune reactions in humans. There is also a potential risk of contamination with viruses or prions, which can lead to serious health complications. In contrast, recombinant collagens are virtually identical to their native counterparts, effectively minimizing the risks of inflammation, immune reactions, and disease transmission associated with animal-derived collagen.
Bovine Collagen-IV is a naturally occurring protein extracted from bovine placenta. This collagen type is purified using proprietary chromatographic methods.
This product appears as a white lyophilized (freeze-dried) powder that has been filtered.
Collagen-IV undergoes lyophilization without any additional ingredients.
For reconstitution of the lyophilized Collagen-IV, it is advisable to dissolve it in 20 mM acetic acid at a concentration of at least 1 mg/mL. Further dilutions can be made using other aqueous solutions.
Lyophilized Collagen-IV demonstrates stability at room temperature for a period of 3 weeks; however, it is recommended to store it in a dry environment below -18°C. Upon reconstitution, Collagen-IV can be stored at 4°C for 2 to 7 days. For extended storage, it is recommended to store it below -18°C. To ensure optimal long-term storage, the addition of a carrier protein (0.1% HSA or BSA) is advised. It is important to avoid repeated freeze-thaw cycles.
Greater than 90.0% pure.
Bovine placenta.
Q&A
What is the structural organization of bovine Collagen-IV?
Bovine Collagen-IV is organized into three distinct functional domains: the triple-helical collagenous domain, the 7S domain, and the NC1 (non-collagenous 1) domain. The collagenous domain provides the structural framework, while the 7S and NC1 domains are crucial for network formation through intermolecular interactions. The NC1 domain, particularly in bovine placental basement membrane, consists predominantly of crosslinked α1 and α2 NC1 subunits forming dimers, while other tissue sources show different subunit compositions . The protein forms a complex meshwork within basement membranes that offers both flexibility and tensile strength to tissues.
How does bovine Collagen-IV differ from other basement membrane components?
Collagen-IV differs from fibrillar collagens (like types I and III) in its ability to form network-like structures rather than fibrils. In bovine tissues, Collagen-IV is specifically localized to basement membranes underlying all endometrial epithelia and surrounding smooth muscle cells of blood vessels, whereas Collagen I forms a fine meshwork of thin fibers directly below the surface epithelium, and Collagen III forms the bulk of connective tissue fibers arranged in fine aggregates within the superficial endometrial stroma . This distinctive distribution pattern allows Collagen-IV to provide specialized support for epithelial and endothelial structures.
What is the source material typically used for bovine Collagen-IV isolation?
Bovine placenta is the primary source material for Collagen-IV isolation due to its rich basement membrane content. Bovine Collagen-IV is purified from bovine placental basement membrane (bPBM) using proprietary chromatographic techniques following selective proteolytic degradation of the triple-helical part of collagen IV networks by purified bacterial collagenase . Alternative bovine sources include basement membranes from eye lens (bLBM), kidney glomeruli (bGBM), lung alveoli, seminiferous tubules of testis (bSTBM), and aorta, each requiring specific pre-processing steps before collagenase treatment .
What physical characteristics define purified bovine Collagen-IV?
Purified bovine Collagen-IV typically appears as a filtered white lyophilized (freeze-dried) powder. The protein is generally lyophilized without additives to maintain its native structure . Due to extensive intermolecular crosslinking and noncovalent associations with other basement membrane components, Collagen-IV exhibits limited solubility, which presents challenges for isolation and experimental manipulation . The physical characteristics may vary between different tissue sources due to variations in post-translational modifications and molecular associations.
How does the FAK signaling pathway regulate Collagen-IV in bovine tissues?
The Focal Adhesion Kinase (FAK) signaling pathway plays a crucial role in regulating Collagen-IV through modulation of matrix metalloproteinases (MMPs). Research on bovine placental tissues has demonstrated that FAK regulates the hydrolysis of Collagen-IV by affecting the activity of MMP-2 and MMP-9 . In placental tissues from cows with retained fetal membranes (RFM), expression levels of FAK, Src, MMP-2, and MMP-9 were significantly downregulated, while Collagen-IV was upregulated compared to healthy cows . Experimental manipulation of FAK signaling in bovine endometrial epithelial cells has confirmed this relationship:
| Treatment Group | FAK Pathway Components | Collagen-IV Expression |
|---|---|---|
| FAK Inhibitor | Significantly decreased | Significantly increased |
| FAK Activator | Significantly increased | Significantly decreased |
| Control | Baseline levels | Baseline levels |
These findings indicate that FAK in maternal endometrial epithelial cells regulates Collagen-IV degradation, with implications for placental expulsion and potentially other tissue remodeling processes .
What are the composition differences of NC1 hexamers across various bovine basement membranes?
NC1 hexamers from different bovine basement membranes exhibit significant variations in composition and degree of crosslinking, which affects their structural and functional properties:
| Basement Membrane Source | NC1 Hexamer Composition | Crosslinking Characteristics |
|---|---|---|
| Placenta (bPBM) | Predominantly α1 and α2 NC1 subunits | High proportion of crosslinked dimers |
| Eye Lens (bLBM) | Enriched in NC1 monomers | Lower degree of crosslinking |
| Kidney Glomeruli (bGBM) | Enriched in α3, α4, and α5 NC1 subunits | Significant crosslinking |
| Seminiferous Tubules (bSTBM) | Enriched in α3, α4, and α5 NC1 subunits | Significant crosslinking |
These compositional differences influence both the extractability of Collagen-IV from different tissues and the biophysical characteristics of the isolated protein, contributing to significant lot-to-lot variability when using tissue-derived material .
How can we quantify Collagen-IV turnover dynamics in developing tissues?
Recent advances using fluorescently tagged Collagen-IV have enabled quantitative assessment of Collagen-IV turnover. Studies using eGFP-COL4A2 mouse models and fluorescence recovery after photobleaching (FRAP) techniques have revealed unexpectedly rapid turnover rates in developing tissues, challenging the traditional view of collagens as static, slowly replaced molecules . In hair follicle development, the turnover rates vary significantly by region:
| Hair Follicle Region | Fluorescence Recovery Time (50%) | Recovery Rate at 3h30min |
|---|---|---|
| Tip Region | ~3h 25min (205 min) | 54% |
| Lower Stalk Region | Not reached within timeframe | 23% |
| Junction Region | Not reached within timeframe | 7% |
This spatial heterogeneity in turnover rates suggests that basement membrane expansion during morphogenesis involves active incorporation of new Collagen-IV protein, with rates correlated to the degree of tissue remodeling activity .
What is the optimal protocol for isolating 7S and NC1 domains from bovine placental tissues?
The isolation of Collagen-IV domains from bovine placental basement membrane involves several critical steps:
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Tissue preparation: Bovine placental tissue is minced and homogenized in buffer containing protease inhibitors.
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Basement membrane isolation: The homogenate undergoes differential centrifugation and washing steps to isolate basement membrane-enriched fractions.
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Collagenase digestion: Purified bacterial collagenase is used for selective proteolytic degradation of the triple-helical part of Collagen-IV networks while preserving the 7S and NC1 domains.
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Domain separation: The digested material is subjected to size-exclusion chromatography to separate the 7S and NC1 domains.
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Further purification: Additional chromatographic techniques (e.g., ion-exchange chromatography) are employed to achieve higher purity.
This methodological approach can be adapted, with minor modifications, for isolation from other bovine basement membrane sources, though tissue-specific pre-processing steps may be required .
How can recombinant expression systems be utilized to produce Collagen-IV fragments?
Recombinant expression offers an alternative to tissue extraction for obtaining Collagen-IV fragments with several advantages:
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Expression system: Human embryonic kidney HEK-293 cells are commonly used for expressing recombinant Collagen-IV fragments, particularly NC1 domains.
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Construct design: cDNA encoding the desired Collagen-IV fragment (e.g., NC1 domains) is cloned into an expression vector (such as pRc-X) with an amino-terminal FLAG tag for detection and purification.
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Transfection and selection: Stable transfection is achieved via calcium phosphate precipitation, followed by selection with geneticin (G418).
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Protein purification: Recombinant proteins are purified from conditioned medium using affinity chromatography on anti-FLAG agarose and elution with FLAG peptide.
This approach has enabled the identification of target antigens for human autoantibodies in Goodpasture's disease, mapping of pathogenic epitopes, and the development of heterotrimeric fragments for studying molecular mechanisms of Collagen-IV assembly .
What imaging techniques can be employed to study Collagen-IV dynamics in live tissues?
Recent developments in mouse models expressing fluorescently tagged endogenous Collagen-IV have revolutionized the ability to visualize basement membrane dynamics in real-time:
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Genetic models: The eGFP-Col4a2 knock-in mouse model expresses the eGFP protein inserted into the 7S domain of the Collagen-IV α2 chain, allowing for direct visualization of Collagen-IV in living tissues .
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Fluorescence recovery after photobleaching (FRAP): This technique involves photobleaching a region of interest and monitoring the recovery of fluorescence over time, providing quantitative data on protein turnover rates.
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Live tissue imaging: Using confocal or multiphoton microscopy, researchers can visualize Collagen-IV dynamics in developing tissues such as hair follicles.
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Quantitative analysis: Software-based analysis of fluorescence recovery curves enables calculation of half-recovery times and comparison of turnover rates across different tissue regions.
These advanced imaging approaches have revealed that basement membrane expansion during morphogenesis involves active incorporation of new Collagen-IV protein at rates that vary significantly between different tissue regions .
How can the issue of lot-to-lot variability in bovine Collagen-IV preparations be addressed?
Animal-derived Collagen-IV exhibits significant lot-to-lot variability due to extensive post-translational modifications that continue over the life of the molecule in the extracellular space. These modifications influence both the extractability of collagens from tissue and their biophysical characteristics . Several approaches can mitigate this variability:
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Standardized source material: Utilizing placental tissue from animals of similar age and physiological state.
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Consistent isolation protocols: Employing rigorously standardized extraction and purification protocols.
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Comprehensive quality control: Implementing analytical techniques to verify protein integrity, domain composition, and functional properties.
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Recombinant alternatives: Using recombinantly expressed Collagen-IV fragments, which are essentially identical to the native collagen protein but with reduced variability.
Recombinant approaches offer the additional benefit of reducing the risk of inflammation, immune response, and disease transmission compared to animal-sourced collagen .
What are the immunohistochemical methods for distinguishing Collagen-IV from other collagen types in bovine tissues?
Immunohistochemical detection of collagens in bovine tissues requires specific methodological considerations:
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Tissue preparation: Unfixed cryostat sections are recommended to preserve antigenic epitopes that may be masked by fixation.
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Antibody selection: Use of highly specific antibodies against Collagen-IV that do not cross-react with other collagen types.
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Detection method: An indirect FITC (fluorescein isothiocyanate) method is effective for visualizing the distribution pattern.
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Comparative analysis: Parallel staining for Collagen I, III, and VI helps distinguish the unique distribution pattern of Collagen-IV.
In bovine uterine tissues, Collagen-IV is distinctively localized to basement membranes underlying all endometrial epithelia and surrounding smooth muscle cells of blood vessels, while other collagen types show different distribution patterns . The day of the estrous cycle may influence the amounts and organization of Collagen-IV, with variations observed between cycle days 1, 8, 15, and 19 .
How might the eGFP-Col4a2 mouse model advance our understanding of Collagen-IV in development and disease?
The recently developed eGFP-Col4a2 mouse model (February 2025) offers unprecedented opportunities to investigate basement membrane dynamics:
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Real-time visualization: The model enables direct observation of basement membrane formation, expansion, and remodeling during development and in disease states.
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Quantitative dynamics: FRAP studies with this model have already revealed unexpectedly rapid turnover rates of Collagen-IV in developing tissues, challenging previous assumptions .
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Disease modeling: The model could be crossed with disease models to investigate how basement membrane dynamics are altered in conditions like Alport syndrome or diabetic nephropathy.
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Drug screening: Visualization of Collagen-IV dynamics could facilitate screening of compounds that modulate basement membrane remodeling for therapeutic applications.
The mouse model has already demonstrated that basement membrane expansion during morphogenesis involves active incorporation of new Collagen-IV protein, with turnover rates that vary significantly between different tissue regions .
What is the potential impact of understanding FAK-mediated regulation of Collagen-IV in bovine reproductive disorders?
Understanding the FAK signaling pathway's role in regulating Collagen-IV degradation has significant implications for bovine reproductive health:
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Diagnostic markers: Aberrant expression levels of FAK pathway components and Collagen-IV could serve as diagnostic markers for conditions like retained fetal membranes (RFM).
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Therapeutic targets: The FAK signaling pathway presents potential therapeutic targets for preventing or treating RFM and other placental disorders.
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Breeding selection: Genetic variants affecting the FAK-Collagen-IV regulatory axis could inform breeding selection for improved reproductive outcomes.
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Model development: The bovine system could serve as a model for understanding similar processes in human pregnancy complications involving basement membrane remodeling.
Research has already established that in RFM cases, the expression of FAK, Src, MMP-2, and MMP-9 is significantly downregulated while Collagen-IV is upregulated compared to healthy cows, suggesting a failure in the normal mechanisms of placental separation .
Product Science Overview
Bovine Collagen-IV is a type of collagen derived from cows. It is primarily sourced from the hide, bones, and tendons of cattle . This type of collagen is known for its ability to support the structural integrity of various tissues in the body, making it a popular ingredient in supplements and skincare products .
The production of bovine collagen involves boiling cow bones, connective tissues, cartilage, or hides in water to extract the collagen . The extracted collagen is then dried and powdered to form a supplement . This process ensures that the collagen retains its beneficial properties, making it suitable for various applications.
Bovine collagen primarily contains Type I and Type III collagen, which are essential for maintaining the health and elasticity of the skin, bones, and connective tissues . However, it also includes Type IV collagen, which is crucial for the formation of basement membranes that support the structure of tissues and organs .
- Skin Health: Bovine Collagen-IV helps improve skin elasticity and hydration, reducing the appearance of wrinkles and fine lines .
- Joint Health: It supports joint function by maintaining the integrity of cartilage, reducing joint pain and stiffness .
- Bone Health: Bovine Collagen-IV contributes to bone strength and density, reducing the risk of fractures and osteoporosis .
- Gut Health: It aids in maintaining the health of the gut lining, preventing issues like leaky gut syndrome .
