FKBP43 Antibody

Basic Research Questions

• What are the key characteristics of the FKBP protein family?

  FKBP (FK506 binding protein) family members are immunophilins with peptidylprolyl cis-trans isomerase (PPIase) activity. These proteins contain functional domains including the PPIase domain and domains binding to immunosuppression. FKBP3 (also known as FKBP25) is primarily located in the nucleus and functions as a molecular chaperone for regulatory proteins like MDM2, affecting p53 and p21 expression . Other family members like FKBP52 (FKBP4) demonstrate co-chaperone activities and interact with heat-shock protein 90 (HSP90) .

• What are the typical subcellular localizations of FKBP family proteins?

  FKBP family proteins exhibit diverse subcellular localizations related to their functions. FKBP3 is predominantly nuclear, where it can regulate gene expression and protein interactions . In contrast, FKBP52 is involved in intracellular trafficking of steroid hormone receptors between cytoplasmic and nuclear compartments, indicating a more dynamic localization pattern . When designing experiments to study FKBP43, researchers should consider potential compartmentalization effects on antibody accessibility and function.

• What functional domains should researchers target when developing FKBP-specific antibodies?

  When developing antibodies against FKBP family proteins, researchers should consider targeting:

  • The peptidylprolyl cis-trans isomerase domain (highly conserved)

  • Protein-specific domains that bind to immunosuppression

  • C-terminal regions that may contain unique epitopes

  For example, the FKBP52 antibody described in the literature targets a synthetic peptide within the C-terminus (aa 400 to C-terminus) of human FKBP4 . This approach helps ensure specificity while maintaining functional recognition.

Advanced Research Applications

• How do FKBP proteins influence viral latency mechanisms?

  FKBP3 has been identified as a critical regulator of HIV-1 latency. Research demonstrates that FKBP3 knockout significantly induces reactivation of latent HIV-1 by approximately 30% in C11 cells containing an HIV-1 proviral DNA with a GFP reporter gene . Similar activation effects were observed in J-Lat 10.6 and ACH2 cell lines. During HIV-1 infection of primary CD4+ T lymphocytes, FKBP3 expression increases significantly, suggesting its involvement in the immune response to infection . These findings indicate FKBP family proteins may be relevant targets in viral latency research beyond HIV.

• What experimental approaches are most effective for studying FKBP knockout effects?

  For studying FKBP knockout effects, the following approaches have demonstrated effectiveness:

  • CRISPR/Cas9 gene editing: Using specific guide RNAs targeting FKBP genes followed by puromycin selection (2 μg/ml for 14 days) has proven successful for stable knockout models .

  • Primary cell models: For primary cells such as CD4+ T lymphocytes, electroporation-based delivery of Cas9-sgRNA targeting FKBP genes provides effective knockout .

  • Validation methods: Confirming knockout through genomic DNA sequencing of target sites and protein level detection ensures experimental validity .

  • Functional readouts: Reporter systems (like GFP or luciferase) provide quantifiable measures of functional effects following FKBP knockout .

• How can researchers design FKBP-ligand interactions for targeted applications?

  Designing specific FKBP-ligand interactions involves strategic interface remodeling:

  • Specificity pocket engineering: By modifying the FKBP-ligand interface to introduce a specificity binding pocket, researchers have achieved 1,000-fold selectivity for mutant FKBPs over wild-type proteins .

  • Structural modifications: Substituting an ethyl group for a carbonyl group in ligands can enhance binding to mutant FKBPs with compensating truncations of specific amino acid residues (e.g., phenylalanine) .

  • Dimerizer development: Homodimers of modified ligands (like AP1903) can be used to control cellular activities in genetically modified cells without interference from endogenous FKBP .

  These principles can guide the development of FKBP43-specific interaction systems for controlled cellular processes.

Methodological Guidance

• What are the recommended protocols for validating FKBP antibody specificity?

  Comprehensive validation of FKBP antibody specificity should include:

  • Western blot analysis: Comparing reactivity against wild-type and knockout cell lysates to confirm target specificity

  • Cross-reactivity testing: Examining reactivity against multiple FKBP family members to ensure target selectivity

  • Immunoprecipitation: Verifying ability to pull down the target protein and known interaction partners

  • Species compatibility: Systematic testing across species with strong homology, as demonstrated with the FKBP52 antibody which reacts with both mouse and human samples

• How can mass spectrometry approaches be applied to analyze FKBP antibody-antigen interactions?

  Mass spectrometry offers powerful approaches for analyzing FKBP antibody-antigen interactions:

  • LC-MS-based profiling: Proteins can be separated and analyzed using nanoLC coupled to mass spectrometers like Orbitrap or timsTOF platforms .

  • Intact protein analysis: For antibody fragments like Fabs, charge state distributions typically range from z=22 to z=42, enabling precise mass determination with errors around 6.8 ppm .

  • Sample preparation: For antibody studies, IgGs can be affinity-enriched from serum, with Fab fragments cleaved from Fc parts using bacterial hinge-directed proteases like IgdE .

  • Data analysis workflows: Software like Bruker Compass DataAnalysis, combined with customized scripts, can process complex antibody repertoire data effectively .

• What controls are essential when evaluating FKBP family protein functions in cellular pathways?

  Essential controls for studying FKBP family protein functions include:

  • Cell viability assessment: Monitoring proliferation (e.g., using CCK-8 assays) and apoptosis (e.g., TUNEL staining) following FKBP manipulation ensures observed effects aren't due to general cellular toxicity .

  • Off-target effect evaluation: Examining effects on related cellular pathways confirms specificity of observed phenotypes

  • Rescue experiments: Re-expressing wild-type or mutant FKBP proteins in knockout cells can confirm direct causality

  • Parallel pathway assessment: For instance, when studying FKBP3's role in HIV latency, researchers confirmed it did not act by suppressing cellular immune responses by examining expression of immune-related genes

Table 1: Comparison of Key FKBP Family Members Relevant to Antibody Research

*Note: Specific information about FKBP43 was not available in the provided search results. The information presented for other FKBP family members may provide guidance for FKBP43 research based on conserved family characteristics.

Table 2: Common Applications of FKBP Family Antibodies in Research