FKBP14 Antibody

What is FKBP14 and why is it important to study?

FKBP14, also known as FK506-binding protein 14 or peptidyl-prolyl cis-trans isomerase, is a 211 amino acid enzyme that plays a critical role in protein folding during synthesis. This process is essential for maintaining cellular function and homeostasis. FKBP14 is primarily localized within the endoplasmic reticulum lumen, where it assists in the proper folding of nascent polypeptides, preventing misfolding and aggregation that can lead to cellular stress and disease . The protein contains two EF-hand domains and one PPIase FKBP-type domain, with the latter being crucial for enzymatic activity . FKBP14 has a preference for substrates containing 4-hydroxylproline modifications, including type III collagen, and may also target type VI and type X collagens . Importantly, mutations in FKBP14 are associated with Ehlers-Danlos syndrome with progressive kyphoscoliosis, myopathy, and hearing loss, highlighting its significance in both normal physiology and pathological conditions .

What types of FKBP14 antibodies are available and how do they differ?

Several types of FKBP14 antibodies are available for research purposes, each with distinct characteristics:

• Mouse Monoclonal Antibodies: Such as AT18E2, an IgG1 kappa light chain antibody that detects FKBP14 protein of human origin across multiple applications including western blotting, immunofluorescence, flow cytometry, and ELISA .

• Rabbit Polyclonal Antibodies: These recognize multiple epitopes on the FKBP14 protein, offering potentially higher sensitivity but with increased background potential. Examples include ab251703, which targets amino acids 100-200 of human FKBP14 , and ab176795, which recognizes the full-length recombinant protein corresponding to amino acids 20-211 .

The choice between monoclonal and polyclonal antibodies depends on the specific experimental requirements. Monoclonal antibodies offer higher specificity and reproducibility, while polyclonal antibodies may provide greater sensitivity for detection of low-abundance proteins or denatured epitopes.

What are the validated applications for FKBP14 antibodies?

FKBP14 antibodies have been validated for multiple experimental applications:

When selecting an application, consider the specific experimental question, sample type, and whether the target epitope remains accessible in the chosen application format .

What are the optimal storage conditions for FKBP14 antibodies?

For maximum stability and performance, FKBP14 antibodies should be stored according to manufacturer recommendations. Common storage guidelines include:

• Short-term storage (1-2 weeks): Store at 4°C .

• Long-term storage: Store at -20°C .

• Avoid freeze/thaw cycles: Repeated freezing and thawing can denature antibodies and reduce activity .

• Aliquoting: For antibodies used frequently, aliquoting is recommended to avoid repeated freeze/thaw cycles .

• Buffer composition: Many FKBP14 antibodies are supplied in PBS with 0.02% sodium azide and 40-50% glycerol at pH 7.2-7.3 .

Proper storage ensures antibody stability and consistent experimental results. For some formulations, such as the 20μl sizes of 15884-1-AP, the product contains 0.1% BSA, which helps stabilize the antibody during storage .

How can I optimize FKBP14 antibody detection in collagen binding studies?

Detecting FKBP14 bound to collagen requires careful consideration of assay design. Based on research findings:

For optimal results in collagen binding studies, use His-tagged FKBP14/FKBP22 with His-tag antibodies rather than relying on HDEL detection.

What are the structural determinants of FKBP14 function and how do they influence experimental design?

Understanding FKBP14's structural elements is crucial for designing experiments that interrogate its function:

• Domain architecture: FKBP14 contains two EF-hand domains and one PPIase FKBP-type domain . The PPIase domain is critical for enzymatic activity and must be positioned at the N-terminus for optimal function .

• N-terminal significance: Truncation of the amino-terminus significantly diminishes peptidyl prolyl cis-trans isomerase activity, indicating this region's critical role in enzyme function . When designing recombinant constructs or selecting antibodies, avoid disrupting the N-terminal region.

• ER localization signals: FKBP14 contains a C-terminal HDEL endoplasmic reticulum retention signal that determines its subcellular localization . This signal can interfere with antibody detection when FKBP14 is bound to substrates like collagen .

• Substrate specificity determinants: FKBP14 has a preference for 4-hydroxylproline-containing substrates like type III collagen . This specificity should inform substrate selection in enzymatic assays.

• Collagen binding region: Evidence suggests the HDEL region may be in or near the collagen-binding site , which has implications for experimental design when studying FKBP14-collagen interactions.

When designing experiments to study FKBP14 function, consider these structural elements and their potential impact on antibody binding, enzymatic activity, and protein-protein interactions.

How do FKBP14 mutations relate to disease pathology, and how can antibodies help study these mechanisms?

FKBP14 mutations are associated with serious genetic disorders, and antibodies can be valuable tools for investigating the underlying molecular mechanisms:

• Disease associations: FKBP14 mutations lead to Ehlers-Danlos syndrome with progressive kyphoscoliosis, myopathy, and hearing loss . This gene is located on human chromosome 7, a region also associated with other genetic disorders including osteogenesis imperfecta and Williams-Beuren syndrome .

• Mechanism of pathogenesis: FKBP14 mutations typically lead to loss of the FKBP22 protein, usually through nonsense-mediated decay of mRNA . This loss disrupts proper collagen folding and processing.

• Cellular consequences: FKBP14 deficiency leads to enlarged endoplasmic reticulum cisterns in dermal fibroblasts in vivo , suggesting ER stress and disrupted protein homeostasis.

• Antibody applications in disease research:

  • Using antibodies to detect residual mutant FKBP14 protein expression

  • Immunohistochemistry to analyze tissue-specific effects of FKBP14 deficiency

  • Western blotting to examine effects on downstream proteins in the collagen biosynthesis pathway

  • Co-immunoprecipitation to identify altered protein interactions in disease states

• Novel disease mechanisms: Recent research has identified additional roles for FKBP14 beyond connective tissue disorders. For example, the miR-577/FKBP14 axis has been implicated in colorectal cancer, affecting cell proliferation, invasion, and migration .

When designing studies of FKBP14-related diseases, selecting antibodies that can detect minimal amounts of protein or specific disease-associated variants is critical.

What methodological considerations are important when analyzing FKBP14 expression in different tissues?

Tissue-specific analysis of FKBP14 expression requires attention to several methodological factors:

• Tissue preparation: For IHC-P applications, antigen retrieval conditions can significantly impact results. Some antibodies (e.g., 15884-1-AP) perform optimally with TE buffer pH 9.0, while others may work better with citrate buffer pH 6.0 .

• Tissue-specific expression patterns: FKBP14 antibodies have been validated in various human tissues, including:

  • Pancreas (ab251703)

  • Fetal stomach (ab176795)

  • Brain (15884-1-AP)

  • Ovarian cancer tissue (15884-1-AP)

• Cell line controls: Positive controls for validating FKBP14 antibody reactivity include:

  • HEK-293 cells

  • HeLa cells

  • Cell lysates with FKBP14 overexpression provide excellent positive controls

• Detection of native versus recombinant protein: When analyzing tissues, consider that the observed molecular weight may differ slightly from the calculated weight (24 kDa) due to post-translational modifications. The observed weight is typically around 28 kDa .

• Application-specific dilutions: Optimization is essential, with recommended starting dilutions varying by application and tissue type:

  • WB: 1:500-1:2400

  • IHC: 1:50-1:500

Always perform preliminary experiments to determine optimal conditions for each specific tissue type and application.

What are common troubleshooting strategies for nonspecific binding of FKBP14 antibodies?

Nonspecific binding can compromise experimental results. Here are methodological approaches to address this issue:

• Antibody validation: Confirm specificity using positive and negative controls. For instance, vector-only transfected HEK-293T cells serve as an excellent negative control compared to FKBP14 overexpression lysates .

• Blocking optimization: Adjust blocking conditions (time, temperature, blocking agent) to reduce background. Some FKBP14 antibody formulations (e.g., 15884-1-AP in small sizes) already contain 0.1% BSA, which may affect optimal blocking conditions .

• Dilution optimization: Titrate antibody concentrations to find the optimal signal-to-noise ratio. For example:

  • For WB applications, test a range from 1:200 to 1:2400

  • For IHC applications, test a range from 1:50 to 1:500

• Washing stringency: Increase wash steps or detergent concentration in wash buffers to reduce nonspecific binding.

• Secondary antibody selection: Choose secondary antibodies with minimal cross-reactivity to the species of your sample.

• Fixation and permeabilization: For immunofluorescence or IHC, optimize fixation and permeabilization conditions as these can affect epitope accessibility.

• Preabsorption: For particularly troublesome antibodies, consider preabsorption with non-relevant proteins to remove cross-reactive antibodies.

If nonspecific binding persists despite these measures, consider switching to a different FKBP14 antibody with validated specificity in your application of interest.

How can I validate the specificity of a new FKBP14 antibody?

Rigorous validation is essential when working with a new FKBP14 antibody. A comprehensive validation approach includes:

• Western blot analysis with positive controls: Test the antibody against samples with confirmed FKBP14 expression. Multiple antibodies show reactivity with HEK-293 cells, HeLa cells, and human brain tissue .

• Negative controls: Include samples where FKBP14 is absent or knocked down. Vector-only transfected HEK-293T cells serve as an effective negative control .

• Overexpression systems: Compare detection in normal cells versus cells overexpressing FKBP14. This approach reveals the expected band size and confirms specificity, as demonstrated with FKBP14 overexpression in HEK-293T cells .

• Cross-reactivity testing: Some antibodies, like 15884-1-AP, have been validated specifically for human samples with predicted reactivity for mouse and rat samples . Always confirm cross-reactivity experimentally before proceeding with non-validated species.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to demonstrate that binding is specifically blocked.

• Multiple detection methods: Validate the antibody across different applications (WB, IHC, IF) to ensure consistent results. For example, ab176795 has been validated for IHC-P, WB, and ELISA applications .

• Protein array screening: Some manufacturers perform specificity verification on protein arrays containing the target protein plus hundreds of non-specific proteins .

Thorough validation ensures confidence in experimental results and prevents misinterpretation of data due to antibody cross-reactivity.

What factors affect the detection of FKBP14 in different experimental systems?

Several factors can influence FKBP14 detection across experimental platforms:

• Protein conformation and epitope accessibility: The detection of FKBP14 can be affected by protein folding and interaction with binding partners. For example, HDEL epitopes become inaccessible when FKBP14 binds to collagen III, significantly reducing detection efficiency with HDEL antibodies .

• Tag interference: When using tagged recombinant FKBP14, the position of tags can affect antibody binding and protein function. N-terminal tags may interfere with the PPIase domain function, while C-terminal tags may affect ER localization or substrate binding .

• Fixation methods: For IHC applications, antigen retrieval conditions are critical. Some antibodies perform better with TE buffer pH 9.0, while others may require citrate buffer pH 6.0 .

• Expression levels: FKBP14 expression varies across tissues and cell types. Human brain, HEK-293, and HeLa cells are confirmed to express detectable levels of endogenous FKBP14 .

• Post-translational modifications: The calculated molecular weight of FKBP14 is 24 kDa, but the observed molecular weight is often around 28 kDa, suggesting post-translational modifications that may affect antibody binding .

• Binding kinetics: Fast on/off binding rates between FKBP14 and its substrates can complicate detection in interaction studies .

• Buffer conditions: The composition of lysis buffers, including detergent types and concentrations, can affect protein extraction and subsequent detection.

Understanding these factors allows researchers to optimize experimental conditions for reliable FKBP14 detection across different experimental systems.

How should I approach experimental design when studying FKBP14 interactions with collagens?

Studying FKBP14-collagen interactions requires careful experimental design:

• Selection of detection method: Different methods offer complementary insights:

  • Surface plasmon resonance (SPR) provides direct protein-protein binding measurements and is suitable for untagged FKBP22

  • ELISA-like assays require tagged proteins and indirect detection via antibodies

• Tag selection and antibody compatibility: His-tagged FKBP22 (His-FKBP22) can be effectively detected using anti-His antibodies when bound to collagen III, while detection via HDEL epitopes is problematic due to accessibility issues when the protein is bound to collagen .

• Concentration-dependent binding analysis: Test multiple protein concentrations to establish binding curves. Research shows a concentration-dependent increase in signal when using His-FKBP22 at 10μM (A450 = 0.137 ± 0.092) versus 40μM (A450 = 0.328 ± 0.04) .

• Collagen preparation considerations: Pepsin-treated, purified, full-length collagen III has been successfully used in FKBP14 binding studies . The preparation method can affect protein conformation and binding properties.

• Controls and baseline subtraction: Include controls without FKBP14 to establish baselines and subtract background signals .

• Replicate experiments: Perform at least three replicate experiments for each condition to account for variability in protein-protein interactions .

• Consider binding kinetics: Fast on/off rates between FKBP14 and collagens may affect experimental results and interpretation .

By carefully considering these factors, researchers can design robust experiments to study the physiologically relevant interactions between FKBP14 and collagen substrates.

What are emerging research areas involving FKBP14 antibodies?

FKBP14 research continues to expand beyond traditional roles in collagen processing:

• Cancer biology: Recent findings suggest FKBP14 involvement in colorectal cancer through the miR-577/FKBP14 axis, affecting cancer cell proliferation, invasion, and migration . FKBP14 antibodies will be essential tools for exploring these oncogenic mechanisms.

• Structural biology: Understanding the precise interaction between FKBP14 and its substrates, particularly the role of the protein's net charge state in determining collagen binding specificity .

• Therapeutic targeting: As connections between FKBP14 and disease pathways become clearer, antibodies will be crucial for validating therapeutic approaches targeting this protein.

• Disease biomarker development: Exploring FKBP14 expression patterns across pathological conditions may reveal new biomarker applications requiring sensitive and specific antibodies.

• Genetic disorder mechanisms: Further investigation into how FKBP14 mutations lead to Ehlers-Danlos syndrome and related disorders will require antibodies capable of detecting mutant variants and studying downstream effects.