FCHO1 Antibody, Biotin conjugated

What is FCHO1 and why is it important in immunological research?

FCHO1 is a protein involved in the early stages of clathrin-mediated endocytosis, a major pathway by which eukaryotic cells internalize cell-surface cargo proteins and extracellular molecules. It contains an N-terminal F-BAR domain that binds to phosphatidylinositol 4,5-biphosphate on the inner side of the cell membrane, inducing and stabilizing membrane curvature . The protein is critically important in immunological research because mutations in FCHO1 have been identified as a novel cause of combined immune deficiency in humans, characterized by T and B cell lymphopenia . FCHO1 deficiency results in impaired T-cell proliferation, increased activation-induced T-cell death, and defective CME . Studying FCHO1 provides insights into fundamental mechanisms of lymphocyte development and function.

What are the typical applications of FCHO1 antibodies in immunological studies?

FCHO1 antibodies are valuable tools in several research applications:

• Protein detection: Western blotting to quantify FCHO1 expression in different cell types and patient samples

• Immunophenotyping: Flow cytometry to analyze FCHO1 expression in lymphocyte subpopulations

• Microscopy: Immunofluorescence studies to visualize FCHO1 localization during clathrin-coated pit formation

• Functional studies: Investigation of protein-protein interactions between FCHO1 and binding partners

• Patient diagnosis: Potential diagnostic tool for identifying FCHO1 deficiency in patients with combined immunodeficiency

For biotin-conjugated variants specifically, these antibodies offer enhanced detection sensitivity through streptavidin-based amplification systems and compatibility with multi-labeling experiments .

What cell types most abundantly express FCHO1?

FCHO1 shows differential expression across cell types. Quantitative PCR analysis has revealed that FCHO1 is predominantly expressed in lymphoid cells, including CD4+ and CD8+ T cells, CD19+ B cells, and CD56+ natural killer cells . In contrast, its paralog FCHO2 is more abundantly expressed in fibroblasts and non-lymphoid cell lines like K562 erythroleukemic cells . This differential expression pattern suggests specialized roles for FCHO1 in immune cell function, which explains why FCHO1 deficiency primarily manifests as an immunological disorder rather than affecting multiple organ systems.

How can I validate the specificity of an FCHO1 antibody?

To validate FCHO1 antibody specificity, employ these methodological approaches:

• Positive and negative controls:

  • Use cell lines with confirmed high (lymphoid cells) and low (fibroblasts) FCHO1 expression

  • Include FCHO1-knockout cells generated by CRISPR-Cas9 as negative controls

• Western blot validation:

  • Confirm a single band at the expected molecular weight (~80 kDa for wild-type FCHO1)

  • Test truncated FCHO1 mutants to confirm epitope specificity (as demonstrated with HA- or FLAG-tagged FCHO1 constructs)

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide before application to samples

  • Signal should be substantially reduced or eliminated

• Cross-reactivity assessment:

  • Test against related proteins, particularly FCHO2, to ensure specificity

How can biotin-conjugated FCHO1 antibodies be used to study T-cell receptor internalization?

Biotin-conjugated FCHO1 antibodies offer significant advantages in studying T-cell receptor (TCR) internalization due to their compatibility with streptavidin-based detection systems. Research has demonstrated that FCHO1 deficiency severely impairs TCR internalization, which is critical for proper T-cell function .

A methodological approach to study this phenomenon includes:

• Dual-color flow cytometry: Use biotin-conjugated FCHO1 antibody with streptavidin-fluorophore conjugates along with fluorochrome-conjugated CD3 antibodies to simultaneously track FCHO1 expression and TCR internalization.

• Chase experiments: As demonstrated in patient studies, add biotinylated transferrin followed by streptavidin-phycoerythrin (PE) to measure internalization kinetics . This approach revealed that while transferrin internalization occurs readily in control T cells, it was minimally detectable in FCHO1-deficient T cells.

• Confocal microscopy: Use biotin-conjugated FCHO1 antibodies with streptavidin-fluorophore conjugates to visualize FCHO1 localization relative to TCR complexes during internalization.

Importantly, research has shown that while TCR/CD3 internalization is impaired in FCHO1-deficient cells, this process can be rescued by expression of wild-type FCHO1, confirming the specific role of FCHO1 in this critical T-cell function .

What experimental considerations are important when using FCHO1 antibodies to differentiate FCHO1 from FCHO2 function?

Differentiation between FCHO1 and FCHO2 functions requires careful experimental design, particularly since these proteins have partially overlapping functions in clathrin-mediated endocytosis:

• Expression pattern analysis:

  • Quantitative PCR should be performed to determine the relative expression levels of FCHO1 and FCHO2 in the cell type of interest

  • FCHO1 is predominantly expressed in lymphoid cells, while FCHO2 is more abundant in fibroblasts and other cell types

• Domain-specific antibodies:

  • Select antibodies targeting non-conserved regions between FCHO1 and FCHO2

  • The linker region of FCHO1 is particularly distinctive and acts as an allosteric activator of the adaptor protein 2 complex

• Functional assays:

  • Compare transferrin receptor internalization (affected by both FCHO1 and FCHO2) with T-cell-specific functions (more dependent on FCHO1)

  • Conduct rescue experiments with selective expression of each protein in knockout models

• Co-immunoprecipitation studies:

  • Use biotin-conjugated FCHO1 antibodies with streptavidin beads to pull down FCHO1-specific protein complexes

  • Compare interacting partners with those of FCHO2 to identify unique functions

How can FCHO1 antibodies be applied in studying clathrin-coated pit formation dynamics?

FCHO1 antibodies are valuable tools for investigating the temporal and spatial dynamics of clathrin-coated pit (CCP) formation:

• Live-cell imaging:

  • Use fluorescently labeled FCHO1 antibody fragments to track FCHO1 during CCP nucleation and maturation

  • Research has demonstrated that mutations in FCHO1 lead to impaired formation of CCPs as visualized by live-cell imaging

• Super-resolution microscopy:

  • Biotin-conjugated FCHO1 antibodies used with streptavidin-conjugated quantum dots provide precise localization within forming CCPs

  • This approach allows visualization of FCHO1's role in inducing and stabilizing membrane curvature during early endocytosis

• Quantitative analysis of pit formation:

  • Track the rate and efficiency of CCP formation in normal vs. FCHO1-deficient cells

  • Assess how mutations in different domains affect CCP dynamics

• Dual-labeling experiments:

  • Combine biotin-conjugated FCHO1 antibodies with antibodies against other CCP components (clathrin, AP2) to analyze recruitment kinetics

  • Studies have shown that FCHO1 functions as an allosteric activator that converts AP2 into an open, active conformation

What are the methodological approaches for using FCHO1 antibodies in investigating combined immunodeficiency?

FCHO1 antibodies serve as essential tools in characterizing the immunological phenotype of patients with FCHO1 mutations:

• Patient lymphocyte immunophenotyping:

  • Flow cytometry panel including biotin-conjugated FCHO1 antibody alongside CD4, CD8, CD19, and activation markers

  • Patients with FCHO1 deficiency exhibit variable T and B cell lymphopenia

• Functional T-cell assays:

  • Analyze T-cell proliferation responses following TCR stimulation with anti-CD3 antibodies

  • FCHO1-deficient T cells show impaired proliferation and increased activation-induced cell death

• Endocytosis functional assays:

  • Measure transferrin receptor internalization kinetics using biotinylated transferrin and streptavidin-PE

  • Research shows significantly reduced transferrin internalization in patient T cells compared to controls

• Mutation analysis correlation:

  • Compare FCHO1 protein expression levels (detected by antibodies) with specific mutations

  • Western blotting analysis of HA- or FLAG-tagged FCHO1 constructs has revealed expression of truncated products corresponding to specific patient mutations

How can one optimize FCHO1 antibody-based assays for measuring therapeutic responses in FCHO1-deficient patients?

Optimizing FCHO1 antibody-based assays for monitoring therapeutic interventions requires methodical approaches:

• Establishing baseline measurements:

  • Quantify FCHO1 expression in patient cells pre-treatment

  • Create functional baselines of clathrin-mediated endocytosis efficiency

  • Documented cases show that allogeneic hematopoietic stem cell transplantation (HSCT) has been successful in treating FCHO1 deficiency

• Post-transplantation monitoring:

  • Monitor FCHO1 expression in lymphocytes at regular intervals post-HSCT

  • Compare with donor chimerism analysis to correlate functional recovery

  • Assess normalization of T-cell proliferation in response to stimuli

• Endocytosis functional recovery assessment:

  • Use biotinylated transferrin internalization assays to quantify recovery of CME function

  • Multiplex with T-cell activation markers to correlate with clinical improvement

• Technical optimization considerations:

  • Standardize antibody concentrations and incubation conditions (30 minutes at 4°C in dark conditions has been used successfully)

  • Include appropriate controls from both healthy donors and pre-treatment samples

  • Perform wash steps with PBS twice to ensure specific binding