FKBP15-1 Antibody

What is FKBP15 and what cellular functions does it perform?

FKBP15, also known as FKBP133, belongs to the FK506-binding protein family of immunophilins. It is primarily expressed in the developing nervous system and contains both an FK506-binding protein motif and a domain similar to Wiskott-Aldrich syndrome protein homology region 1 (WH1). FKBP15 is distributed along axonal shafts and partially co-localizes with F-actin in growth cones of dorsal root ganglion neurons. Research has demonstrated that FKBP15 modulates growth cone behavior, as overexpression results in altered numbers of filopodia in transfected neurons. Additionally, FKBP15 associates with both microtubule and actin filament systems, playing a critical role in early endosome transport—disruption of its expression via RNAi delays early endosome transport in HeLa cells .

What are the known isoforms of FKBP15 and how do they differ?

Current research has identified at least three distinct isoforms of FKBP15. These isoforms result from alternative splicing of the FKBP15 gene. While all isoforms share the core FK506-binding protein domain, they may differ in their subcellular localization patterns and potentially their functional activities. The canonical protein in humans has a reported length of 1219 amino acid residues and a molecular mass of approximately 133.6 kDa. The protein's subcellular localization is primarily cytoplasmic, and it is widely expressed across many tissue types .

What criteria should I consider when selecting an anti-FKBP15 antibody for my research?

When selecting an anti-FKBP15 antibody, consider these critical parameters:

• Epitope specificity: Determine which region of FKBP15 the antibody targets. Available options include antibodies targeting:

  • N-terminal regions (amino acids 30-80 or 50-100)

  • Middle regions (amino acids 960-1020)

  • C-terminal regions (amino acids 1175-1234)

• Validated applications: Verify that the antibody has been validated for your intended application:

  • Western blot (typical dilutions: 1:2000-1:10000)

  • Immunoprecipitation (typical usage: 1-4 μg/mg of lysate)

  • Immunohistochemistry (typical dilutions: 1:20-1:200)

  • Immunofluorescence

  • ELISA

• Species reactivity: Confirm compatibility with your experimental model:

  • Human (UniProt ID: Q5T1M5)

  • Mouse (UniProt ID: Q6P9Q6)

  • Rat (Gene ID: 362528)

  • Other reported species include bovine, frog, chimpanzee, and chicken

• Validation data: Examine published validation data, including Western blot images showing expected band size (~134 kDa) .

What are the optimal protocols for using anti-FKBP15 antibodies in Western blot applications?

For optimal Western blot results with anti-FKBP15 antibodies, follow this methodological approach:

• Sample preparation:

  • Prepare whole cell lysates using NETN lysis buffer (recommended for HeLa, 293T, and NIH3T3 cells)

  • Use 25-50 μg of total protein per lane

• Antibody dilution:

  • Primary antibody: 0.1-1 μg/mL (1:2000-1:10000 dilution)

  • For Novus Biologicals antibody NB100-422: Use at 0.1 μg/mL

• Detection considerations:

  • Expected molecular weight: 133-134 kDa

  • Recommended exposure time: 3-10 minutes for chemiluminescence detection

  • Cell lines with confirmed detection: HeLa, 293T, NIH3T3

• Controls:

  • Positive controls: HeLa nuclear extracts, NIH/3T3 cell lysate

  • Consider using FKBP15 knockout/knockdown samples as negative controls

How should I optimize immunoprecipitation experiments using FKBP15 antibodies?

For successful immunoprecipitation of FKBP15:

• Starting material:

  • Use 0.5-1.0 mg of whole cell lysate per IP reaction

  • NETN lysis buffer is recommended for sample preparation

• Antibody amounts:

  • Use 2-10 μg of antibody per mg of lysate

  • For Novus Biologicals antibody NB100-422: 6 μg per reaction is optimal

• Protocol optimization:

  • Load 20-25% of immunoprecipitated material for Western blot detection

  • For blotting immunoprecipitated FKBP15, use antibody at 1 μg/mL

  • HeLa cells have been successfully used for FKBP15 immunoprecipitation

• Antibody recommendations:

  • NB100-422, NB100-423, and NB100-424 have all been validated for FKBP15 immunoprecipitation

  • Note: NB100-424 is not recommended for Western blot detection

What are the best practices for immunohistochemical detection of FKBP15?

For optimal immunohistochemical detection of FKBP15:

• Tissue preparation:

  • Both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues are suitable

  • For FFPE tissues, antigen retrieval is critical

• Antigen retrieval methods:

  • Option 1: TE buffer pH 9.0 (recommended)

  • Option 2: Citrate buffer pH 6.0 (alternative)

• Antibody dilutions:

  • Typical range: 1:20-1:200

  • For PAB16735: 2.5 μg/mL has been validated

• Validated tissues:

  • Mouse brain tissue has shown positive staining

  • Human tissues with confirmed expression should be used as positive controls

• Visualization systems:

  • DAB (3,3'-diaminobenzidine) detection systems are commonly used

  • Fluorescence-based detection can be used for co-localization studies

How can I investigate FKBP15's role in neuronal development and growth cone dynamics?

To investigate FKBP15's role in neuronal development and growth cone dynamics:

• Experimental models:

  • Primary dorsal root ganglion neurons

  • Neuronal cell lines with FKBP15 expression

  • Transgenic mouse models with FKBP15 modifications

• Methodological approaches:

  • Overexpression studies: Transfect neurons with FKBP15 constructs and quantify changes in filopodia number and morphology

  • RNAi knockdown: Use siRNA or shRNA to reduce FKBP15 expression and analyze impacts on growth cone architecture

  • Co-localization analysis: Perform dual immunofluorescence labeling with FKBP15 antibodies and F-actin markers

  • Live imaging: Track FKBP15-GFP fusion proteins to monitor dynamic localization in growth cones

• Key measurements:

  • Filopodia number and length

  • Growth cone area and complexity

  • Neurite extension rate

  • Co-localization coefficient with F-actin and microtubules

What approaches can be used to study FKBP15's involvement in endosomal transport?

To investigate FKBP15's role in endosomal transport:

• Experimental design:

  • RNAi-mediated knockdown: Use siRNA (transient) or shRNA (stable) targeting FKBP15

  • CRISPR-Cas9 gene editing: Generate FKBP15 knockout cell lines

  • Domain mutation analysis: Create constructs with mutations in specific FKBP15 domains

• Functional assays:

  • Endosome tracking: Label early endosomes with fluorescent markers (e.g., Rab5-GFP) and analyze transport kinetics

  • Cargo trafficking: Monitor the transport of specific endosomal cargo proteins

  • Cytoskeletal co-dependence: Use cytoskeleton-disrupting drugs to determine FKBP15's role at the intersection of actin and microtubule dynamics

• Analytical approaches:

  • Measure endosome velocity, directionality, and processivity

  • Quantify endosome size and distribution patterns

  • Assess co-localization with cytoskeletal markers and motor proteins

How does FKBP15 differ from other FKBP family members like FKBP51 in cellular functions?

FKBP15 differs from other family members like FKBP51 in several key aspects:

• Structural distinctions:

  • FKBP15 contains a WH1-like domain not found in other FKBPs

  • FKBP15 (133 kDa) is significantly larger than FKBP51 (51 kDa)

• Functional differences:

  • FKBP15: Primarily involved in cytoskeletal organization and endosomal transport

  • FKBP51/FKBP5: Functions as a negative regulator of glucocorticoid receptor (GR) activity and is involved in stress responses

• Expression patterns:

  • FKBP15: Predominantly expressed in developing nervous system

  • FKBP51: More broadly expressed in multiple tissues

• Stress response roles:

  • Recent research shows FKBP51/FKBP5 mediates resilience to inflammation-induced anxiety

  • FKBP51 is involved in systemic inflammation-induced neuroinflammation and hippocampal GR activation

  • FKBP51 contributes to enhancement of GAD65 expression for GABA synthesis in the ventral hippocampus

• Experimental approaches to compare functions:

  • Generate double knockdown/knockout models

  • Perform reciprocal domain swapping experiments

  • Conduct differential interactome analysis

What are the most common issues encountered when using FKBP15 antibodies and how can they be resolved?

For Western blot specifically, verify that the expected molecular weight (133-134 kDa) is being detected. If using the NB100-424 antibody, note that it is not recommended for Western blot applications, and NB100-422 or NB100-423 should be used instead .

How can I validate the specificity of my FKBP15 antibody?

To validate FKBP15 antibody specificity:

• Genetic approaches:

  • Use FKBP15 knockout/knockdown samples as negative controls

  • Perform rescue experiments with exogenous FKBP15 expression

• Biochemical validation:

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide

  • Multiple antibody approach: Test multiple antibodies targeting different FKBP15 epitopes

  • Mass spectrometry: Confirm identity of immunoprecipitated protein bands

• Cross-reactivity assessment:

  • Test antibody against recombinant FKBP family proteins

  • Validate in multiple species if working with non-human models

• Application-specific controls:

  • For IHC/IF: Include isotype control antibodies at equivalent concentrations

  • For WB: Include molecular weight markers and positive control samples

  • For IP: Perform parallel experiments with non-immune IgG

What are the considerations for performing cross-species detection of FKBP15?

When working with FKBP15 across different species:

• Sequence homology analysis:

  • Human FKBP15 (UniProt ID: Q5T1M5)

  • Mouse FKBP15 (UniProt ID: Q6P9Q6)

  • Rat FKBP15 (Gene ID: 362528)

  • Check epitope sequence conservation across target species

• Antibody selection for cross-species applications:

  • Novus Biologicals NB100-422: Validated for human and mouse

  • Abnova PAB16735: Validated for human, mouse, and rat

  • ProSci 5135_S: Validated for human, mouse, and rat

• Protocol adjustments:

  • May require species-specific optimization of dilutions

  • Consider increased antibody concentration for less homologous species

  • Adjust incubation times and temperatures for optimal binding

• Species-specific controls:

  • Include positive control samples from each species

  • Consider species-specific blocking reagents to reduce background

How is FKBP15 being studied in the context of neurodevelopmental disorders?

Research into FKBP15's role in neurodevelopmental disorders is an emerging field with several methodological approaches:

• Genetic association studies:

  • Analyze FKBP15 gene variants in patient cohorts with neurodevelopmental disorders

  • Perform whole exome/genome sequencing to identify rare FKBP15 variants

• Functional studies in model systems:

  • Generate transgenic mouse models with FKBP15 mutations identified in patients

  • Use iPSC-derived neurons from patients to study FKBP15 expression and function

  • Apply CRISPR-Cas9 to create isogenic cell lines with specific FKBP15 mutations

• Neuronal development assays:

  • Analyze neurite outgrowth, branching complexity, and synapse formation

  • Study growth cone dynamics in neurons with altered FKBP15 expression

  • Examine axon guidance and target innervation in animal models

• Molecular interaction studies:

  • Identify FKBP15-interacting proteins in developing neurons

  • Investigate links between FKBP15 and established neurodevelopmental risk factors

What is known about the relationship between FKBP15 and inflammation or immune responses?

While FKBP15 itself has not been extensively studied in inflammation, insights can be drawn from related FKBP family members:

• Comparison to FKBP51/FKBP5:

  • FKBP51 has been directly implicated in mediating resilience to inflammation-induced anxiety

  • Research shows FKBP51 is involved in systemic inflammation-induced neuroinflammation

  • FKBP51 contributes to hippocampal glucocorticoid receptor activation during inflammatory responses

• Potential research directions for FKBP15:

  • Investigate FKBP15 expression changes during neuroinflammation

  • Examine FKBP15's potential role in microglial activation and function

  • Study possible interactions between FKBP15 and inflammatory signaling pathways

• Experimental approaches:

  • Lipopolysaccharide (LPS) challenge models to induce inflammation

  • Analysis of FKBP15 expression in inflammatory conditions

  • Co-localization studies with inflammatory markers in brain tissue

How can new antibody technologies be applied to study FKBP15 dynamics in live cells?

Advanced antibody technologies for studying FKBP15 dynamics include:

• Intrabodies and nanobodies:

  • Develop FKBP15-specific nanobodies for intracellular expression

  • Fuse nanobodies to fluorescent proteins for live-cell imaging

  • Engineer cell-permeable antibody fragments for temporal studies

• Proximity labeling approaches:

  • Create FKBP15 fusion proteins with BioID or APEX2

  • Identify proximal proteins in different cellular compartments

  • Map dynamic interaction networks during endosomal transport

• Super-resolution microscopy applications:

  • Apply STORM/PALM imaging with FKBP15 antibodies

  • Perform live-cell STED microscopy with fluorescently tagged nanobodies

  • Combine with lattice light-sheet microscopy for 4D visualization

• Optogenetic approaches:

  • Develop optogenetic tools to manipulate FKBP15 localization

  • Create light-inducible FKBP15 degradation systems

  • Engineer photoactivatable FKBP15 variants for localized activation

How conserved is FKBP15 across species and what are the implications for research models?

FKBP15 shows significant evolutionary conservation:

• Phylogenetic analysis:

  • FKBP15 orthologs have been reported in mammals (human, mouse, rat, bovine)

  • Also identified in other vertebrates (frog, chimpanzee, chicken)

• Domain conservation:

  • The FK506-binding domain shows highest conservation

  • WH1-like domain also maintains structural conservation

  • C-terminal regions may show more species-specific variations

• Implications for research model selection:

  • Mouse models are appropriate for studying conserved functions

  • Species-specific functions may require homologous models

  • Consider domain conservation when designing cross-species experiments

• Antibody selection for comparative studies:

  • Choose antibodies targeting highly conserved epitopes

  • Validate antibody reactivity in each species prior to comparative studies

  • Consider using multiple antibodies targeting different epitopes