FN1 Recombinant Monoclonal Antibody

What is FN1 and why is it significant in biomedical research?

Fibronectin (FN1) is a multifunctional glycoprotein present in the extracellular matrix of tissues. It plays critical roles in cell adhesion, tissue development, wound healing, blood clot formation, and cell signaling pathways. As a key structural component, fibronectin maintains tissue integrity and supports various cellular activities . The protein exists in multiple forms - a soluble dimeric form secreted by hepatocytes in plasma, and dimeric or cross-linked multimeric forms produced by fibroblasts, epithelial cells, and other cell types that are deposited as fibrils in the extracellular matrix . Due to its involvement in numerous physiological and pathological processes, FN1 is an important research target in cancer, cardiovascular disease, developmental biology, stem cell research, and signal transduction studies .

What distinguishes recombinant monoclonal antibodies against FN1 from conventional monoclonal antibodies?

Recombinant monoclonal antibodies against FN1 are synthetically generated in vitro, starting with the harvest of FN1 antibody genes from immunoreactive B cells. These genes are amplified, cloned into phage vectors, and introduced into mammalian cell lines for functional antibody production . In contrast, conventional monoclonal antibodies are typically produced through the traditional hybridoma technique, where antibody-producing B cells are fused with myeloma cells to create hybridomas that secrete antibodies continuously .

The recombinant approach offers several advantages:

Recombinant antibodies demonstrate superior lot-to-lot consistency and provide a more sustainable supply chain for long-term research projects .

How should researchers select the appropriate FN1 antibody for their specific experimental needs?

Selection of the appropriate FN1 antibody depends on multiple experimental factors:

• Target species reactivity: Verify the antibody's reactivity with your species of interest. Available FN1 antibodies show varying cross-reactivity with human, mouse, and rat samples .

• Application compatibility: Confirm the antibody has been validated for your intended application:

  • Western blotting (WB): Most FN1 antibodies work at dilutions between 1:500-1:2000

  • Immunohistochemistry (IHC): Typically used at 1:50-1:400 dilutions

  • Immunofluorescence (IF): Often effective at 1:100-1:800 dilutions

  • Flow cytometry (FC): Usually requires 1:50-1:200 dilutions

  • Functional studies: Some antibodies like HFN 7.1 have function-blocking capabilities

• Epitope specificity: Consider which domain of FN1 you need to target. Some antibodies recognize specific regions, such as HFN 7.1 which targets the region between the PHSRN synergy and RGD sites spanning type III repeats 9 and 10 .

• Clonality and isotype: Mouse IgG1 is common for many FN1 monoclonal antibodies, but rabbit IgG alternatives are also available .

• Validation data: Assess the quality of validation data provided by manufacturers, including verified samples (e.g., HeLa cells, rat liver, human appendix) and observed molecular weight (typically 262-285 kDa) .

What is the optimal protocol for Western blot analysis using FN1 antibodies?

A standardized Western blot protocol for FN1 detection should account for the large molecular weight of the protein (262-285 kDa) and potential multiple isoforms:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • Load 20-50 μg of total protein per lane

• SDS-PAGE optimization:

  • Use low percentage (6-8%) gels to resolve high molecular weight FN1

  • Run the gel at lower voltage (80-100V) for better resolution of large proteins

• Transfer conditions:

  • Employ wet transfer at 4°C overnight at low voltage (30V)

  • Use PVDF membrane with 0.45 μm pore size for larger proteins

• Antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Dilute primary FN1 antibody at 1:500-1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash 3-5 times with TBST

  • Use appropriate HRP-conjugated secondary antibody at 1:2000-1:5000

  • Incubate for 1 hour at room temperature

  • Wash thoroughly before detection

• Detection and analysis:

  • Use enhanced chemiluminescence with exposure times optimized for your sample

  • Be prepared to observe a band around 285 kDa, which may differ from the calculated MW (263 kDa) due to post-translational modifications

  • Multiple bands may be detected due to alternative splicing (FN1 has three regions subject to alternative splicing with potential for 20 different transcript variants)

How can researchers optimize immunohistochemistry protocols for FN1 detection in tissue samples?

For optimal FN1 detection in tissue sections:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Cut sections at 4-5 μm thickness

• Antigen retrieval methods:

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

  • Pressure cooker treatment for 20 minutes is often effective for FN1

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Block non-specific binding with 5-10% normal serum from the same species as the secondary antibody

  • Apply primary FN1 antibody at dilutions of 1:50-1:300 (for manual IHC) or 1:400-1:1600 (for automated systems like Leica Bond)

  • Incubate overnight at 4°C in a humidified chamber

  • Use appropriate detection systems (e.g., HRP/DAB)

• Controls and validation:

  • Include positive control tissues with known FN1 expression (e.g., rat liver tissue has been verified for many FN1 antibodies)

  • Include negative controls by omitting primary antibody

  • Consider dual staining with markers of ECM or fibrosis for co-localization studies

• Signal amplification for weak expression:

  • Employ tyramide signal amplification (TSA) if standard methods yield weak signals

  • Consider polymer-based detection systems for enhanced sensitivity

What steps are critical for validating the specificity of a new FN1 antibody?

Thorough validation of FN1 antibody specificity requires multiple complementary approaches:

• Western blot validation:

  • Confirm single band or expected pattern of bands at the predicted molecular weight (262-285 kDa)

  • Include positive control lysates from cells known to express FN1 (e.g., HeLa cells)

  • Compare with established FN1 antibodies as reference standards

  • Perform peptide competition assays using the immunogen peptide to confirm specificity

• Immunoprecipitation cross-validation:

  • Immunoprecipitate with the FN1 antibody and probe with a different FN1 antibody targeting a separate epitope

  • Verify protein identity using mass spectrometry after immunoprecipitation

• Genetic manipulation controls:

  • Test antibody reactivity in FN1 knockdown/knockout models

  • Compare staining patterns in cell lines with varying FN1 expression levels

• Cross-reactivity assessment:

  • Evaluate antibody performance across multiple species if cross-reactivity is claimed

  • Verify reactivity with recombinant FN1 protein

• Application-specific validation:

  • For IHC/IF: Compare staining patterns with known FN1 distribution (extracellular matrix localization)

  • For function-blocking antibodies: Perform cell adhesion assays to confirm inhibitory activity

Why do researchers often observe unexpected bands or molecular weight variations when detecting FN1 by Western blot?

Several factors contribute to the complex banding patterns and molecular weight variations observed when detecting FN1:

• Alternative splicing: FN1 has three regions subject to alternative splicing, potentially producing 20 different transcript variants, resulting in proteins of varying molecular weights .

• Post-translational modifications: FN1 undergoes extensive glycosylation and other modifications that can alter its electrophoretic mobility, causing the observed molecular weight (approximately 285 kDa) to differ from the calculated weight (262-263 kDa) .

• Proteolytic processing: FN1 can be cleaved into functional fragments by various proteases during sample preparation or in biological processes, generating lower molecular weight bands (including 69-71 kDa isoforms and shorter cleavage products) .

• Dimer formation: Fibronectin naturally exists as dimers held together by disulfide bonds, which may not be completely reduced during sample preparation, resulting in very high molecular weight bands.

• Cross-reactivity: Some antibodies may show weak cross-reactivity with other extracellular matrix proteins with similar domains.

To address these issues:

• Ensure complete protein denaturation with adequate SDS and reducing agents

• Use freshly prepared samples with protease inhibitors

• Consider native gel electrophoresis if studying intact FN1 complexes

• Validate bands using multiple antibodies targeting different epitopes

What are effective strategies for minimizing background in immunofluorescence studies with FN1 antibodies?

Reducing background in FN1 immunofluorescence requires addressing several potential sources of non-specific signals:

• Optimized blocking:

  • Use 5-10% normal serum from the species of the secondary antibody

  • Add 0.1-0.3% Triton X-100 for better penetration in fixed cells

  • Consider using commercial blocking reagents specifically designed for fluorescence applications

  • Extend blocking time to 1-2 hours at room temperature or overnight at 4°C

• Antibody dilution optimization:

  • Titrate antibody concentrations; start with manufacturer recommendations (typically 1:100-1:300 for FN1 antibodies) and adjust as needed

  • Use antibody diluent containing 1-3% BSA and 0.05-0.1% Tween-20

• Washing procedures:

  • Increase washing steps (5-6 times) with PBS containing 0.05-0.1% Tween-20

  • Extend wash times to 5-10 minutes per wash

  • Use gentle agitation during washes

• Sample preparation considerations:

  • Optimize fixation protocol (4% paraformaldehyde for 10-15 minutes is typically suitable)

  • Consider light autofluorescence quenching (e.g., 0.1% sodium borohydride treatment)

  • For tissue sections, treat with Sudan Black B (0.1-0.3%) to reduce autofluorescence

• Secondary antibody selection:

  • Use highly cross-adsorbed secondary antibodies

  • Consider directly conjugated primary antibodies to eliminate secondary antibody issues

  • Include a secondary-only control to assess non-specific binding

• Advanced countermeasures:

  • For tissue with high extracellular matrix content, pre-incubate sections with unconjugated Fab fragments

  • Use image acquisition settings that minimize autofluorescence detection

How should researchers address inconsistent results when comparing FN1 antibodies from different sources?

Inconsistent results between different FN1 antibodies can be systematically addressed through:

• Epitope mapping comparison:

  • Identify the specific epitopes recognized by each antibody

  • Antibodies targeting different domains of FN1 may yield distinct staining patterns or detect different subsets of FN1 isoforms

  • Some antibodies like HFN 7.1 target functional domains (between PHSRN and RGD sites) , while others may target conserved regions

• Protocol standardization:

  • Develop standardized protocols optimized for each antibody

  • Document and control variables that might affect results (fixation time, antigen retrieval method, incubation conditions)

  • Process samples in parallel when comparing antibodies

• Reference standards usage:

  • Include the same positive control samples across experiments

  • Consider using recombinant FN1 protein as a standard

  • Compare results with published data for established antibodies

• Cross-validation approach:

  • Employ multiple detection methods (WB, IHC, IF) with each antibody

  • Verify results with orthogonal techniques (qPCR, mass spectrometry)

  • Use genetic manipulation (siRNA knockdown) to confirm specificity

• Quantitative comparison:

  • Develop quantitative metrics for comparing antibody performance

  • Document sensitivity, specificity, and signal-to-noise ratio for each antibody

  • Create a decision matrix for selecting the most appropriate antibody for specific applications

How can function-blocking FN1 antibodies be utilized to study cell-ECM interactions in 3D culture systems?

Function-blocking FN1 antibodies, such as HFN 7.1 which interferes with fibronectin binding to cell surface receptors , offer powerful tools for studying cell-ECM interactions in 3D culture:

• Gradient inhibition studies:

  • Create spatial gradients of function-blocking antibodies within 3D matrices

  • Analyze directional cell migration in response to regional FN1 inhibition

  • Quantify cell morphology changes along the gradient using live cell imaging

• Temporal regulation of cell adhesion:

  • Add function-blocking antibodies at specific timepoints to disrupt established adhesions

  • Monitor real-time changes in cell behavior using time-lapse microscopy

  • Correlate changes with activation/deactivation of downstream signaling pathways

• Receptor specificity analysis:

  • Combine with specific integrin-blocking antibodies to dissect the contribution of different adhesion receptors

  • Map the functional domains of FN1 involved in specific cellular processes

  • Compare HFN 7.1 (which blocks the region between PHSRN and RGD sites) with antibodies targeting other domains

• Matrix assembly inhibition:

  • Study fibrillogenesis in 3D culture by adding function-blocking antibodies during matrix formation

  • Analyze the structural consequences of impaired FN1 fibrillogenesis on other ECM components

  • Evaluate the mechanical properties of matrices formed under FN1 inhibition conditions

• Controlled release systems:

  • Develop hydrogel or nanoparticle systems for controlled release of function-blocking antibodies

  • Create temporally defined windows of FN1 inhibition

  • Study the reversibility of cellular phenotypes after temporary FN1 function blockade

What insights can FN1 expression analysis provide in cancer research and how should such studies be designed?

FN1 expression analysis in cancer research provides multiple insights when properly designed:

• Tumor microenvironment assessment:

  • Compare FN1 expression in tumor cells versus stromal components using dual immunostaining

  • Analyze correlation between stromal FN1 deposition and cancer progression

  • Examine co-localization with other ECM proteins to characterize matrix remodeling

• EMT and metastasis investigation:

  • Monitor FN1 expression during epithelial-to-mesenchymal transition

  • Correlate FN1 levels with expression of EMT markers (E-cadherin, vimentin)

  • Track FN1 isoform switching during cancer progression using domain-specific antibodies

• Therapeutic resistance mechanisms:

  • Evaluate FN1-mediated cell adhesion as a mechanism of drug resistance (cell adhesion-mediated drug resistance, CAM-DR)

  • Compare FN1 expression before and after treatment failure

  • Test combination of FN1-targeting strategies with conventional therapies

• Experimental design considerations:

  • Use tissue microarrays for high-throughput analysis across multiple patient samples

  • Employ multi-parameter IHC/IF to correlate FN1 with other biomarkers

  • Validate findings in patient-derived xenografts and 3D organoid models

  • Complement protein-level analysis with transcriptomic data on FN1 splice variants

• Clinical correlation approach:

  • Stratify patient cohorts based on FN1 expression patterns

  • Correlate FN1 expression with clinical outcomes (survival, recurrence, treatment response)

  • Develop standardized scoring systems for FN1 expression in clinical samples

How can researchers effectively use FN1 antibodies in stem cell differentiation and developmental biology studies?

FN1 antibodies offer valuable tools for studying stem cell biology and development:

• Lineage specification monitoring:

  • Track temporal changes in FN1 expression during differentiation of pluripotent stem cells

  • Correlate FN1 isoform switching with key developmental transitions

  • Use function-blocking antibodies to determine critical windows when FN1-integrin interactions regulate cell fate decisions

• Spatial patterning analysis:

  • Employ whole-mount immunostaining with FN1 antibodies to visualize ECM organization during embryonic development

  • Create high-resolution maps of FN1 distribution across developing tissues

  • Correlate FN1 gradients with morphogen distribution and cell migration patterns

• Engineered microenvironments for directed differentiation:

  • Conjugate FN1 antibodies to defined regions of culture substrates to create patterned surfaces

  • Analyze how spatial restriction of FN1 function affects stem cell differentiation

  • Compare results with genetic knockdown approaches to validate antibody effects

• Multi-parameter temporal analysis:

  • Combine live cell imaging with fixed-timepoint immunostaining for FN1

  • Use pulse-chase experiments with ECM labeling to track matrix turnover during differentiation

  • Correlate changes in FN1 organization with cytoskeletal remodeling and cell shape changes

• Experimental recommendations:

  • Include careful validation of antibody specificity, as FN1 structure changes during development

  • Optimize fixation protocols to preserve ECM structure while allowing antibody penetration

  • Use super-resolution microscopy to resolve nanoscale organization of FN1 fibrils

  • Complement antibody-based studies with reporter systems for live tracking of FN1 dynamics

How should researchers quantify and interpret changes in FN1 expression patterns across different experimental conditions?

Rigorous quantification and interpretation of FN1 expression changes require systematic approaches:

• Western blot quantification:

  • Use internal loading controls appropriate for your experimental system

  • Employ densitometry software to quantify band intensity

  • Account for potential molecular weight variants (262-285 kDa)

  • Present data as fold-change relative to control conditions

  • Perform statistical analysis across multiple biological replicates (minimum n=3)

• Immunofluorescence quantification methods:

  • Develop standardized image acquisition parameters (exposure time, gain, offset)

  • Analyze both signal intensity and pattern (fibrillar vs. diffuse)

  • Quantify parameters such as:

    • Mean fluorescence intensity (cellular or regional)

    • Fibril length, thickness, and orientation using specialized software

    • Colocalization coefficients with cell surface markers or other ECM proteins

  • Use automated image analysis algorithms to reduce bias

• Interpretation frameworks:

  • Consider both cellular and matrix-associated FN1 separately

  • Distinguish between changes in expression level versus altered localization

  • Account for the extracellular nature of FN1 when interpreting results

  • Remember that plasma FN1 (soluble dimeric form) is primarily secreted by hepatocytes, while cellular FN1 is produced by fibroblasts, epithelial cells, and other cell types

• Contextual analysis:

  • Interpret FN1 changes in context of other ECM components

  • Consider the role of FN1 in your specific biological system (wound healing, development, disease)

  • Validate findings with complementary techniques (qPCR for transcript levels, proteomics)

• Reporting standards:

  • Clearly describe quantification methods in publications

  • Include representative images alongside quantitative data

  • Report both mean values and measures of variability

  • Present raw data points alongside statistical summaries

What approaches can help distinguish between cellular FN1 production and matrix-incorporated FN1 in complex tissue samples?

Distinguishing cellular production from matrix incorporation of FN1 requires specialized techniques:

• Differential extraction protocols:

  • Sequential extraction to separate soluble, membrane-associated, and matrix-bound FN1

  • Compare extracts using Western blotting with the same FN1 antibody

  • Quantify the distribution across different fractions

• Cell-type specific markers in dual immunostaining:

  • Co-stain for FN1 and cell-type specific markers

  • Use nuclear counterstains to identify cellular boundaries

  • Employ confocal microscopy with z-stack analysis to distinguish intracellular from extracellular signals

  • Analyze colocalization with ER/Golgi markers to identify actively producing cells

• In situ hybridization combined with immunostaining:

  • Detect FN1 mRNA using in situ hybridization to identify producing cells

  • Follow with FN1 immunostaining on the same section

  • Compare patterns to distinguish cells producing FN1 from areas of matrix incorporation

• Pulse-chase experimental design:

  • Label newly synthesized proteins using click chemistry approaches

  • Track the fate of labeled FN1 from intracellular to extracellular compartments

  • Analyze incorporation into the existing matrix over time

• Functional biochemical discrimination:

  • Use domain-specific antibodies that distinguish cellular and plasma FN1 isoforms

  • Analyze deoxycholate-soluble (newly synthesized) versus deoxycholate-insoluble (matrix-incorporated) FN1

  • Consider the relationship between fibril formation and FN1 incorporation into stable matrix structures

How can researchers accurately analyze post-translational modifications of FN1 using antibody-based approaches?

Analysis of FN1 post-translational modifications requires specialized strategies:

• Modification-specific antibodies:

  • Use antibodies that specifically recognize phosphorylated, glycosylated, or otherwise modified FN1

  • Validate specificity using enzymatic treatment (e.g., deglycosylation, dephosphorylation)

  • Combine with pan-FN1 antibodies to determine modified fraction

• Sequential immunoprecipitation approach:

  • First immunoprecipitate total FN1 using a general FN1 antibody

  • Then probe the immunoprecipitate with modification-specific antibodies

  • Compare ratios across experimental conditions

• Mass spectrometry validation:

  • Immunoprecipitate FN1 and perform LC-MS/MS analysis

  • Map identified modifications to specific domains

  • Correlate antibody-based detection with MS results for validation

• Functional correlation studies:

  • Correlate detection of specific modifications with functional assays

  • Analyze how modifications affect FN1-dependent cell behaviors

  • Use site-directed mutagenesis of modification sites to confirm functional relevance

• Cautions and controls:

  • Include samples treated with modifying or demodifying enzymes as controls

  • Consider the effects of sample preparation on preserving labile modifications

  • Validate findings using multiple antibody clones when available

  • Remember that modifications may affect antibody accessibility to epitopes

This approach allows for detailed characterization of FN1 post-translational modifications, providing insights into how these changes affect protein function in different physiological and pathological contexts.