FKBP62 Antibody

What are FKBP proteins and why are they important in research?

FK506-binding proteins (FKBPs) belong to the immunophilin family and are characterized by their ability to bind immunosuppressive drugs such as FK506 (tacrolimus) and rapamycin. Many FKBPs possess peptidyl-prolyl isomerase (PPIase) activity, enabling them to catalyze the cis-trans isomerization of peptide bonds preceding proline residues, an essential step in protein folding. Their significance in research stems from their diverse biological roles in immunoregulation, signal transduction, and protein folding. These proteins are increasingly being linked to various pathological processes, making them important targets for therapeutic intervention . For example, FKBP12 interacts with multiple intracellular calcium release channels and coordinates multi-protein complex formation of the tetrameric skeletal muscle ryanodine receptor, with its deletion in mice causing congenital heart disorders .

What are the major differences between FKBP12 and FKBP12.6?

While FKBP12 and FKBP12.6 share significant sequence homology, they exhibit distinct binding specificities and physiological roles. FKBP12.6 displays selectivity for binding to the ryanodine receptor type 2 (RyR2), which is primarily expressed in cardiac tissue. This selectivity is attributed to three specific amino acids (Gln 31, Asn 32, and Phe 59). Research has demonstrated that triple mutants of FKBP12 incorporating these amino acids gain RyR2 selectivity, while the corresponding triple mutant of FKBP12.6 loses this selectivity . Functionally, FKBP12.6 plays a critical role in regulating calcium release in cardiomyocytes, with FKBP12.6 deficiency or disruption by FK506/rapamycin resulting in enhanced Ca²⁺ spark frequency and altered calcium channel-RyR2 coupling .

How do antibodies against different FKBP proteins achieve specificity?

Antibodies against different FKBP proteins achieve specificity through careful immunogen selection targeting unique epitopes. For example, the PA1-026A antibody against FKBP12 is generated using a synthetic peptide corresponding to the N-terminal residues G(1)VQVETISPGDGR(13) of human FKBP12 . The specificity of anti-FKBP12.6 antibodies has been demonstrated in Western blot analyses showing specific binding to FKBP12.6 without cross-reactivity to other FKBP family members, including FKBP12, FKBP13, FKBP25, FKBP38, and FKBP52 . This high degree of specificity is crucial for researchers investigating the distinct roles of closely related FKBP family members in various physiological and pathological processes.

What are the optimal applications for FKBP antibodies in cellular research?

FKBP antibodies are versatile research tools applicable across multiple experimental techniques. For immunodetection, antibodies like PA1-026A (anti-FKBP12) have been successfully employed in Western blot, immunohistochemistry, and immunoprecipitation procedures . When designing experiments, researchers should consider the specific isoform they are targeting and select antibodies with demonstrated specificity. For instance, when investigating FKBP12.6 in brain tissue, researchers have successfully used antigen affinity-purified polyclonal antibodies to detect the approximately 13 kDa protein in human cortex, human hippocampus, and mouse brain tissues via Western blot . The experimental design should incorporate appropriate positive controls (such as recombinant FKBP proteins) and negative controls to validate antibody specificity.

How can researchers distinguish between different FKBP isoforms in complex tissue samples?

Distinguishing between different FKBP isoforms in complex tissue samples requires a multi-faceted approach:

• Antibody selection: Use highly specific antibodies with validated cross-reactivity profiles. For example, anti-FKBP12.6 antibodies that show no reactivity with FKBP12, FKBP13, FKBP25, FKBP38, or FKBP52 .

• Molecular weight discrimination: Different FKBP isoforms have distinct molecular weights (e.g., FKBP12 at 12 kDa, FKBP12.6 at approximately 13 kDa) that can be resolved using high-resolution SDS-PAGE .

• Expression pattern analysis: Some FKBPs show tissue-specific expression patterns. For instance, FKBP11 is specifically localized to antibody-producing plasma cells, providing another parameter for discrimination .

• Functional assays: Leveraging the differential binding properties of FKBPs to ligands like FK506 or their differential effects on binding partners (e.g., FKBP12.6's selective binding to RyR2) can provide functional confirmation of isoform identity .

• Genetic approaches: RNA interference or gene editing targeting specific FKBP isoforms can provide additional confirmation of antibody specificity.

What considerations are important when using FKBP antibodies in co-immunoprecipitation studies?

When designing co-immunoprecipitation experiments with FKBP antibodies, researchers should consider:

• Binding partners: FKBPs interact with multiple proteins. For example, FKBP12 interacts with type I TGF-beta receptors and intracellular calcium release channels . Consider whether the antibody epitope might interfere with these protein-protein interactions.

• Drug complexes: FKBP proteins can form complexes with drugs like FK506 and rapamycin, which can alter protein-protein interactions. The FKBP12-FK506 complex binds calcineurin while the FKBP12-rapamycin complex binds to the FRB domain of mTOR . These drug-induced complexes may affect co-immunoprecipitation results.

• Buffer conditions: Optimize lysis and washing buffers to preserve physiologically relevant interactions while minimizing non-specific binding.

• Controls: Include appropriate negative controls (such as isotype-matched control antibodies) and positive controls (known interacting proteins) to validate specificity.

• Validation: Confirm co-immunoprecipitation results with reciprocal experiments (i.e., immunoprecipitate with antibodies against the binding partner and detect the FKBP protein).

How do FKBP proteins participate in the unfolded protein response pathway?

FKBP proteins, particularly FKBP11, play significant roles in the unfolded protein response (UPR) pathway. Research has demonstrated that FKBP11 expression is induced during ER stress in an X-box-binding protein 1 (XBP1)-dependent manner . This induction is particularly evident during B cell to plasma cell differentiation, suggesting a specialized role in antibody production. Functionally, FKBP11 has been shown to act as an antibody peptidyl-prolyl cis-trans isomerase, capable of refolding IgG antibodies in vitro—an activity that can be inhibited by FK506 .

What is the role of FKBP proteins in disease pathogenesis and potential therapeutic targets?

FKBP proteins have been implicated in various pathological processes and represent potential therapeutic targets:

• Immunosuppression: The FKBP12-FK506 and FKBP12-rapamycin complexes mediate immunosuppressive effects by inhibiting calcineurin and mTOR signaling pathways, respectively. These mechanisms are exploited therapeutically to prevent allograft rejection in post-transplantation patients .

• Fibrosis and tissue remodeling: FKBP65 associates with lysyl hydroxylase 2 (LH2), promoting its dimerization and enhancing collagen cross-linking activity. This has implications for fibrotic disorders and cancer metastasis, with FKBP65 inhibition representing a potential therapeutic strategy .

• Cardiac disorders: FKBP12.6 regulates calcium release via the ryanodine receptor in cardiomyocytes. Disruption of this interaction may contribute to cardiac arrhythmias and other heart disorders . In mice, deletion of the FKBP12 gene causes congenital heart disorder known as noncompaction of left ventricular myocardium .

• Connective tissue diseases: FKBP12 binding to the GS domain of activin receptor-like kinase-2 (ALK2) stabilizes the kinase in an inactive conformation. Mutations in ALK2 that abolish FKBP12 binding are associated with fibrodysplasia ossificans progressiva (FOP) .

• Autoimmune diseases: Understanding the role of FKBP11 in antibody folding may provide new therapeutic approaches for autoimmune diseases where autoantibodies play a pathogenic role, potentially offering alternatives to B-cell depletion strategies .

How do different FKBP ligands affect antibody-based detection methods?

FKBP ligands like FK506 (tacrolimus) and rapamycin can significantly impact antibody-based detection methods through several mechanisms:

• Epitope masking: Binding of FK506 or rapamycin to the FKBP protein may occlude antibody epitopes, potentially reducing detection efficiency. This is particularly relevant for antibodies targeting the ligand-binding domain.

• Conformational changes: FKBP ligands can induce conformational changes in the protein that may expose or hide certain epitopes, altering antibody recognition profiles.

• Protein-protein interactions: FKBP-ligand complexes form novel protein-protein interactions (e.g., FKBP12-FK506 with calcineurin, FKBP12-rapamycin with mTOR) . These interactions may interfere with antibody binding or create novel epitopes.

• Subcellular redistribution: Ligand binding may alter the subcellular localization of FKBP proteins, affecting immunolocalization results.

• Enzymatic activity inhibition: FK506 inhibits the peptidyl-prolyl isomerase activity of FKBPs, including the antibody refolding activity of FKBP11 . This functional inhibition should be considered when interpreting results from activity-based assays.

Researchers should carefully consider these potential effects when designing experiments involving both FKBP ligands and antibody-based detection methods, particularly in studies examining the physiological roles of FKBP-ligand interactions.

What are the optimal conditions for using FKBP antibodies in Western blot applications?

Optimizing Western blot conditions for FKBP antibodies requires attention to several key parameters:

When analyzing brain or heart tissues, researchers have successfully detected FKBP12.6 using 1 μg/mL of antigen affinity-purified polyclonal antibody followed by HRP-conjugated secondary antibody . For FKBP12 detection in rat heart homogenate, similar conditions have proven effective .

How can researchers validate the specificity of their FKBP antibodies?

Validating FKBP antibody specificity is crucial for reliable research outcomes. A comprehensive validation approach should include:

• Recombinant protein panel testing: As demonstrated for anti-FKBP12.6 antibodies, test against a panel of recombinant FKBP proteins (FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP38, FKBP52) to confirm specificity .

• Peptide competition: Use the immunizing peptide (e.g., PEP-012 for PA1-026A anti-FKBP12 antibody) in neutralization experiments to confirm epitope-specific binding .

• Knockout/knockdown controls: Samples from FKBP-deficient models or cells with FKBP knockdown provide definitive negative controls.

• Cross-species reactivity: Confirm expected cross-reactivity with samples from different species (e.g., human, mouse, rat) based on epitope conservation.

• Multiple detection methods: Validate antibody performance across multiple techniques (Western blot, immunohistochemistry, immunoprecipitation) to ensure consistent specificity.

• Expected molecular weight: Confirm detection at the expected molecular weight (approximately 12 kDa for FKBP12, 13 kDa for FKBP12.6) .

• Tissue expression patterns: Verify detection in tissues with known expression (e.g., FKBP11 in plasma cells, FKBP12.6 in cardiac tissue) .

What approaches can resolve common issues when working with FKBP-targeting antibodies?

When troubleshooting experiments with FKBP antibodies, consider these common issues and solutions:

• Low signal intensity:

  • Increase antibody concentration or incubation time

  • Enhance signal with more sensitive detection systems

  • Enrich target protein with immunoprecipitation before analysis

  • Use fresh tissue samples with minimal proteolytic degradation

• Non-specific banding:

  • Optimize blocking conditions (type and concentration of blocking agent)

  • Increase washing stringency

  • Pre-absorb antibody with non-relevant tissues

  • Validate with peptide competition assays

• Inconsistent results across tissue types:

  • Consider tissue-specific post-translational modifications

  • Optimize extraction buffers for different tissue types

  • Account for potential binding partners that may mask epitopes

  • Consider potential splicing variants with altered epitopes

• Cross-reactivity issues:

  • Use antibodies validated against multiple FKBP family members

  • Confirm results with a second antibody targeting a different epitope

  • Employ genetic approaches (siRNA, CRISPR) to validate specificity

• Functional interference by FK506/rapamycin:

  • Design experiments to account for potential ligand effects on antibody binding

  • Include appropriate drug-treated and untreated controls

  • Consider the impact of endogenous immunophilin ligands

What are emerging applications for FKBP antibodies in disease research?

Emerging applications for FKBP antibodies in disease research span multiple fields:

• Autoimmune disease mechanisms: FKBP11's role in antibody folding opens new avenues for investigating autoimmune pathologies. Rather than complete B-cell depletion (e.g., with rituximab), targeting specific components of the antibody folding machinery might provide more nuanced therapeutic approaches .

• Fibrosis research: FKBP65's association with lysyl hydroxylase 2 (LH2) and its role in collagen cross-linking stability position it as a potential target in fibrotic disorders. Antibodies against FKBP65 could help elucidate mechanisms underlying fibrosis in various tissues and potentially guide development of anti-fibrotic therapies .

• Cancer metastasis: The FKBP65-LH2 axis has been implicated in cancer metastasis. Antibody-based detection of these proteins in tumor samples may provide prognostic information and reveal therapeutic vulnerabilities .

• Cardiac pathophysiology: FKBP12.6's role in regulating ryanodine receptor function in cardiomyocytes makes it relevant to cardiac disease research. Antibodies distinguishing between FKBP12 and FKBP12.6 can help elucidate isoform-specific functions in normal and pathological cardiac conditions .

• Neurodegeneration: Given the detection of FKBP12.6 in brain tissues, including human cortex and hippocampus, antibodies against this protein may facilitate investigation of its roles in neuronal calcium signaling and potential contributions to neurodegenerative disorders .

How might advances in antibody engineering impact FKBP research?

Advances in antibody engineering technologies are poised to significantly impact FKBP research through:

• Enhanced specificity: Next-generation recombinant antibodies with engineered complementarity-determining regions (CDRs) may achieve even greater discrimination between highly homologous FKBP family members.

• Intrabodies and nanobodies: Smaller antibody formats capable of functioning within cells could allow real-time tracking of FKBP proteins and their interactions in living cells.

• Bispecific antibodies: Dual-targeting antibodies could simultaneously recognize an FKBP protein and one of its binding partners, providing tools to study specific protein-protein interactions.

• Conditionally active antibodies: Antibodies that become active only under specific cellular conditions (pH, redox state, proteolytic activation) could enable compartment-specific detection of FKBP proteins.

• Antibody-drug conjugates: Beyond research tools, engineered antibodies conjugated to small molecule inhibitors could potentially deliver FKBP-modulating compounds to specific cell types for therapeutic applications.

• Proximity-labeling antibodies: Antibodies conjugated to enzymes that catalyze proximity-dependent labeling could help identify novel FKBP binding partners and better characterize FKBP interactomes.

What methodological advances are needed to better understand FKBP isoform-specific functions?

Despite significant progress, several methodological challenges remain in understanding FKBP isoform-specific functions:

• Selective inhibitors: Development of isoform-selective chemical probes that can distinguish between closely related FKBP family members would complement antibody-based approaches and enable functional studies in complex systems.

• Tissue-specific knockout models: Generation of conditional knockout models for specific FKBP isoforms would help delineate their functions in different tissues while avoiding developmental complications observed in some global knockouts.

• Improved structural resolution: Higher resolution structural studies of FKBP proteins in complex with their cellular targets (beyond FK506/rapamycin complexes) would guide development of more specific detection reagents and modulators.

• Quantitative proteomics: More sensitive mass spectrometry-based approaches to quantify FKBP isoform expression and post-translational modifications across tissues and disease states would complement antibody-based detection.

• Single-cell analysis: Integration of FKBP antibodies into single-cell proteomic workflows would reveal cell-type specific expression patterns and potential heterogeneity within tissues.

• In vivo imaging: Development of antibody-based imaging probes for non-invasive detection of FKBP expression or activity in living organisms would facilitate translational research.

• Domain-specific antibodies: Generation of antibodies targeting specific functional domains within multi-domain FKBP proteins (e.g., the four FKBP-like domains in FKBP65) would help distinguish their individual contributions to protein function.