FURIN Antibody

What is FURIN and why are FURIN antibodies critical for research?

FURIN is a ubiquitous endoprotease belonging to the proprotein convertase family, capable of cleaving proteins at the RX(K/R)R consensus motif . It plays essential roles in processing numerous precursor proteins and is involved in diverse biological processes including viral infection mechanisms, cellular signaling, and tumorigenesis . FURIN antibodies enable researchers to detect, quantify, and localize this protein in various experimental systems, making them invaluable tools for investigating FURIN's multifaceted biological functions. These antibodies help researchers elucidate FURIN's involvement in normal cellular processes and pathological conditions.

What technical applications can FURIN antibodies support in laboratory research?

FURIN antibodies support multiple research applications including Western blotting (WB), immunocytochemistry (ICC), immunohistochemistry (IHC), and immunofluorescence (IF) . For Western blotting, antibodies like ab3467 have been validated at dilutions between 1:500 to 1:1000, producing specific bands at the expected molecular weight of 87 kDa in cell lines such as HeLa, HEK-293, and K-562 . For immunofluorescence applications, these antibodies can visualize FURIN's subcellular localization in fixed cells, typically showing distribution patterns in the Golgi apparatus, trans-Golgi network, endosomes, and cell membrane . The versatility of these antibodies allows researchers to investigate both quantitative expression levels and spatial distribution of FURIN in experimental systems.

How can researchers validate the specificity of FURIN antibodies in their experimental systems?

Validating antibody specificity is critical for obtaining reliable research outcomes. The gold standard approach involves comparing antibody signals between wild-type and FURIN knockout samples. As demonstrated with ab3467, Western blot analysis shows a clear 87 kDa band in wild-type A549 cells that is completely absent in FURIN knockout A549 cells . Additional validation methods include:

• RNA interference approach - comparing signals in cells treated with FURIN-specific siRNA versus scrambled controls

• Overexpression validation - observing increased signal in cells transfected with FURIN expression constructs

• Peptide competition assays - pre-incubating the antibody with immunizing peptide to block specific binding

• Multiple antibody approach - verifying similar patterns with antibodies targeting different FURIN epitopes

These complementary validation strategies ensure that observed signals genuinely represent FURIN rather than cross-reactive proteins.

What are the optimal protocols for detecting FURIN using Western blotting?

Optimizing Western blot protocols for FURIN detection requires careful consideration of several experimental parameters:

When troubleshooting, researchers should include positive controls such as HeLa or HEK-293 cell lysates and, when available, FURIN knockout cells as negative controls to validate signal specificity .

How should researchers interpret differences in FURIN detection patterns between different antibodies?

When different FURIN antibodies yield varying results, researchers should consider several technical and biological factors:

• Epitope recognition - Antibodies targeting different domains of FURIN (N-terminal, catalytic domain, C-terminal) may produce distinct patterns due to differential epitope accessibility or post-translational modifications

• Validation status - Compare antibody validation data (knockout/knockdown controls) to assess reliability; antibodies like ab3467 demonstrate specificity through absence of signal in FURIN knockout cells

• Technical variables - Different antibodies may require specific optimization for buffer conditions, incubation times, and detection methods

• Biological interpretation - Variations might reflect biologically relevant phenomena such as:

  • Detection of different FURIN isoforms

  • Capture of various processing states (zymogen vs. active enzyme)

  • Differential recognition of conformational states

When faced with discrepancies, researchers should conduct side-by-side comparisons under identical conditions and validate findings with complementary non-antibody methods, such as mass spectrometry or activity-based assays.

What approaches can researchers use to quantify FURIN expression levels across different tissue or cell types?

Accurate quantification of FURIN expression requires methodological rigor and appropriate controls:

• Western blot-based quantification:

  • Normalize FURIN signals to validated housekeeping proteins (GAPDH, β-actin)

  • Include standard curves of recombinant FURIN for absolute quantification

  • Use digital imaging systems with linear detection ranges

• Immunohistochemical/immunofluorescence quantification:

  • Employ standardized image acquisition parameters across samples

  • Use automated image analysis software for unbiased quantification

  • Score both staining intensity and percentage of positive cells

• Flow cytometry:

  • Optimize permeabilization conditions for intracellular FURIN detection

  • Use fluorescence intensity calibration beads for standardization

  • Consider dual staining with organelle markers to assess compartment-specific expression

• ELISA-based approaches:

  • Develop sandwich ELISA systems using capture and detection antibodies

  • Include recombinant FURIN standards for calibration curves

When comparing across tissues or cell types, researchers must account for tissue-specific matrix effects and validate findings using multiple methodological approaches.

How are FURIN antibodies utilized in SARS-CoV-2 infection research?

FURIN antibodies have become essential tools in SARS-CoV-2 research, driven by the critical role of FURIN in processing the viral spike protein:

• Mechanistic studies - FURIN antibodies help visualize and quantify FURIN's interaction with the SARS-CoV-2 spike protein, elucidating how the virus exploits this host protease for its infection cycle

• Therapeutic development - Engineered antibodies like FuG1 represent innovative approaches that directly target the FURIN-mediated activation of viral proteins. FuG1 incorporates an Fc-extended peptide that specifically interferes with host furin function, limiting spike activation and viral transmissibility

• Experimental methods using FURIN antibodies in coronavirus research:

  • Co-localization studies to visualize FURIN and viral proteins in infected cells

  • Biochemical assays to monitor FURIN-mediated cleavage of viral proteins

  • Cell-based infection assays to assess how FURIN inhibition affects viral entry and propagation

Research has demonstrated that FURIN plays a crucial role in SARS-CoV-2 infection by cleaving the spike protein into S1 and S2 subunits, making the virus highly transmissible between cells .

How does the engineered FuG1 antibody differ from conventional FURIN antibodies in viral research applications?

The FuG1 antibody represents an innovative advancement beyond conventional detection antibodies:

What experimental approaches can assess how FURIN inhibition affects viral infection cycles?

Researchers can employ several complementary approaches to investigate FURIN inhibition effects:

• Viral entry assays:

  • Pseudovirus systems expressing viral envelope proteins

  • Cell-cell fusion assays monitoring FURIN-dependent membrane fusion

  • Single-cycle infection assays with reporter viruses

• Protein processing analysis:

  • Western blotting to assess viral protein cleavage patterns with and without FURIN inhibition

  • Pulse-chase experiments to track processing kinetics

  • Mass spectrometry to identify precise cleavage sites

• Viral replication assessment:

  • Plaque assays to quantify infectious virus production

  • qPCR measurement of viral genome replication

  • Immunofluorescence visualization of viral spread in cell culture

• Inhibition approaches besides antibodies:

  • Small molecule FURIN inhibitors as comparison tools

  • FURIN gene knockout/knockdown to complement antibody studies

  • Peptide inhibitors mimicking FURIN cleavage sites

Research with FuG1 demonstrated that targeting the furin-mediated cleavage step specifically interferes with SARS-CoV-2's ability to spread from cell to cell, validating this as a critical point in the viral life cycle .

How can researchers optimize immunofluorescence protocols to visualize FURIN's subcellular distribution?

Effective immunofluorescence visualization of FURIN requires protocol optimization:

• Fixation methods:

  • Paraformaldehyde (4%) preserves FURIN epitopes while maintaining cellular architecture

  • Methanol fixation (shown effective with ab3467) may provide better access to certain epitopes

  • Avoid harsh fixatives that might destroy conformational epitopes

• Permeabilization considerations:

  • 0.1-0.2% Triton X-100 provides access to intracellular FURIN compartments

  • Digitonin (0.01%) offers selective plasma membrane permeabilization for distinguishing surface vs. intracellular pools

• Blocking optimization:

  • 1-5% BSA combined with 5% serum matching secondary antibody species

  • Addition of 0.1% gelatin can reduce non-specific binding in some cell types

• Co-localization markers:

  • TGN46 or Golgin-97 for trans-Golgi network

  • EEA1 for early endosomes

  • LAMP1 for late endosomes/lysosomes

  • Na⁺/K⁺-ATPase for plasma membrane

• Signal amplification:

  • Tyramide signal amplification for low-abundance detection

  • Super-resolution microscopy techniques for precise localization studies

Published immunofluorescence images using ab3467 show characteristic perinuclear Golgi staining pattern with some cytoplasmic vesicular distribution, consistent with FURIN's known trafficking pathways .

What considerations are important when using FURIN antibodies in primary cells versus established cell lines?

Working with FURIN antibodies across different cellular systems requires tailored approaches:

• Primary cell considerations:

  • Often express lower levels of FURIN requiring signal amplification methods

  • May show cell-type specific FURIN distribution patterns

  • Require optimization of fixation protocols to preserve delicate primary cell architecture

  • Display donor-to-donor variability necessitating more biological replicates

  • Often benefit from longer antibody incubation times (overnight at 4°C)

• Established cell line considerations:

  • Cell lines like HeLa, HEK-293, and A549 show reliable FURIN expression suitable for positive controls

  • Transformed cells may have altered FURIN expression or localization compared to primary counterparts

  • Allow for efficient protocol optimization before moving to valuable primary samples

  • FURIN knockout variants provide excellent negative controls for antibody validation

• Comparative analysis methods:

  • Normalize FURIN expression to total protein or housekeeping genes

  • Consider relative differences in subcellular distribution patterns

  • Document passage number of cell lines to account for expression drift

Researchers should validate FURIN antibody performance in each cell system rather than assuming transferability of protocols between established lines and primary cells.

What advanced techniques can researchers employ to study FURIN-substrate interactions?

Understanding FURIN's interactions with substrates requires sophisticated methodological approaches:

• Proximity Ligation Assay (PLA):

  • Combines FURIN antibody with substrate-specific antibody

  • Generates fluorescent signals only when proteins are within 40nm

  • Provides spatial information about interactions within cellular compartments

  • Quantifiable by counting interaction spots per cell

• FRET/FLIM analysis:

  • Requires fluorophore-conjugated antibodies or fusion proteins

  • Detects direct molecular interactions (1-10nm distance)

  • Can provide real-time interaction information in living cells

  • Particularly useful for studying dynamic FURIN-substrate processing events

• Co-immunoprecipitation strategies:

  • Use FURIN antibodies validated for immunoprecipitation

  • Can be coupled with mass spectrometry for unbiased substrate identification

  • Requires careful optimization of lysis conditions to preserve interactions

• Activity-based probes with immunostaining:

  • Biotinylated or fluorescent inhibitors that bind active FURIN

  • Combine with substrate antibodies to correlate enzyme activity with substrate processing

  • Provides functional information beyond simple co-localization

• Engineered reporter substrates:

  • Fluorogenic or luminescent substrates containing FURIN cleavage sites

  • Can be targeted to specific cellular compartments

  • Allows real-time monitoring of FURIN activity in living cells

These advanced techniques move beyond simple detection to provide mechanistic insights into FURIN's proteolytic activity and substrate specificity.

How are engineered FURIN antibodies being developed as potential therapeutic agents?

The development of engineered FURIN antibodies as therapeutic agents represents an emerging frontier:

• Therapeutic design strategies:

  • Antibodies like FuG1 incorporate functional peptides that interfere with FURIN enzymatic activity

  • Bi-specific antibodies targeting both FURIN and disease-relevant substrates

  • Antibody-drug conjugates delivering inhibitors specifically to FURIN-expressing cells

• Viral infection applications:

  • The FuG1 antibody demonstrates proof-of-concept for targeting FURIN to disrupt SARS-CoV-2 transmission

  • Similar approaches could target other FURIN-dependent viruses (influenza, HIV)

  • Combination therapies with direct antiviral agents show potential for synergistic effects

• Cancer therapeutic potential:

  • FURIN overexpression in multiple cancer types presents targeting opportunities

  • Inhibitory antibodies could block processing of cancer-promoting growth factors

  • Tumor microenvironment modulation through FURIN pathway interference

• Evaluation metrics:

  • Efficacy at blocking substrate processing in cellular and animal models

  • Specificity for FURIN versus related proprotein convertases

  • Safety profile considering FURIN's roles in normal physiology

The FuG1 antibody exemplifies this approach, showing how a rationally designed antibody can specifically disrupt FURIN's role in viral infection cycles while maintaining target specificity .

What are the key considerations when developing and validating novel FURIN antibodies for specialized research applications?

Developing specialized FURIN antibodies requires rigorous characterization:

• Target epitope selection:

  • Catalytic domain targeting for functional inhibition

  • Regulatory domain targeting for activation-state specific detection

  • Species-conserved regions for cross-species applications

  • Unique regions for isoform-specific detection

• Comprehensive validation approach:

  • Binding kinetics measurement via Surface Plasmon Resonance

  • Epitope mapping using peptide arrays or hydrogen-deuterium exchange MS

  • Cross-reactivity testing against related proprotein convertases

  • Functional validation in cell-based assays

• Application-specific optimization:

  • Buffer compatibility with live-cell imaging

  • Thermal stability for challenging applications

  • Conjugation chemistry for specialized detection methods

  • Fragment generation (Fab, scFv) for improved tissue penetration

• Documentation requirements:

  • Detailed validation protocols and positive/negative controls

  • Application-specific performance characteristics

  • Batch-to-batch consistency metrics

  • Sharing of validation data with research community

The scientific community benefits from transparent reporting of antibody development and validation, ensuring reproducibility and accelerating research progress.

How might multi-omics approaches complement FURIN antibody-based research?

Integrating antibody-based FURIN research with multi-omics approaches provides comprehensive insights:

• Integrative proteomics approaches:

  • Combine FURIN immunoprecipitation with mass spectrometry to identify interaction partners

  • Correlate FURIN protein levels (antibody-detected) with global proteome changes

  • Use quantitative proteomics to map FURIN-dependent processing events

• Transcriptomics integration:

  • Align antibody-detected FURIN protein expression with mRNA expression profiles

  • Identify discordant protein-mRNA relationships suggesting post-transcriptional regulation

  • Discover co-regulated genes in FURIN-high versus FURIN-low populations

• Functional genomics connections:

  • Correlate CRISPR-based FURIN perturbations with antibody-detected phenotypes

  • Link genetic variants affecting FURIN expression to protein-level changes

  • Use genetic screens to identify modifiers of FURIN function

• Spatial multi-omics:

  • Combine FURIN immunohistochemistry with spatial transcriptomics

  • Map FURIN protein distribution in tissue context alongside substrate expression

  • Correlate FURIN localization with local proteome composition

These integrated approaches transform FURIN antibodies from simple detection tools into components of comprehensive biological understanding, connecting protein expression and localization to broader cellular processes and disease mechanisms.