FCHO1 Antibody, HRP conjugated

What is FCHO1 and why is it significant for immunological research?

FCHO1 is a modular protein that functions in the early stages of clathrin-mediated endocytosis (CME), a fundamental cellular process for internalizing molecules across cell membranes. It contains three main functional components: an N-terminal F-BAR domain responsible for membrane binding/bending, a central linker region that interacts with adaptor protein-2 (AP-2), and a C-terminal μ homology domain (μHD) that binds to endocytic proteins like EPS15 and EPS15L1 . FCHO1's significance in immunology emerged when mutations were discovered in patients with T and B cell lymphopenia, revealing its crucial role in T-cell development and function. Patient T cells with FCHO1 mutations show unresponsiveness to T cell receptor (TCR) triggering, with severely perturbed TCR internalization . These findings establish FCHO1 as a critical link between clathrin-mediated endocytosis and immune system function, making it an important target for immunological research.

What experimental techniques are compatible with HRP-conjugated FCHO1 antibodies?

HRP-conjugated FCHO1 antibodies are versatile tools compatible with multiple experimental approaches in molecular and cellular biology. These antibodies are particularly well-suited for western blotting, providing enhanced sensitivity through enzymatic signal amplification without requiring secondary antibodies. For immunohistochemistry on paraffin-embedded tissues (IHC-P), they offer direct visualization with appropriate substrates like DAB or TMB . In immunocytochemistry and immunofluorescence (ICC/IF), HRP-conjugated antibodies can be used with tyramide signal amplification systems to substantially increase detection sensitivity when studying endogenous FCHO1 localization at clathrin-coated pits . For flow cytometry applications, researchers should consider using protein transport inhibitors before cell fixation to enhance detection of intracellular FCHO1. ELISA applications benefit from the direct detection capabilities of HRP-conjugated antibodies, eliminating additional incubation steps and reducing background signal.

What are optimal sample preparation methods for FCHO1 detection in cellular studies?

For optimal FCHO1 detection in cellular studies, sample preparation methods should preserve both protein structure and spatial distribution while maximizing antibody accessibility. When working with adherent cells for ICC/IF applications, 4% paraformaldehyde fixation for 10-15 minutes at room temperature preserves FCHO1's membrane association and punctate distribution pattern . For membrane proteins interacting with FCHO1, such as clathrin or adaptor proteins, adding 0.1% glutaraldehyde can help maintain complex protein structures. Permeabilization should be performed with 0.1-0.2% Triton X-100 for 5-10 minutes, as harsher detergents may disrupt the delicate clathrin-coated structures. When preparing samples for co-localization studies, it's essential to use control staining to verify that HRP signal is not bleeding into other channels. For tissue sections, antigen retrieval using citrate buffer (pH 6.0) followed by overnight primary antibody incubation at 4°C typically yields optimal results for FCHO1 visualization in lymphoid tissues where its expression is physiologically relevant .

How can researchers validate the specificity of FCHO1 antibodies in their experimental systems?

Validation of FCHO1 antibody specificity requires a multi-faceted approach to ensure reliable experimental results. Researchers should first perform western blot analysis to confirm detection of a single band at the expected molecular weight (~90 kDa for full-length human FCHO1, with truncated versions at ~70 kDa for mutations like p.Stop687) . Knockout or knockdown controls are particularly valuable; CRISPR/Cas9-mediated FCHO1 knockout cells, as described in Lehmann et al.'s work, provide excellent negative controls to verify antibody specificity . Competitive blocking experiments using recombinant FCHO1 protein (preferably the immunogen fragment corresponding to aa 250-400) can confirm binding specificity . For additional validation, researchers can perform immunoprecipitation followed by mass spectrometry to verify that the antibody pulls down FCHO1 and its known interaction partners like EPS15 and EPS15L1. Expression of GFP-tagged FCHO1 in cells can serve as a positive control for co-localization studies, allowing comparison between antibody staining patterns and GFP signal distribution .

How can HRP-conjugated FCHO1 antibodies be used to investigate clathrin-mediated endocytosis dynamics?

HRP-conjugated FCHO1 antibodies enable sophisticated investigation of clathrin-mediated endocytosis dynamics through both temporal and spatial analyses. For live-cell imaging studies, researchers can employ HRP-conjugated FCHO1 antibodies with membrane-permeable substrates to visualize the temporal recruitment of FCHO1 to nascent clathrin-coated pits. This approach can be enhanced by combining with SK-MEL-2 cells expressing RFP-tagged clathrin light chains to simultaneously track FCHO1 and clathrin dynamics . For ultrastructural studies, the HRP moiety can be leveraged for electron microscopy via DAB precipitation, allowing precise localization of FCHO1 at different stages of clathrin-coated pit formation with nanometer resolution.

Pulse-chase experiments using cell-surface biotinylation followed by immunoprecipitation with HRP-conjugated FCHO1 antibodies can quantitatively assess endocytic rates and protein interactions during vesicle formation. To investigate the role of FCHO1 in TCR internalization specifically, researchers can combine HRP-conjugated FCHO1 antibodies with anti-CD3 stimulation in primary T cells or Jurkat cell models, using the enzymatic activity for quantitative readouts of FCHO1 recruitment to TCR signaling complexes . This multi-modal approach provides comprehensive insights into the spatial and temporal dynamics of FCHO1 during clathrin-mediated endocytosis.

What methodological approaches can resolve contradictory data regarding FCHO1's role in clathrin coat assembly?

Resolving contradictory data regarding FCHO1's role in clathrin coat assembly requires methodologically rigorous approaches that account for protein interactions, temporal dynamics, and functional redundancy. Total internal reflection fluorescence (TIRF) microscopy using cells expressing fluorescently-tagged FCHO1 variants alongside HRP-conjugated antibodies against endogenous proteins enables precise temporal resolution of protein recruitment events at the plasma membrane. This approach has revealed that FCHO1 arrives early at surface clathrin assemblies, potentially acting as a coat nucleator .

To address contradictory findings regarding FCHO1's interaction with AP-2, researchers should employ a combination of biochemical and imaging techniques. In vitro binding assays with purified components can determine direct protein interactions, while cellular studies using TALEN or CRISPR-edited cells lacking FCHO proteins (as described in search result 2) provide physiologically relevant contexts . The use of domain-specific mutations (affecting either the F-BAR domain or μHD domain) helps dissect the specific contributions of each functional region to clathrin coat assembly .

The table below summarizes key methodological approaches to resolve contradictory data regarding FCHO1 function:

How do patient-derived FCHO1 mutations affect antibody epitope recognition and experimental design?

Patient-derived FCHO1 mutations present unique considerations for antibody recognition and experimental design that researchers must carefully address. The documented mutations in FCHO1 (p.R679P, p.A34P, p.Stop687) affect different domains and produce structurally altered proteins that may impact epitope accessibility . For antibodies targeting regions within amino acids 250-400, such as the commercial antibody mentioned in the search results, epitope recognition should remain intact for most patient mutations as they affect either the F-BAR domain (p.A34P) or the μHD domain (p.R679P, p.Stop687) .

When designing experiments involving patient-derived mutations, researchers should consider using multiple antibodies targeting different epitopes to ensure comprehensive detection. For truncation mutations like p.Stop687, C-terminal targeting antibodies would fail to detect the protein, while N-terminal antibodies would recognize the shortened form. Western blotting can verify this differential recognition pattern, as demonstrated by Lehmann et al., who observed a shorter protein product with the p.Stop687 mutation .

Immunoprecipitation experiments require special attention when working with mutated FCHO1, as the mutations may disrupt protein-protein interactions. For instance, co-immunoprecipitation assays showed that μHD domain mutants (p.R679P and p.Stop687) exhibited reduced interaction with EPS15 and EPS15R, while the F-BAR domain mutant (p.A34P) maintained these interactions . These findings inform experimental design by highlighting the need for appropriate positive controls (wild-type FCHO1) and careful interpretation of negative results, which may reflect either antibody failure or genuine biological effects of the mutations.

What approach should be taken to investigate FCHO1's role in T-cell receptor trafficking using HRP-conjugated antibodies?

Investigating FCHO1's role in T-cell receptor (TCR) trafficking using HRP-conjugated antibodies requires a sophisticated experimental approach that integrates biochemical, microscopic, and functional analyses. Researchers should begin with Jurkat T cells or primary human T lymphocytes as experimental models, as FCHO1 has been specifically implicated in TCR internalization in these cell types . For biochemical trafficking studies, cells can be surface-biotinylated and stimulated with anti-CD3/CD28 antibodies to trigger TCR internalization, followed by immunoprecipitation with HRP-conjugated FCHO1 antibodies at various time points to capture and analyze the dynamic protein complexes formed during endocytosis.

For imaging studies, dual-label experiments using HRP-conjugated FCHO1 antibodies (with appropriate substrates) and fluorescently-labeled TCR components can reveal colocalization patterns during receptor triggering. Super-resolution microscopy techniques such as STORM or PALM would provide the necessary spatial resolution to distinguish between closely associated proteins at clathrin-coated pits. To directly assess FCHO1's functional role, CRISPR/Cas9-mediated knockout of FCHO1 in T cells followed by rescue experiments with wild-type or mutant FCHO1 constructs would establish causality in TCR trafficking defects.

The key scientific control in these experiments is the comparison between unstimulated and TCR-stimulated conditions, as FCHO1's involvement may be specific to activation-induced TCR internalization. Additionally, comparing FCHO1-deficient patient-derived T cells with those from healthy donors provides physiologically relevant validation of experimental findings . This comprehensive approach would effectively delineate FCHO1's specific contributions to TCR trafficking while distinguishing them from its general endocytic functions.

How can researchers quantitatively assess FCHO1's membrane remodeling activity using antibody-based approaches?

Quantitative assessment of FCHO1's membrane remodeling activity using antibody-based approaches requires sophisticated methodologies that can detect both structural changes in membranes and FCHO1's spatial distribution. Researchers can employ a combination of in vitro and cellular systems to comprehensively evaluate this activity. In vitro liposome-based assays using purified FCHO1 protein (wild-type or mutant variants) in conjunction with PtdIns(4,5)P2-containing liposomes can demonstrate direct membrane binding and tubulation capabilities . The membrane remodeling can be quantified using electron microscopy to measure tubule diameter and frequency, while immunogold labeling with HRP-conjugated FCHO1 antibodies can verify protein localization along the tubules.

For cellular studies, quantitative immunofluorescence microscopy using HRP-conjugated FCHO1 antibodies with tyramide signal amplification provides sufficient sensitivity to detect endogenous FCHO1 at membrane invaginations. This approach can be coupled with membrane-specific dyes or markers to measure parameters such as membrane curvature, invagination depth, and FCHO1 density at these sites. High-content imaging platforms enable automated quantification across thousands of cells, providing robust statistical analysis of membrane remodeling events.

To establish functional relevance, researchers should conduct comparative studies between wild-type FCHO1 and F-BAR domain mutants like p.A34P, which form large plasma membrane-dissociated agglomerations rather than the scattered bright puncta associated with normal membrane binding . This approach directly links structural observations to functional outcomes in clathrin-coated pit formation. Statistical analysis should include measurements of puncta size, density, membrane association, and colocalization with known endocytic markers to comprehensively assess FCHO1's membrane remodeling activity in physiologically relevant contexts.

What are the critical parameters for optimizing HRP-conjugated FCHO1 antibody signal-to-noise ratio?

Optimizing signal-to-noise ratio for HRP-conjugated FCHO1 antibodies requires careful attention to several critical parameters throughout the experimental workflow. Fixation conditions significantly impact epitope preservation and accessibility; for FCHO1 detection, 4% paraformaldehyde for 10-15 minutes represents an optimal balance between structural preservation and antibody penetration. Over-fixation, particularly with glutaraldehyde, can mask epitopes and reduce specific signal . Blocking solutions should contain both protein components (3-5% BSA or normal serum) and detergents (0.1% Triton X-100) to minimize non-specific binding while maintaining membrane structure.

Antibody dilution optimization is essential; while manufacturer recommendations provide starting points (typically 1:100-1:500 for ICC/IF applications), titration experiments should be performed for each specific application and cell type . For HRP-conjugated antibodies, substrate development timing is crucial - excessive development increases background noise while insufficient development reduces detection sensitivity. When working with tissues or cells expressing low levels of FCHO1, tyramide signal amplification can enhance sensitivity without proportionally increasing background.

Temperature and incubation time also significantly impact performance; while room temperature incubations (1-2 hours) work well for many applications, overnight incubation at 4°C often improves signal specificity by favoring high-affinity binding interactions. For challenging samples, antigen retrieval methods such as citrate buffer (pH 6.0) heat treatment can dramatically improve signal detection by unmasking epitopes without increasing background staining. These parameters should be systematically optimized for each experimental system to achieve maximum signal-to-noise ratio.

How should researchers address potential cross-reactivity between FCHO1 and FCHO2 in experimental systems?

Addressing potential cross-reactivity between FCHO1 and FCHO2 requires rigorous validation strategies due to their structural similarities, with the N-terminal EFC domains sharing 58% sequence identity . Researchers should begin by examining the immunogen sequence used to generate their FCHO1 antibody; antibodies targeting the more divergent μHD domain or linker region (such as those corresponding to amino acids 250-400) are less likely to cross-react than those targeting the highly conserved EFC domain . Western blot analysis using recombinant FCHO1 and FCHO2 proteins side-by-side can directly assess cross-reactivity, with FCHO1 appearing at approximately 90 kDa and FCHO2 at approximately 80 kDa.

For definitive validation, CRISPR/Cas9-generated knockout cells lacking either FCHO1, FCHO2, or both proteins provide ideal systems to test antibody specificity . Immunoprecipitation followed by mass spectrometry analysis can identify whether an antibody pulls down both proteins or exclusively FCHO1. In imaging applications, co-staining with independently validated antibodies against each protein can reveal distinct or overlapping localization patterns.

When cross-reactivity cannot be eliminated, researchers should employ RNA interference to selectively deplete either FCHO1 or FCHO2 and assess changes in antibody signal. Alternatively, expression of tagged versions of each protein enables selective identification. For quantitative applications where absolute specificity is required, researchers may need to develop custom antibodies against uniquely divergent epitopes or employ genetic tagging strategies to circumvent the cross-reactivity issue entirely.

How can FCHO1 antibodies be used to investigate immunodeficiency disorders associated with FCHO1 mutations?

FCHO1 antibodies provide powerful tools for investigating immunodeficiency disorders associated with FCHO1 mutations through multiple experimental approaches. For diagnostic applications, immunohistochemistry using HRP-conjugated FCHO1 antibodies can assess protein expression patterns in lymphoid tissues from patients with suspected FCHO1 deficiency . This approach can reveal altered expression levels, subcellular localization, or complete absence of protein, particularly in T and B cell populations within lymphoid organs. Flow cytometry with permeabilized peripheral blood mononuclear cells (PBMCs) enables quantitative analysis of FCHO1 expression across different immune cell subsets, helping to correlate genotype with cell-type-specific phenotypes.

For functional studies, patient-derived T cells can be analyzed using HRP-conjugated FCHO1 antibodies in conjunction with TCR stimulation assays to assess FCHO1 recruitment to the immunological synapse and subsequent TCR internalization . Biochemical approaches such as immunoprecipitation followed by mass spectrometry can identify altered protein interaction networks in cells expressing mutant FCHO1 variants, potentially revealing compensatory mechanisms or disrupted signaling pathways.

The table below summarizes key disease-associated FCHO1 mutations and their immunological consequences, providing a framework for antibody-based investigations:

What methodological approaches can determine FCHO1's role in regulating T-cell development?

Determining FCHO1's role in T-cell development requires integrated methodological approaches spanning from molecular analyses to functional immunology. Researchers should begin with expression profiling using HRP-conjugated FCHO1 antibodies to characterize protein levels across T-cell developmental stages in thymic tissue sections or sorted thymocyte populations (double-negative, double-positive, and single-positive cells). This provides crucial information about stage-specific expression patterns that may correlate with developmental checkpoints .

In vitro T-cell development assays using OP9-DL1 co-culture systems offer controlled environments to study the impact of FCHO1 manipulations. Here, CRISPR/Cas9-mediated knockout of FCHO1 in hematopoietic progenitors, followed by tracking of T-cell developmental progression, can reveal specific blockades. These genetic approaches can be complemented by pharmacological inhibition of clathrin-mediated endocytosis, which has been shown to cause marked delays in T-cell differentiation .

For mechanistic insights, researchers should investigate how FCHO1 affects receptor internalization for key developmental signals like Notch, IL-7R, and pre-TCR using flow cytometry and imaging approaches. Patient-derived cells from individuals with FCHO1 mutations provide physiologically relevant models to validate findings from experimental systems . Rescue experiments introducing wild-type or structure-guided mutant FCHO1 variants into FCHO1-deficient cells can establish causal relationships between specific protein functions and developmental phenotypes. This comprehensive approach enables robust determination of FCHO1's precise contributions to T-cell development while accounting for potential compensatory mechanisms.

What emerging technologies might enhance FCHO1 antibody applications in future research?

Emerging technologies promise to significantly enhance FCHO1 antibody applications in future research through improvements in specificity, sensitivity, and functional analysis capabilities. Nanobody and single-domain antibody technologies offer smaller binding molecules with superior tissue penetration and reduced background compared to conventional antibodies, potentially improving detection of FCHO1 at clathrin-coated pits. These smaller antibody formats, when conjugated with HRP, would provide higher spatial resolution for ultrastructural studies of endocytic processes.

CRISPR-based protein tagging systems, such as CRISPR-APEX, could enable endogenous FCHO1 visualization without exogenous antibodies by inserting enzyme tags that generate electron-dense precipitates visible by electron microscopy. This approach would circumvent potential issues with antibody specificity while maintaining physiological expression levels. Proximity labeling techniques like BioID or TurboID fused to FCHO1 could identify novel interaction partners involved in clathrin-mediated endocytosis and T-cell receptor trafficking networks.

Advanced imaging technologies, particularly lattice light-sheet microscopy combined with adaptive optics, would enable long-term live-cell imaging of FCHO1 dynamics with minimal phototoxicity. This could reveal previously unobservable temporal patterns in FCHO1 recruitment during endocytosis. For structural studies, cryo-electron tomography with gold-conjugated FCHO1 antibodies could visualize FCHO1's molecular organization within native cellular environments at near-atomic resolution. These technological advances collectively promise to deepen our understanding of FCHO1's precise roles in membrane trafficking and immune cell function, potentially revealing new therapeutic targets for immunodeficiency disorders associated with FCHO1 mutations.

How might understanding FCHO1's role in clathrin-mediated endocytosis inform potential therapeutic approaches for associated immunodeficiencies?

Understanding FCHO1's role in clathrin-mediated endocytosis could inform several potential therapeutic approaches for associated immunodeficiencies through targeted interventions at multiple levels. Gene therapy approaches targeting FCHO1 mutations represent the most direct strategy; viral vector-mediated delivery of functional FCHO1 to hematopoietic stem cells could potentially restore normal T-cell development and function in patients with genetic deficiencies . The identification of specific mutations affecting distinct protein domains (F-BAR vs. μHD) enables precision medicine approaches tailored to particular molecular defects.

Small molecule modulators of clathrin-mediated endocytosis could provide pharmacological alternatives to genetic approaches. Compounds that enhance the efficiency of residual FCHO1 activity or activate compensatory endocytic pathways might partially restore T-cell receptor internalization and signaling. Structure-based drug design targeting the interaction between FCHO1 and key binding partners like EPS15 or AP-2 could yield molecules that stabilize these interactions even in the presence of destabilizing mutations .