LRRFIP2 Antibody

What is LRRFIP2 and why is it important in cellular signaling research?

LRRFIP2 (Leucine-rich repeat flightless-interacting protein 2) is a protein with a canonical length of 721 amino acid residues and a mass of 82.2 kDa in humans. It functions as an activator of the canonical Wnt signaling pathway, acting in association with DVL3 upstream of CTNNB1/beta-catenin. LRRFIP2 is widely expressed throughout the body, with highest expression levels observed in heart and skeletal muscle tissues . Recent research has also identified LRRFIP2 as a negative regulator of the NLRP3 inflammasome, indicating its importance in regulating inflammatory responses . This dual functionality makes LRRFIP2 a significant target for researchers studying both developmental biology through Wnt signaling and inflammatory disease mechanisms through inflammasome regulation.

What species reactivity can be expected from commercially available LRRFIP2 antibodies?

Commercial LRRFIP2 antibodies demonstrate variable species reactivity profiles depending on the specific product. Based on available data, many LRRFIP2 antibodies show reactivity toward human (Homo sapiens) samples, with some cross-reacting with mouse (Ms) and rat (Rt) LRRFIP2 . This cross-reactivity is supported by the evolutionary conservation of LRRFIP2, with orthologs identified in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken . When selecting an antibody for experiments involving non-human species, researchers should carefully verify the documented species reactivity and consider performing validation experiments to confirm cross-reactivity in their specific experimental system.

What are the primary applications for LRRFIP2 antibodies in research?

LRRFIP2 antibodies are utilized across several experimental techniques, with Western Blot being the most common application. Additional validated applications include:

• Western Blotting (WB): For detecting LRRFIP2 protein expression levels and molecular weight

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of LRRFIP2

• Immunohistochemistry (IHC): For visualization of LRRFIP2 in tissue sections

• Immunocytochemistry (ICC): For cellular localization studies

• Immunofluorescence (IF): For subcellular localization and co-localization studies

• Dot Blotting (DB): For rapid screening of samples

Working dilutions vary by application and specific antibody product, typically ranging from 1:100 for immunocytochemistry to 1:1,000 for Western blotting applications . Researchers should optimize dilutions for their specific experimental conditions.

How can LRRFIP2 antibodies be used to investigate NLRP3 inflammasome regulation?

LRRFIP2 has been identified as a negative regulator of NLRP3 inflammasome activation through co-immunoprecipitation and mass spectrometry studies. To investigate this regulatory mechanism, researchers can employ the following approach:

• Co-immunoprecipitation protocol: Use anti-NLRP3 antibody with total cell lysates of macrophages primed with lipopolysaccharide (LPS) and then stimulated with ATP to identify LRRFIP2 as an interacting partner .

• Temporal interaction analysis: LRRFIP2 co-precipitates with NLRP3 approximately 10 minutes after ATP stimulation or 30 minutes after Nigericin stimulation, suggesting stimulus-specific timing for this interaction .

• Domain mapping: N-terminal and coil motifs of LRRFIP2 are required for its inhibitory effect on NLRP3 inflammasome activation. Researchers can use truncated mutants of LRRFIP2 to identify which domains are necessary for its regulatory function .

• Functional validation: Silencing LRRFIP2 in macrophages enhances NLRP3 inflammasome-mediated caspase-1 cleavage and IL-1β production, while overexpression of LRRFIP2 reduces these inflammatory outputs .

This methodological approach allows researchers to thoroughly investigate LRRFIP2's role in regulating inflammatory responses mediated by the NLRP3 inflammasome.

What experimental design is optimal for investigating LRRFIP2's role in the Wnt signaling pathway?

To investigate LRRFIP2's role as an activator of the canonical Wnt signaling pathway, researchers can implement the following experimental design:

• Expression analysis: Use LRRFIP2 antibodies for Western blotting to determine basal expression levels across various cell types with known Wnt pathway activity.

• Gain/loss-of-function experiments:

  • Overexpress LRRFIP2 using expression vectors and confirm using LRRFIP2 antibodies via Western blot

  • Silence LRRFIP2 using siRNA technology and verify knockdown efficiency

• Pathway activation assessment:

  • Quantify β-catenin nuclear translocation using nuclear/cytoplasmic fractionation followed by Western blotting

  • Measure Wnt target gene expression by qRT-PCR

  • Employ TOP/FOP flash reporter assays to measure TCF/LEF-dependent transcription

• Protein interaction studies:

  • Perform co-immunoprecipitation with LRRFIP2 antibodies to detect interaction with DVL3

  • Use proximity ligation assays to visualize LRRFIP2-DVL3 interactions in situ

• Domain mapping: Create truncated constructs of LRRFIP2 to identify which domains are essential for Wnt pathway activation, similar to approaches used in inflammasome studies .

This comprehensive experimental approach enables researchers to establish both the necessity and sufficiency of LRRFIP2 in Wnt signaling activation and define its precise mechanistic role in the pathway.

How can LRRFIP2 antibodies be utilized in studies of inflammatory disease models?

For investigating LRRFIP2's role in inflammatory disease models, researchers can employ the following methodological approach:

• In vivo silencing: Utilize siRNA technology to silence LRRFIP2 expression in mouse models. This has been demonstrated to enhance NLRP3 inflammasome activation and increase inflammatory cell recruitment in peritonitis models .

• Tissue analysis protocol:

  • Collect peritoneal exudate cells (PECs) after inflammatory challenge

  • Perform flow cytometry to quantify total inflammatory cell recruitment

  • Specifically analyze neutrophil and Ly6C+ monocyte populations

  • Use LRRFIP2 antibodies for immunohistochemistry to assess expression patterns in inflamed tissues

• Cytokine profiling:

  • Measure IL-1β production using ELISA in tissue samples and culture supernatants

  • Assess other inflammatory cytokines to understand broader effects

• Cellular mechanism studies:

  • Use immunofluorescence with LRRFIP2 antibodies to track protein localization during inflammation

  • Perform co-immunoprecipitation studies to identify LRRFIP2 interactions with Flightless-I and NLRP3 in disease contexts

This methodological framework allows researchers to establish the functional significance of LRRFIP2 in inflammatory disease processes while providing mechanistic insights into its regulatory role.

What are the optimal protocols for Western blot detection of LRRFIP2?

For optimal Western blot detection of LRRFIP2, researchers should consider the following protocol guidelines:

• Sample preparation:

  • Cell lysis buffer: RIPA buffer supplemented with protease inhibitors

  • Protein concentration: 20-40 μg total protein per lane

  • Denaturation: 95°C for 5 minutes in Laemmli buffer with β-mercaptoethanol

• Gel electrophoresis parameters:

  • Gel percentage: 8-10% SDS-PAGE (due to LRRFIP2's 82.2 kDa size)

  • Running conditions: 100V through stacking gel, 120V through resolving gel

• Transfer conditions:

  • Wet transfer recommended: 100V for 90 minutes or 30V overnight at 4°C

  • PVDF membrane preferred over nitrocellulose for higher sensitivity

• Antibody incubation:

  • Blocking: 5% non-fat milk in TBST, 1 hour at room temperature

  • Primary antibody: 1:1,000 dilution in 5% BSA in TBST, overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5,000 dilution, 1 hour at room temperature

• Detection system:

  • Enhanced chemiluminescence (ECL) substrate

  • Expected band size: 82.2 kDa for canonical isoform, with potential detection of up to 5 different isoforms

• Validation controls:

  • Positive control: A431 cell lysate has been validated for LRRFIP2 detection

  • Loading control: β-actin or GAPDH antibodies

This optimized protocol significantly enhances detection specificity and sensitivity for LRRFIP2 in Western blotting applications.

What considerations are important when using LRRFIP2 antibodies for immunohistochemistry and immunofluorescence?

When performing immunohistochemistry (IHC) or immunofluorescence (IF) with LRRFIP2 antibodies, researchers should consider these methodological aspects:

• Fixation method comparison:

• Antigen retrieval considerations:

  • Heat-induced epitope retrieval (HIER): Citrate buffer (pH 6.0) for 20 minutes

  • Enzymatic retrieval: Consider proteinase K treatment if HIER is ineffective

• Antibody optimization:

  • Dilution range: Start with 1:100 for IHC/IF applications

  • Incubation conditions: Overnight at 4°C for maximum sensitivity

  • Secondary antibody selection: Use highly cross-adsorbed secondary antibodies to reduce background

• Signal detection systems:

  • IHC: DAB (3,3'-diaminobenzidine) substrate for permanent staining

  • IF: Alexa Fluor conjugates (488, 555, 647) for fluorescence detection

• Counterstaining options:

  • IHC: Hematoxylin for nuclear visualization

  • IF: DAPI for nuclear counterstaining, phalloidin for F-actin visualization

• Controls:

  • Negative controls: Primary antibody omission and isotype controls

  • Positive controls: Heart or skeletal muscle tissue (high LRRFIP2 expression)

  • Blocking peptide controls: Pre-incubation with immunizing peptide

These methodological considerations ensure optimal detection of LRRFIP2 in tissue and cellular samples while minimizing background and non-specific staining.

What are the technical considerations for co-immunoprecipitation experiments with LRRFIP2 antibodies?

For successful co-immunoprecipitation (co-IP) experiments involving LRRFIP2, researchers should consider these technical aspects:

• Lysis buffer optimization:

  • For protein-protein interactions: Non-denaturing buffers (e.g., 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8.0)

  • Protease inhibitor cocktail: Essential to prevent degradation

  • Phosphatase inhibitors: Important when studying signaling pathways

• Pre-clearing protocol:

  • Incubate lysates with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation before adding the specific antibody

• Antibody amounts and ratios:

  • LRRFIP2 immunoprecipitation: 2-5 μg antibody per 500 μg total protein

  • For co-IP with NLRP3: Similar amounts to maintain stoichiometric balance

• Incubation conditions:

  • Antibody binding: 2-4 hours or overnight at 4°C with gentle rotation

  • Protein A/G bead binding: 1-2 hours at 4°C with gentle rotation

• Washing stringency optimization:

  • Less stringent: PBS with 0.1% Tween-20

  • More stringent: Increasing salt concentration (150-500 mM NaCl)

  • Number of washes: 3-5 times, 5 minutes each

• Elution methods:

  • Denaturing: SDS sample buffer at 95°C for 5 minutes

  • Non-denaturing: Glycine buffer (pH 2.5) followed by neutralization

• Detection strategy:

  • Western blot using antibodies against suspected interaction partners

  • Mass spectrometry for unbiased identification of novel interactors

• Timing considerations:

  • For NLRP3 interaction: Perform co-IP 10 minutes after ATP stimulation or 30 minutes after Nigericin stimulation

This detailed co-IP protocol enhances the detection of physiologically relevant LRRFIP2 protein interactions while minimizing non-specific binding.

What are common issues in Western blot detection of LRRFIP2 and how can they be resolved?

Researchers often encounter these challenges when detecting LRRFIP2 by Western blot:

When facing persistent issues, researchers may consider:

• Using recombinant LRRFIP2 protein as a positive control

• Comparing results with different LRRFIP2 antibody clones

• Validating with knockdown/knockout samples if available

• Optimizing transfer conditions for proteins >80 kDa

These troubleshooting approaches address the most common technical challenges in LRRFIP2 Western blot detection.

How can researchers address specificity concerns when using LRRFIP2 antibodies?

To address specificity concerns with LRRFIP2 antibodies, researchers should implement the following validation strategies:

• Genetic validation approaches:

  • siRNA/shRNA knockdown: Compare antibody signal in control vs. LRRFIP2-depleted samples

  • CRISPR/Cas9 knockout: Generate complete LRRFIP2 knockout for definitive negative control

  • Overexpression: Transfect cells with LRRFIP2 expression constructs to confirm signal increase

• Epitope competition assays:

  • Pre-incubate antibody with immunizing peptide (if available)

  • Compare signal with and without peptide competition

  • A specific antibody should show significant signal reduction after peptide competition

• Multiple antibody validation:

  • Use antibodies targeting different LRRFIP2 epitopes

  • Compare staining/binding patterns across antibodies

  • Consistent patterns across antibodies suggest specificity

• Cross-reactivity testing:

  • Test antibody in species with known LRRFIP2 homology

  • Perform Western blot on lysates from various species

  • Results should align with expected cross-reactivity based on epitope conservation

• Biophysical validation:

  • Perform affinity binding assays (as demonstrated with one antibody showing KD of 5.4 x 10-6)

  • Consider surface plasmon resonance for precise affinity measurements

• Application-specific controls:

  • For ICC/IF: Include secondary-only controls

  • For IHC: Use isotype controls at matching concentrations

  • For IP: Include IgG control immunoprecipitations

These comprehensive validation approaches ensure that experimental observations genuinely reflect LRRFIP2 biology rather than antibody artifacts.

How can conflicting results between different LRRFIP2 antibodies be reconciled?

When researchers encounter conflicting results between different LRRFIP2 antibodies, these systematic approaches can help reconcile discrepancies:

• Epitope mapping analysis:

  • Determine the exact epitopes recognized by each antibody

  • Epitopes in highly conserved regions may yield more consistent results

  • Epitopes in regions subject to alternative splicing may detect different isoforms

• Isoform-specific detection:

  • LRRFIP2 has up to 5 reported isoforms

  • Different antibodies may preferentially detect specific isoforms

  • Use RT-PCR to determine which isoforms are expressed in your experimental system

• Post-translational modification interference:

  • Some epitopes may be masked by phosphorylation, glycosylation, or other modifications

  • Treatment with phosphatases or glycosidases can reveal if modifications affect antibody binding

  • Consider antibodies specifically targeting modified forms if relevant

• Context-dependent expression:

  • LPS treatment significantly upregulates LRRFIP2 expression after 8 hours

  • Different cell activation states may result in variable detection

  • Standardize cell treatment conditions when comparing antibodies

• Methodological validation:

  • Perform side-by-side comparisons using identical protocols

  • Systematically vary antibody concentrations to determine optimal working ranges

  • Consider cell/tissue-specific optimization

• Reconciliation strategy:

  • For multiple positive signals: Accept overlapping patterns as likely true positives

  • For contradictory signals: Prioritize antibodies with genetic validation

  • Consider implementing orthogonal detection methods (e.g., mass spectrometry)

This systematic approach helps researchers determine which antibody is most appropriate for their specific experimental context and biological question.

What emerging applications of LRRFIP2 antibodies are being developed for inflammation research?

Several emerging applications of LRRFIP2 antibodies show promise for advancing inflammation research:

• In vivo imaging applications:

  • Development of near-infrared fluorophore-conjugated LRRFIP2 antibodies for non-invasive imaging

  • Tracking LRRFIP2 expression dynamics during inflammatory disease progression

  • Potential for monitoring therapeutic efficacy in real-time

• Single-cell analysis techniques:

  • Adapting LRRFIP2 antibodies for mass cytometry (CyTOF) applications

  • Single-cell Western blotting to detect LRRFIP2 in rare cell populations

  • Incorporating LRRFIP2 into multiparameter flow cytometry panels for immune cell profiling

• Spatial transcriptomics integration:

  • Combining LRRFIP2 antibody staining with spatial transcriptomics

  • Correlating protein expression with localized gene expression patterns

  • Creating tissue atlases of LRRFIP2 expression in inflammatory diseases

• Therapeutic target validation:

  • Using LRRFIP2 antibodies to validate small molecule modulators of LRRFIP2 function

  • Developing blocking antibodies to manipulate LRRFIP2-mediated inflammasome regulation

  • Screening for compounds that enhance LRRFIP2's anti-inflammatory properties

• Biomarker development:

  • Investigating LRRFIP2 expression patterns in inflammatory diseases

  • Correlating LRRFIP2 levels with disease severity and therapeutic responses

  • Developing standardized assays for clinical research applications

These emerging applications leverage LRRFIP2's role as a negative regulator of NLRP3 inflammasome activation , potentially opening new avenues for therapeutic intervention in inflammatory diseases.

How might LRRFIP2 antibodies contribute to understanding cross-talk between Wnt signaling and inflammation pathways?

LRRFIP2 antibodies can provide unique insights into the cross-talk between Wnt signaling and inflammation pathways through these methodological approaches:

• Dual pathway visualization:

  • Multiplex immunofluorescence combining LRRFIP2 with both Wnt pathway markers (β-catenin, TCF/LEF) and inflammasome components (NLRP3, ASC)

  • Super-resolution microscopy to visualize nanoscale protein interactions

  • Live-cell imaging to track dynamic interactions during pathway activation

• Protein complex analysis:

  • Sequential co-immunoprecipitation to identify multi-protein complexes

  • Blue native PAGE to preserve native protein complexes for Western blot analysis

  • Proximity-dependent labeling approaches (BioID, APEX) with LRRFIP2 as bait

• Cellular response mapping:

  • Stimulate cells with both Wnt ligands and inflammasome activators

  • Use LRRFIP2 antibodies to track redistribution and modification states

  • Perform time-course analyses to determine sequence of events in dual pathway activation

• Context-dependent interactions:

  • Compare LRRFIP2 interaction partners in different cell types (epithelial vs. immune cells)

  • Analyze interaction networks under homeostatic vs. inflammatory conditions

  • Investigate tissue-specific LRRFIP2 functions in models of inflammatory disease

• Mechanistic dissection:

  • Use domain-specific LRRFIP2 antibodies to determine which regions mediate Wnt vs. inflammasome interactions

  • Employ conformation-specific antibodies to detect activation states

  • Develop phospho-specific antibodies if regulatory phosphorylation sites are identified

These approaches can help elucidate how LRRFIP2 might integrate or segregate signals between developmental Wnt pathways and inflammatory response pathways, potentially revealing new therapeutic targets at this intersection.

What methodologies are being developed to study LRRFIP2 in the context of macrophage polarization and function?

Emerging methodologies for studying LRRFIP2 in macrophage polarization and function include:

• Single-cell analysis protocols:

  • Single-cell RNA-seq combined with LRRFIP2 protein detection (CITE-seq)

  • Mass cytometry (CyTOF) panels incorporating LRRFIP2 antibodies

  • Correlation of LRRFIP2 levels with macrophage polarization markers (M1 vs. M2)

• Advanced imaging techniques:

  • Lattice light-sheet microscopy for long-term tracking of LRRFIP2 dynamics during polarization

  • Multi-color STORM or PALM super-resolution imaging to visualize nanoscale protein complexes

  • Correlative light and electron microscopy (CLEM) to connect LRRFIP2 localization with ultrastructural features

• Functional genomics approaches:

  • CRISPR-Cas9 screening to identify genes that modify LRRFIP2 function in macrophages

  • CRISPRi/CRISPRa for tunable modulation of LRRFIP2 expression

  • CRISPR base editing to introduce specific mutations for structure-function studies

• Macrophage-specific in vivo models:

  • Development of macrophage-specific LRRFIP2 conditional knockout mice

  • Adoptive transfer studies with LRRFIP2-modified macrophages

  • Tissue-specific deletion to examine organ-specific functions

• Secretome analysis:

  • Quantitative proteomics of macrophage secretomes after LRRFIP2 manipulation

  • Cytokine profiling comparing wild-type and LRRFIP2-deficient macrophages

  • Extracellular vesicle characterization to identify LRRFIP2-dependent secretory pathways

These methodologies will provide deeper insights into how LRRFIP2 influences macrophage polarization states and effector functions, particularly in the context of its role as a negative regulator of NLRP3 inflammasome activation .