FKBPL Antibody

What is FKBPL and why are antibodies against it valuable for research?

FKBPL (FK506 Binding Protein Like) is a divergent member of the immunophilin protein superfamily with potent anti-tumor activity through inhibition of angiogenesis and cancer stemness. FKBPL has been identified as a critical regulator of both developmental and pathological angiogenesis, while also playing important roles in inflammation through modulation of NF-κB signaling .

FKBPL antibodies are essential research tools for:

• Detecting FKBPL expression in various tissues and cellular compartments

• Investigating FKBPL's role in angiogenesis and vascular integrity

• Studying inflammatory pathways regulated by FKBPL

• Examining FKBPL as a prognostic biomarker in cancer (particularly breast and ovarian cancer)

• Validating the effects of FKBPL-derived therapeutic peptides (AD-01, ALM201)

Importantly, FKBPL homozygous knockout mice (fkbpl-/-) are embryonic lethal, whereas heterozygous knockdown mice (fkbpl+/-) develop normally but show early signs of endothelial and vascular dysfunction, highlighting the critical nature of this protein in development .

What applications are FKBPL antibodies commonly used for?

FKBPL antibodies support multiple experimental applications across various research disciplines. The table below summarizes common applications and typical reactivity:

For optimal results, antigen retrieval with TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 is recommended for IHC applications . Importantly, working dilutions should be optimized for each specific antibody and experimental system to achieve the best signal-to-noise ratio .

What molecular characteristics should researchers know about FKBPL when using antibodies?

Understanding FKBPL's molecular characteristics is essential for proper antibody selection and experimental design:

• Molecular Weight: The calculated molecular weight of FKBPL is approximately 38 kDa, but the observed molecular weight in Western blotting typically ranges from 42-48 kDa . This discrepancy may be due to post-translational modifications.

• Protein Structure: FKBPL contains tetratricopeptide repeat (TPR) domains involved in protein-protein interactions, particularly with Hsp90 and estrogen receptor alpha .

• Expression Pattern: FKBPL is secreted predominantly by fibroblasts and endothelial cells . Its expression is downregulated by hypoxic conditions but not by angiogenic cytokines like VEGF or IL-8 .

• Functional Domains: The amino acid 34-57 region in the N-terminus (outside the HSP90 binding region) has potent anti-angiogenic activity . This region is the basis for therapeutic peptides AD-01 and ALM201.

• Cellular Localization: FKBPL can be both intracellular and secreted, with distinct functions in each compartment. During development, it appears as "an intense subcellular spot, closely associated with the nucleus" in some cell types .

When selecting FKBPL antibodies, researchers should consider which domain or epitope they target and whether they can detect both intracellular and secreted forms of the protein.

How can researchers validate the specificity of FKBPL antibodies?

Validating antibody specificity is crucial for generating reliable research data. For FKBPL antibodies, consider these methodological approaches:

• Genetic Models:

  • Use FKBPL heterozygous knockdown (fkbpl+/-) tissues, which show reduced FKBPL immunostaining consistent with qPCR data

  • Generate FKBPL knockdown cell lines using siRNA or CRISPR-Cas9 systems

  • Note that complete FKBPL knockout is embryonic lethal, limiting some validation approaches

• Blocking Peptides:

  • Pre-incubate the antibody with the immunizing peptide (if available) - this should abolish specific staining

  • For instance, with ABIN1881347 antibody, the immunizing peptide corresponds to amino acids 264-292 from the C-terminal region

• Multi-application Validation:

  • Verify consistent results across different techniques (WB, IHC, IF) in the same samples

  • Compare staining patterns with published literature describing FKBPL localization

• Multiple Antibodies:

  • Use antibodies targeting different epitopes of FKBPL

  • Compare commercial antibodies raised against different regions (N-terminal, C-terminal, internal)

• Isotype Controls:

  • Include rabbit IgG (for rabbit polyclonal antibodies) or appropriate isotype controls

  • Use secondary-only controls to assess non-specific binding of detection reagents

Antibody validation should be documented and included in research publications to enhance reproducibility and reliability of findings.

How can FKBPL antibodies be used to investigate inflammatory pathways and NF-κB signaling?

FKBPL has recently been identified as a negative regulator of NF-κB activation, offering new research avenues in inflammatory disease models. Here's a methodological approach for studying FKBPL's role in inflammation:

• Phosphorylation Analysis:

  • Use FKBPL antibodies alongside phospho-p65(RelA) antibodies to study NF-κB activation

  • In both endothelial cells and bone marrow-derived macrophages (BMDMs), FKBPL knockdown increases p65 phosphorylation following LPS stimulation

  • The FKBPL peptide AD-01 abrogates LPS-induced phosphorylation of p65

• Cytokine Production Assessment:

  • Use FKBPL antibodies to correlate protein expression with cytokine production

  • FKBPL regulates pro-inflammatory cytokines (TNF, IL-6, IL-1β) and anti-inflammatory cytokines (IL-10)

  • Experimental design should include appropriate stimuli (e.g., LPS) and time-course analysis

• Endothelial Barrier Function:

  • FKBPL knockdown promotes endothelial cell barrier permeability, especially after LPS stimulation

  • Use FKBPL immunostaining to examine co-localization with tight junction proteins (e.g., VE-cadherin)

• In Vivo Inflammation Models:

  • Compare wild-type and FKBPL heterozygous mice in LPS survival models

  • FKBPL+/- mice show reduced survival following LPS injection compared to wild-type mice

  • Use antibodies to assess FKBPL expression in harvested tissues

• Genetic Variant Analysis:

  • FKBPL variants are associated with inflammatory disorders including psoriasis and rheumatoid arthritis

  • Use antibodies to examine how these variants affect protein expression and function

These methodological approaches provide a framework for investigating FKBPL's role in inflammation, with antibodies serving as essential tools throughout this research.

What technical considerations are important when studying FKBPL's role in angiogenesis?

Investigating FKBPL's anti-angiogenic properties requires careful experimental design and consideration of several technical factors:

• Regulation by Hypoxia:

  • FKBPL secretion is downregulated by hypoxic conditions but not by angiogenic cytokines

  • Control oxygen levels carefully in experiments and include appropriate hypoxia markers

• CD44 Interaction:

  • Extracellular FKBPL exerts anti-migratory effects via CD44 receptor

  • Include CD44 neutralizing antibodies or CD44 knockout models in experiments

  • Interestingly, AD-01 can regulate NF-κB signaling independently of CD44, suggesting multiple mechanisms of action

• In Vitro Angiogenesis Models:

  • Endothelial cell migration assays

  • Tube formation assays

  • Endothelial barrier permeability assays (FKBPL regulates vascular integrity)

• In Vivo Angiogenesis Models:

  • Lewis lung carcinoma tumor model shows increased growth rate and tumor vasculature in FKBPL+/- mice

  • CD31 staining of tumor sections reveals increased and more irregular blood vessels in FKBPL+/- mice

• FKBPL Peptide Derivatives:

  • Include AD-01 (preclinical peptide) and/or ALM201 (clinical peptide) as experimental controls

  • These peptides have anti-angiogenic effects that can help validate antibody-based findings

• Antibody Selection:

  • For angiogenesis studies, choose antibodies that can detect the N-terminal region (aa 34-57) responsible for anti-angiogenic activity

  • Consider antibodies that can detect both intracellular and secreted forms

Careful consideration of these factors will enhance the reliability and relevance of angiogenesis research using FKBPL antibodies.

How does FKBPL expression change in disease states, and how can antibodies track these changes?

FKBPL expression is altered in several pathological conditions, providing opportunities for biomarker development and therapeutic intervention. Here's how antibodies can help characterize these changes:

• Cancer:

  • High intra-tumor FKBPL levels correlate with improved prognosis in breast and ovarian cancer

  • Methodological approach: Use IHC with carefully validated antibodies on tumor microarrays

  • Quantify expression using digital pathology and correlate with clinical outcomes

• Inflammatory Disorders:

  • FKBPL expression is downregulated in psoriatic skin lesions compared to non-lesional skin

  • SNPs in FKBPL associate with psoriasis, rheumatoid arthritis, and high lymphocyte count

  • Methodological approach: Compare antibody staining between affected and unaffected tissues

• Vascular Dysfunction:

  • FKBPL regulates endothelial barrier function and vascular integrity

  • Methodological approach: Use dual immunofluorescence to co-localize FKBPL with endothelial markers

• Genetic Variants:

  • The missense mutation rs28732176 is associated with both psoriasis and rheumatoid arthritis

  • This variant falls within the region where the therapeutic peptides are based

  • Methodological approach: Develop antibodies specific to common variants to study their expression patterns

• COVID-19 and ARDS:

  • Vascular integrity breakdown and excessive inflammation are hallmarks of severe COVID-19 and ARDS

  • FKBPL's role in maintaining vascular integrity makes it a potential target in these conditions

  • Methodological approach: Analyze FKBPL expression in patient samples using antibody-based techniques

When studying disease-related changes, researchers should consider using multiple antibodies targeting different epitopes to ensure comprehensive detection, as disease-associated mutations or post-translational modifications might affect antibody binding.

How can I optimize sample preparation for FKBPL detection in immunohistochemistry?

Successful immunohistochemical detection of FKBPL requires careful sample preparation and protocol optimization:

• Fixation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues have been successfully used for FKBPL staining

  • Maintain consistent fixation times to ensure reproducible results

• Antigen Retrieval:

  • TE buffer pH 9.0 is recommended as the primary antigen retrieval method

  • Alternatively, citrate buffer pH 6.0 can be used if TE buffer yields suboptimal results

  • Optimize retrieval time and temperature for your specific tissue type

• Blocking:

  • Use appropriate blocking reagents to reduce background (typically serum from the same species as the secondary antibody)

  • Consider additional blocking steps for tissues with high endogenous biotin or peroxidase activity

• Antibody Dilution and Incubation:

  • Starting dilutions range from 1:100-1:1200 for IHC applications

  • Optimize both concentration and incubation time (typically overnight at 4°C or 1-2 hours at room temperature)

• Detection System:

  • DAB (3,3'-diaminobenzidine) staining has been successfully used for FKBPL visualization

  • Consider amplification systems for tissues with low FKBPL expression

• Controls:

  • Include positive controls (tissues known to express FKBPL, such as testis tissue)

  • Use FKBPL heterozygous tissues as partial negative controls that show reduced staining

  • Include isotype controls to assess non-specific binding

Remember that FKBPL has specific subcellular localization patterns, appearing as "an intense subcellular spot, closely associated with the nucleus" in some cell types , which should be considered when evaluating staining patterns.

What are the challenges in detecting secreted versus intracellular forms of FKBPL?

FKBPL exists in both intracellular and secreted forms, presenting unique challenges for detection and experimental design:

• Secreted FKBPL Detection:

  • FKBPL is secreted primarily by fibroblasts and endothelial cells

  • For Western blotting, concentration methods (TCA precipitation, ultrafiltration) may be needed for conditioned media

  • For ELISA, optimize sample collection timing as secretion is regulated by hypoxia

  • Consider slot blotting or dot blotting for direct detection in conditioned media

• Intracellular FKBPL Detection:

  • Permeabilization is critical for intracellular staining in flow cytometry and immunofluorescence

  • Optimize permeabilization agents (Triton X-100, saponin, methanol) based on the antibody epitope

  • Remember that FKBPL shows specific subcellular localization patterns in some cells

• Antibody Selection:

  • Secreted proteins may undergo processing that alters epitopes

  • Consider using antibodies targeting different regions of FKBPL for comprehensive detection:

    • N-terminal antibodies (aa 1-349 or aa 1-347)

    • C-terminal antibodies (aa 264-292)

    • Internal region antibodies (aa 71-120 or aa 251-300)

• Experimental Design Considerations:

  • For secretion studies, include brefeldin A or monensin controls to block secretion

  • Note that hypoxia specifically downregulates FKBPL secretion

  • Time-course experiments are important as secretion kinetics may vary by cell type and conditions

• Biological Relevance:

  • Intracellular FKBPL interacts with Hsp90 and estrogen receptor alpha

  • Extracellular FKBPL exerts anti-migratory effects via CD44

  • Both pools have distinct functions and should be considered in experimental interpretation

Comprehensive study of FKBPL may require complementary approaches targeting both intracellular and secreted forms to fully understand its biological roles.

How can I troubleshoot weak or nonspecific signals when using FKBPL antibodies in Western blotting?

Western blotting for FKBPL can present challenges that require systematic troubleshooting:

• Sample Preparation:

  • Use fresh tissue/cell lysates with complete protease inhibitor cocktails

  • Consider different lysis buffers (RIPA vs. NP-40) as they may affect epitope accessibility

  • For tissues with low expression, enrich FKBPL by immunoprecipitation before Western blotting

• Antibody Optimization:

  • Test a range of primary antibody dilutions (1:200-1:4000)

  • Extend primary antibody incubation (overnight at 4°C)

  • Try antibodies targeting different epitopes of FKBPL

• Blocking Optimization:

  • Test different blocking agents (5% milk, 5% BSA, commercial blockers)

  • Extend blocking time if background is high

  • Add 0.1-0.3% Tween-20 to reduce nonspecific binding

• Detection System:

  • For weak signals, use more sensitive detection methods (ECL Prime/Plus, femto-level detection)

  • Consider alternative visualization (fluorescent secondary antibodies instead of HRP)

  • Optimize exposure times for digital imaging systems

• Molecular Weight Considerations:

  • The calculated molecular weight of FKBPL is 38 kDa, but observed bands typically appear at 42-48 kDa or 45 kDa

  • Verify you're looking at the correct molecular weight range

  • Use ladder markers that clearly distinguish this range

• Positive Controls:

  • Include lysates from cells known to express FKBPL: HEK-293, HeLa, Jurkat, MCF-7, or human testis tissue

  • Consider running recombinant FKBPL protein as a positive control

• Technical Adjustments:

  • Try PVDF membranes instead of nitrocellulose for better protein retention

  • Adjust transfer conditions (time, voltage, buffer composition)

  • Load more protein (25-50 μg) if FKBPL expression is low

Systematic adjustment of these parameters should help optimize FKBPL detection in Western blotting applications.

How are FKBPL antibodies being used in cancer stem cell research?

FKBPL has emerging roles in cancer stem cell (CSC) biology, offering new research directions:

• CSC Identification and Quantification:

  • FKBPL knockdown expands the cancer stem cell subpopulation

  • Use FKBPL antibodies alongside CSC markers (CD44, CD133, ALDH) in multicolor flow cytometry

  • Employ immunofluorescence to examine FKBPL expression in tumor spheroid models

• Regulatory Mechanisms:

  • FKBPL knockdown increases expression of pluripotency transcription factors (NANOG, OCT4, SOX2)

  • Use ChIP assays with FKBPL antibodies to investigate potential direct or indirect regulation

  • Combine with RNA-seq or qPCR to correlate FKBPL levels with stemness gene expression

• Therapeutic Applications:

  • FKBPL-based peptides (AD-01, ALM201) have anti-CSC activity

  • Use FKBPL antibodies to monitor expression changes during treatment

  • Compare effects of FKBPL peptides with other anti-CSC therapeutics

• Clinical Correlations:

  • High intra-tumor FKBPL levels correlate with improved prognosis in breast and ovarian cancer

  • Use IHC with FKBPL antibodies on patient-derived xenografts or tissue microarrays

  • Correlate FKBPL expression with treatment response and clinical outcomes

• CD44 Interaction:

  • FKBPL exerts anti-CSC activity partially through CD44

  • Use co-immunoprecipitation with FKBPL antibodies to study CD44 interactions

  • Perform competition assays with FKBPL peptides and antibodies targeting different epitopes

This research area represents a promising frontier for FKBPL antibody applications, potentially leading to new cancer therapeutic strategies.

What are the latest developments in FKBPL-based therapeutics and how can antibodies support this research?

FKBPL-based peptide therapeutics show considerable promise, with antibodies playing crucial roles in their development and validation:

• Therapeutic Peptide Development:

  • ALM201, a 23-mer FKBPL-based peptide, has completed Phase 1a clinical trials in oncology

  • AD-01, a preclinical 24-residue peptide (aa 34-58), demonstrates potent anti-angiogenic activity

  • Antibodies can confirm target engagement and mechanistic activity of these peptides

• Safety and Pharmacokinetics:

  • ALM201 displayed good safety and pharmacokinetic profiles in clinical trials

  • Received orphan drug status from FDA for ovarian cancer

  • Antibodies can help measure circulating peptide levels in pharmacokinetic studies

• New Therapeutic Indications:

  • Beyond cancer, FKBPL peptides show anti-inflammatory activity

  • In LPS survival models, ALM201 treatment resulted in 100% survival and reduced pro-inflammatory cytokines

  • Antibodies can track changes in endogenous FKBPL during treatment

• Inflammatory Disease Applications:

  • Potential applications in psoriasis, rheumatoid arthritis, and sepsis

  • FKBPL peptides inhibit p65(RelA) phosphorylation and reduce NF-κB target gene expression

  • Antibodies can assess downstream pathway modulation in response to treatment

• Dual-Targeting Approach:

  • FKBPL peptides uniquely target both angiogenesis and inflammation

  • Particularly relevant for conditions with both components (psoriasis, rheumatoid arthritis)

  • Antibodies can help distinguish between these mechanisms of action

• Genetic Variation Considerations:

  • The missense mutation rs28732176 falls within the region where therapeutic peptides are based

  • Present in approximately 1% of the population

  • Antibodies can help assess how these variants might affect therapeutic response

FKBPL antibodies are essential tools for advancing this promising therapeutic avenue, from mechanism studies to clinical biomarker development.

How can FKBPL antibodies be used to investigate its role in vascular integrity and endothelial function?

FKBPL's emerging role in maintaining vascular integrity opens new research directions:

• Endothelial Tight Junction Analysis:

  • FKBPL knockdown promotes endothelial cell barrier permeability

  • Use immunofluorescence with FKBPL antibodies alongside tight junction proteins (VE-cadherin)

  • The FKBPL peptide AD-01 increases VE-cadherin endothelial tight junctions following LPS stimulation

• Response to Inflammatory Stimuli:

  • FKBPL regulates endothelial permeability particularly in response to LPS

  • Design experiments comparing normal and inflammatory conditions

  • Use permeability assays in combination with FKBPL antibody staining

• In Vivo Vascular Models:

  • FKBPL heterozygous mice (fkbpl+/-) show vascular irregularities

  • Examine blood vessel morphology using CD31 staining in combination with FKBPL antibodies

  • Lewis lung carcinoma tumors in FKBPL+/- mice show increased and more irregular blood vessels

• Developmental Angiogenesis:

  • FKBPL is strongly expressed in endothelial cells during development

  • Use antibodies to track expression changes during vascular development

  • Study embryonic tissues at different stages to understand temporal regulation

• Hypoxia Response:

  • FKBPL secretion is regulated by hypoxia

  • Design experiments comparing normoxic and hypoxic conditions

  • Use antibodies to track changes in FKBPL localization and expression under hypoxia

• Therapeutic Applications:

  • FKBPL peptides act as vascular stabilizers

  • Use antibodies to monitor endogenous FKBPL during peptide treatment

  • Investigate how peptide administration affects endogenous FKBPL expression and function

These approaches provide a framework for investigating FKBPL's critical role in vascular biology, with important implications for diseases characterized by vascular dysfunction.