COL3A1 Monoclonal Antibody

What is COL3A1 and why is it important in scientific research?

COL3A1 (Collagen Type III Alpha 1 Chain) is a gene that encodes the pro-alpha1 chains of type III collagen, a fibrillar collagen found predominantly in extensible connective tissues. Type III collagen functions as a major structural component in hollow organs such as large blood vessels, uterus, and bowel. Beyond its structural role, type III collagen participates in the blood clotting cascade through interactions with platelets and serves as an important signaling molecule in wound healing processes . The significance of COL3A1 in scientific research stems from its association with vascular Ehlers-Danlos syndrome (vEDS) when mutated, making it a crucial target for understanding connective tissue disorders and vascular pathologies .

What are the basic properties of COL3A1 protein?

The COL3A1 protein is a collagen alpha-1(III) chain with a canonical length of 1466 amino acid residues and a molecular weight of approximately 138.6 kDa. The protein is characterized by the following properties:

• Subcellular localization: Secreted into the extracellular matrix

• Protein family: Member of the Fibrillar collagen family

• Number of reported isoforms: Up to 2 different isoforms

• Notable tissue expression: Highly expressed in testis, gall bladder, placenta, bladder, and endometrium

• Biological functions: Cell matrix adhesion and nervous system development

• Cellular identification: Can be used as a marker for Myofibroblast Cells and Type 2 Thymic Fibroblasts

When detected through Western blotting, the observed molecular weight is approximately 138 kDa, which is slightly different from the calculated weight of 139 kDa due to various factors affecting protein mobility during electrophoresis .

What applications are common for COL3A1 monoclonal antibodies?

COL3A1 monoclonal antibodies are versatile tools in molecular and cellular biology research, with multiple validated applications:

These applications enable researchers to detect, localize, and quantify COL3A1 protein in various experimental contexts. The choice of application depends on research objectives, with Western blotting being the most commonly reported method in the scientific literature .

How should COL3A1 monoclonal antibodies be stored and handled?

For optimal antibody performance and longevity, COL3A1 monoclonal antibodies should be stored at -20°C where they remain valid for approximately 12 months. Researchers should follow these handling guidelines:

• Avoid repeated freeze/thaw cycles as they can degrade antibody quality and reduce binding efficacy

• Store in appropriate buffer conditions (typically phosphate buffered solution, pH 7.4, containing stabilizers and protein protectants)

• Upon receipt (typically shipped with ice packs), immediately store at the recommended temperature

• Consider aliquoting the antibody solution to minimize freeze/thaw cycles if multiple experiments are planned

• Follow manufacturer's specific recommendations as formulations may vary slightly between suppliers

How should I optimize Western blot protocols for COL3A1 detection?

When designing Western blot experiments for COL3A1 detection, consider these methodological recommendations:

• Sample preparation: Use appropriate lysis buffers that effectively solubilize extracellular matrix proteins, potentially including detergents like SDS or Triton X-100

• Gel selection: Due to COL3A1's high molecular weight (138-139 kDa), use low percentage (6-8%) SDS-PAGE gels or gradient gels for optimal resolution

• Transfer conditions: Employ longer transfer times or higher voltages for efficient transfer of high molecular weight proteins

• Blocking and antibody dilution:

  • Primary antibody dilution: Begin with 1:1000 (within the recommended 1:500-1:2000 range)

  • Verify specificity using positive controls such as HeLa cell lysates

• Troubleshooting unexpected band patterns:

  • Multiple bands may indicate different modified forms of COL3A1

  • Band size inconsistencies may result from post-translational modifications

  • Always compare with expected molecular weight (139 kDa calculated, typically observed at 138 kDa)

For validation, researchers should consider including appropriate positive controls from tissues known to express COL3A1 highly, such as liver samples.

What methodological considerations are important for immunohistochemistry with COL3A1 antibodies?

For successful immunohistochemical detection of COL3A1, researchers should implement these methodological approaches:

• Tissue preparation:

  • Optimal fixation: 10% neutral buffered formalin for 24-48 hours

  • Paraffin embedding and sectioning (4-6 μm thickness recommended)

  • Antigen retrieval is crucial: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Antibody protocol:

  • Begin with a 1:100 dilution (within recommended 1:50-300 range)

  • Incubation: 1 hour at room temperature or overnight at 4°C

  • Secondary antibody selection should match the host species (mouse) of the COL3A1 primary antibody

  • Include controls: human or mouse liver tissue as positive controls

• Counterstaining and analysis:

  • Use H-score method for semi-quantitative analysis of staining intensity

  • Document both intensity and percentage of positive cells

  • Compare staining patterns with known COL3A1 distribution (extracellular matrix localization)

Researchers should be aware that COL3A1 staining patterns vary by tissue type, with particularly strong expression expected in vascular structures, connective tissues, and organs like the bladder and intestine.

How can I implement COL3A1 immunofluorescence staining for precise cellular localization?

For detailed subcellular localization of COL3A1 using immunofluorescence techniques:

• Sample preparation options:

  • Cultured cells: Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Frozen tissue sections: 10 μm thickness recommended

  • Deparaffinized tissue sections: Require antigen retrieval (as described in IHC)

• Staining protocol optimization:

  • Permeabilization: Use 0.1-0.3% Triton X-100 in PBS (5-10 minutes)

  • Blocking: 1-5% normal serum (from secondary antibody host species) for 30-60 minutes

  • Primary antibody dilution: Start with 1:200 (within recommended 1:100-1:300 range)

  • Overnight incubation at 4°C typically yields best results

  • Include DAPI or similar nuclear counterstain for orientation

• Confocal microscopy settings:

  • Optimal excitation/emission wavelengths based on secondary antibody fluorophore

  • Z-stack imaging recommended for extracellular matrix proteins

  • Use multi-channel imaging to co-localize with other matrix proteins

Expected pattern: COL3A1 should localize to the extracellular space/extracellular matrix, with minimal intracellular staining except in actively synthesizing cells. Mouse liver tissue can serve as a reliable positive control for IF optimization.

How can COL3A1 expression be used as a prognostic biomarker in cancer research?

COL3A1 expression analysis has emerged as a potential prognostic tool in cancer research, particularly in ovarian cancer. Methodological approaches include:

What tissue-specific expression patterns of COL3A1 should be considered when designing experiments?

Understanding tissue-specific expression of COL3A1 is crucial for experimental design, particularly for selecting appropriate controls and interpreting results:

• Human tissue expression profile:

  • Highest expression: Gall bladder, placenta, bladder, endometrium

  • Moderate expression: Gastrointestinal tract, fat, heart, prostate, skin, spleen, testis

  • Notable absence or low expression: Adult brain, liver

• Mouse tissue expression profile:

  • Highest expression: Bladder, mammary gland, placenta, fat, embryonic limbs (E14.5)

  • Moderate expression: Gastrointestinal tract, heart, kidney, lung, ovary, embryonic brain and liver

  • Minimal expression: Adult brain, liver

When designing tissue-specific experiments, researchers should consider these expression patterns for:

• Selecting positive and negative control tissues

• Interpreting antibody specificity

• Understanding potential cross-reactivity

• Contextualizing experimental findings within physiological expression patterns

How can COL3A1 antibodies be used to study Ehlers-Danlos syndrome and vascular pathologies?

COL3A1 monoclonal antibodies provide valuable tools for investigating vascular Ehlers-Danlos syndrome (vEDS) and related pathologies:

• Mutation analysis methodologies:

  • Immunohistochemical detection of abnormal collagen deposition patterns

  • Western blot analysis of COL3A1 protein expression and processing

  • Comparing wild-type and mutant COL3A1 expression and localization

• Experimental approaches for vascular pathology studies:

  • Tissue samples from vEDS patients versus controls

  • Animal models: Col3a1 knockout or mutant mice analysis

  • Cell culture systems with COL3A1 mutations or knockdowns

• Key research findings:

  • Mutations in COL3A1 lead to abnormal collagen structure and function

  • vEDS patients often die suddenly due to large arterial ruptures

  • Col3a1 knockout mice have shortened lifespans due to arterial ruptures, mimicking human vEDS phenotype

For researchers focusing on vEDS, it's crucial to analyze both protein expression patterns and structural abnormalities in collagen fibrils, as both quantity and quality of type III collagen may be affected by pathogenic mutations.

How should results be interpreted when COL3A1 antibody shows unexpected molecular weight bands?

When Western blotting with COL3A1 antibodies yields unexpected band patterns, researchers should consider these interpretative frameworks:

• Common causes of molecular weight discrepancies:

  • Post-translational modifications (glycosylation, phosphorylation)

  • Proteolytic processing (procollagen to mature collagen conversion)

  • Alternative splicing of COL3A1 (two transcript variants reported)

  • Sample preparation conditions affecting protein denaturation

• Methodological verification approaches:

  • Use multiple antibodies targeting different epitopes

  • Include deglycosylation enzyme treatments

  • Compare reducing vs. non-reducing conditions

  • Test different tissue/cell sources with known expression profiles

• Interpretation guidelines:

  • The calculated molecular weight of 139 kDa may differ from observed 138 kDa due to various factors affecting electrophoretic mobility

  • Multiple bands may represent different modified forms of the protein

  • Ensure proper molecular weight standards are used for accurate sizing

Researchers should document and report unexpected band patterns thoroughly, as they may represent biologically relevant modifications or processing events rather than non-specific binding.

What are the most common issues when using COL3A1 antibodies and how can they be resolved?

When working with COL3A1 monoclonal antibodies, researchers frequently encounter several technical challenges. Here are methodological solutions for common problems:

• Low or no signal in Western blotting:

  • Increase protein loading (30-50 μg recommended)

  • Optimize transfer conditions for high molecular weight proteins

  • Extend primary antibody incubation (overnight at 4°C)

  • Verify sample preparation preserves extracellular matrix proteins

  • Check antibody reactivity matches species being tested (Human, Mouse, Rat)

• High background in immunostaining:

  • Increase blocking time and concentration (5% BSA or 10% normal serum)

  • Optimize antibody dilution (start with higher dilutions)

  • Include additional washing steps (5x5 minutes)

  • Use more specific secondary antibodies

  • Test alternative fixation methods

• Inconsistent results between experiments:

  • Standardize protein extraction protocols

  • Establish consistent sample processing timelines

  • Prepare fresh working solutions for each experiment

  • Document lot numbers as antibody performance may vary

  • Implement positive and negative controls in each experiment

How should researchers validate COL3A1 antibody specificity for their experimental system?

Thorough validation of COL3A1 antibody specificity is crucial for generating reliable research data. A systematic validation approach includes:

• Positive and negative control tissues:

  • Positive controls: Human liver, mouse liver, tissues with known high expression (bladder, placenta)

  • Negative controls: Adult brain or primary antibody omission

  • Blocking peptide competition assays to confirm specificity

• Multiple detection methods:

  • Compare results across techniques (WB, IHC, IF)

  • Correlate protein detection with mRNA expression data

  • Use siRNA/shRNA knockdown to confirm antibody specificity in cell culture systems

• Cross-validation with other antibodies:

  • Test multiple antibodies targeting different epitopes

  • Compare monoclonal and polyclonal antibodies

  • Correlate results with known COL3A1 biological functions

  • Document clonality (e.g., clone 5G2) and host information

This multi-faceted validation approach helps ensure experimental findings reflect true COL3A1 biology rather than technical artifacts or cross-reactivity with other proteins.

What considerations are important when comparing COL3A1 expression across different species?

When conducting comparative studies of COL3A1 across species, researchers should account for these methodological considerations:

• Sequence homology and epitope conservation:

  • Verify antibody epitope conservation in target species

  • The monoclonal antibody E-AB-22071 (clone 5G2) shows reactivity with Human, Mouse, and Rat COL3A1

  • Check for species-specific isoforms or processing differences

• Tissue-specific expression variations:

  • Expression patterns differ between species (e.g., mouse vs. human)

  • Developmental stage affects expression (e.g., embryonic vs. adult tissues)

  • Adjust sampling and experimental design accordingly

• Methodological adaptations:

  • Optimize extraction buffers for species-specific tissue composition

  • Adjust antibody dilutions for each species (potentially higher concentrations for less conserved epitopes)

  • Include species-appropriate positive controls

  • Consider species-specific secondary antibodies to minimize background

Researchers should thoroughly document species differences in COL3A1 expression patterns to facilitate accurate cross-species comparisons and translational research applications.

How can COL3A1 antibodies be used in wound healing and tissue regeneration research?

COL3A1 monoclonal antibodies offer valuable tools for investigating wound healing and tissue regeneration processes:

• Temporal expression analysis methodologies:

  • Time-course studies of wound healing with immunohistochemistry

  • Quantification of COL3A1 protein levels during different healing phases

  • Co-localization with other wound healing markers

• Experimental approaches:

  • In vitro wound healing assays with fibroblasts

  • Ex vivo skin explant models

  • In vivo wound models with sequential tissue sampling

  • Analysis of COL3A1 in granulation tissue formation

• Research applications:

  • Monitoring collagen deposition and remodeling during wound healing

  • Evaluating therapeutic interventions targeting ECM synthesis

  • Studying the transition from type III to type I collagen during maturation of scar tissue

Given COL3A1's role as a signaling molecule in wound healing, researchers can use these antibodies to elucidate both structural and signaling functions during tissue repair processes.

What are the best approaches for combining COL3A1 with other biomarkers in multiplex immunostaining?

For multiplex analysis incorporating COL3A1 with other biomarkers, researchers should implement these methodological strategies:

• Panel design considerations:

  • Combine COL3A1 with other ECM components (e.g., COL1A1, fibronectin)

  • Include cell-type specific markers (e.g., α-SMA for myofibroblasts)

  • Consider signaling molecules involved in collagen regulation

• Technical optimization:

  • Carefully select primary antibodies from different host species

  • Choose non-overlapping fluorophores for multiplexed IF

  • For chromogenic IHC, use sequential staining protocols

  • Optimize antigen retrieval conditions compatible with all targets

• Analysis approaches:

  • Implement digital image analysis for quantification

  • Use confocal microscopy for co-localization studies

  • Apply spatial analysis to evaluate distribution patterns

  • Consider single-cell analysis techniques for heterogeneous tissues

Multiplex approaches are particularly valuable for understanding the complex interplay between COL3A1 and other molecular players in tissue homeostasis and disease processes.