FBL13 Antibody

What is FKBP13 and why is it significant in research?

FKBP13 (FK506 binding protein, 13 kilodalton molecular weight, also called FKBP2) is a peptidyl-prolyl isomerase that catalyzes the transition between cis- and trans-proline residues, which is critical for proper protein folding. It is significant in research because it is localized in the endoplasmic reticulum and associated with cytoskeletal elements such as erythrocyte band 4.1. FKBP13 levels increase with treatments affecting Golgi protein processing, suggesting an important role in the unfolded protein response observed during endoplasmic reticulum stress. The immunosuppressants FK506 and rapamycin are potent inhibitors of FKBP13, making it relevant for immunological research .

What species reactivity is available for FKBP13 antibodies?

FKBP13 antibodies are available with cross-reactivity to multiple species, including human, mouse, and rat. This multi-species reactivity is particularly valuable for comparative studies across model organisms. The antibodies are typically generated using E. coli-derived recombinant human FKBP13 (Ala22-Leu142, Accession # P26885) as the immunogen, which shares high sequence homology with mouse and rat orthologs .

What are the primary applications for FKBP13 antibodies?

FKBP13 antibodies are primarily used in Western blotting, immunohistochemistry, and immunofluorescence applications. They are valuable tools for studying protein folding mechanisms, endoplasmic reticulum stress responses, and the unfolded protein response pathway. Researchers typically use these antibodies to detect FKBP13 expression levels in various cell types and to investigate its interactions with other proteins involved in cellular stress responses .

What is the optimal protocol for detecting FKBP13 via Western blot?

For optimal Western blot detection of FKBP13, the following methodology is recommended:

• Prepare cell or tissue lysates using standard lysis buffers containing protease inhibitors

• Separate proteins on 12-15% SDS-PAGE gels (optimal for low molecular weight proteins)

• Transfer to PVDF membrane (preferred over nitrocellulose for small proteins)

• Block with 5% non-fat dry milk or BSA in TBST

• Probe with FKBP13 antibody at 1 μg/mL concentration

• Incubate with appropriate HRP-conjugated secondary antibody

• Develop using enhanced chemiluminescence

FKBP13 will appear as a specific band at approximately 13 kDa. When working with rat embryonic fibroblast cell lines (Rat-2) or mouse neuroblastoma cell lines (Neuro-2A), this protocol has been validated to show specific detection. The use of Immunoblot Buffer Group 1 is recommended for optimal results .

How should researchers optimize antibody dilutions for different applications?

Optimal antibody dilutions should be determined through titration experiments for each specific application and sample type. For Western blotting, start with a concentration of 1 μg/mL and perform a dilution series (0.5-2 μg/mL) to determine optimal signal-to-noise ratio. For immunohistochemistry applications, begin with 10-15 μg/mL based on protocols used for similar antibodies such as BAFF/BLyS/TNFSF13B, where 15 μg/mL was effective for formalin-fixed paraffin-embedded sections . For flow cytometry, follow protocols similar to those used for other intracellular proteins, which typically involve fixation with a flow cytometry fixation buffer and permeabilization with appropriate wash buffers .

What controls should be included when validating FKBP13 antibody specificity?

To validate FKBP13 antibody specificity, researchers should include the following controls:

• Positive control: Cell lines known to express FKBP13 (e.g., Rat-2 or Neuro-2A)

• Negative control: FKBP13 knockout cell lines or cells with siRNA-mediated FKBP13 knockdown

• Isotype control: Appropriate species-matched immunoglobulin at the same concentration

• Peptide competition assay: Pre-incubation of the antibody with excess immunizing peptide

• Cross-reactivity assessment: Testing against related FKBP family members

These controls help distinguish specific from non-specific binding and validate antibody performance across different experimental conditions .

How can FKBP13 antibodies be used to study the unfolded protein response?

FKBP13 antibodies can be used to investigate the unfolded protein response (UPR) through several advanced approaches:

• Induction experiments: Treat cells with UPR inducers (tunicamycin, thapsigargin, heat shock) and monitor FKBP13 expression changes via Western blot or immunofluorescence

• Co-immunoprecipitation: Use FKBP13 antibodies to pull down and identify interaction partners during ER stress

• Chromatin immunoprecipitation (ChIP): Investigate transcription factor binding to the FKBP13 promoter during stress

• Proximity ligation assays: Detect in situ interactions between FKBP13 and other UPR components

• Time-course studies: Monitor FKBP13 expression dynamics throughout the UPR timeline

These approaches can reveal the temporal dynamics of FKBP13 upregulation during ER stress and its functional relationships with other UPR components .

What strategies exist for studying FKBP13 interactions with cytoskeletal elements?

To study FKBP13 interactions with cytoskeletal elements such as erythrocyte band 4.1, researchers can employ several advanced techniques:

• Co-immunoprecipitation with cytoskeletal proteins: Use FKBP13 antibodies to pull down complexes and probe for cytoskeletal proteins

• Immunofluorescence co-localization: Perform dual staining with FKBP13 antibody and cytoskeletal markers

• Subcellular fractionation: Separate cytoskeletal fractions and analyze FKBP13 distribution

• Proximity-dependent biotin labeling (BioID): Identify proteins in close proximity to FKBP13

• FRET/BRET assays: Measure direct interactions between FKBP13 and cytoskeletal proteins

• Super-resolution microscopy: Visualize precise co-localization at nanometer resolution

These approaches can help elucidate the functional significance of FKBP13's association with the cytoskeleton and its potential role in cellular structure and dynamics .

How can biophysics-informed models enhance antibody specificity against FKBP13?

Biophysics-informed models can significantly enhance antibody specificity against FKBP13 through:

• Binding mode analysis: Computational models can identify distinct binding modes specific to FKBP13 versus related proteins

• Energy function optimization: Minimize binding energy functions for FKBP13 while maximizing them for undesired targets

• Sequence optimization: Generate novel antibody sequences with customized specificity profiles

• Epitope mapping: Identify and target unique epitopes on FKBP13 not present in related proteins

• Cross-reactivity prediction: Predict potential cross-reactivity with other FKBP family members

These computational approaches can guide experimental design for generating highly specific antibodies. For example, phage display experiments can be designed based on these predictions to select antibody libraries against specific combinations of ligands. The model can then be trained on experimentally selected antibodies to predict outcomes for new ligand combinations .

What are common causes of non-specific binding with FKBP13 antibodies and how can they be mitigated?

Common causes of non-specific binding and their solutions include:

Methodical optimization of these parameters can significantly improve signal-to-noise ratio and ensure specific detection of FKBP13 .

How should researchers troubleshoot loss of FKBP13 antibody activity over time?

To address loss of FKBP13 antibody activity over time, researchers should:

• Implement proper storage conditions: Store antibodies at -20 to -70°C for long-term storage or at 2-8°C for short-term (up to 1 month) after reconstitution

• Avoid freeze-thaw cycles: Aliquot reconstituted antibody to minimize freeze-thaw cycles

• Use sterile conditions: Maintain sterility during antibody handling and reconstitution

• Add preservatives: Consider adding preservatives like sodium azide (0.02%) for antibodies stored at 2-8°C

• Monitor expiration dates: Use within the recommended shelf life (typically 12 months from receipt if stored properly)

• Check for precipitates: Centrifuge to remove any aggregates before use

• Validate periodically: Routinely test activity using positive control samples

Following these guidelines helps maintain antibody activity and ensures consistent experimental results over time .

What approaches can resolve contradictory results between different detection methods?

When facing contradictory results between different detection methods using FKBP13 antibodies, researchers should:

• Verify antibody specificity: Perform validation experiments including Western blot, knockdown controls, and peptide competition assays

• Consider epitope accessibility: Different fixation or sample preparation methods may differentially affect epitope exposure

• Evaluate subcellular localization: FKBP13 is primarily in the endoplasmic reticulum; confirm proper cell permeabilization for intracellular detection

• Test multiple antibody clones: Different antibodies may recognize different epitopes with varying accessibility

• Compare detection sensitivities: Flow cytometry, Western blot, and immunohistochemistry have different detection thresholds

• Rule out technical artifacts: Systematically troubleshoot each protocol component

• Biological variability: Consider if contradictions reflect actual biological differences in expression or localization

What are optimal storage conditions for maintaining FKBP13 antibody activity?

For optimal maintenance of FKBP13 antibody activity, follow these storage guidelines:

• Lyophilized form: Store at -20 to -70°C for up to 12 months from date of receipt

• Reconstituted antibody:

  • For short-term use (≤1 month): Store at 2-8°C under sterile conditions

  • For long-term storage (≤6 months): Store at -20 to -70°C under sterile conditions

• Working dilutions: Prepare fresh on the day of use or store at 4°C for up to one week

• Avoid repeated freeze-thaw cycles: Use a manual defrost freezer and aliquot antibody upon reconstitution

• Transportation: Ship on ice packs for short transit periods (1-2 days) or on dry ice for longer periods

Proper storage is critical for maintaining antibody activity and ensuring reproducible experimental results .

What reconstitution protocols are recommended for lyophilized FKBP13 antibodies?

For reconstitution of lyophilized FKBP13 antibodies, follow these recommended protocols:

• Equilibration: Allow the lyophilized antibody to reach room temperature before opening

• Reconstitution buffer selection:

  • For most applications: Use sterile PBS

  • For applications requiring carrier protein: Use PBS containing 0.1% BSA

  • For specific applications: Consult buffer recommendations for each antibody

• Reconstitution process:

  • Add buffer slowly to the vial

  • Gently rotate or swirl (do not vortex) to ensure complete dissolution

  • Allow to stand for 5-10 minutes at room temperature

• Concentration calculation: Use the reconstitution calculator provided by the manufacturer to achieve desired concentration

• Aliquoting: Divide into single-use aliquots to avoid freeze-thaw cycles

• Documentation: Record reconstitution date and concentration on each vial

Proper reconstitution ensures maintenance of antibody activity and minimizes variability between experiments .

How should researchers normalize and quantify FKBP13 expression levels in Western blots?

For accurate normalization and quantification of FKBP13 expression in Western blots:

• Loading control selection:

  • Use housekeeping proteins appropriate for your experimental conditions (GAPDH, β-actin, α-tubulin)

  • Consider compartment-specific controls for ER-localized proteins (e.g., calnexin, PDI)

• Quantification methodology:

  • Use digital image analysis software (ImageJ, Image Lab, etc.)

  • Define regions of interest (ROIs) consistently across all lanes

  • Subtract background using a rolling ball algorithm or local background subtraction

• Normalization calculation:

  • Calculate the ratio of FKBP13 band intensity to loading control

  • For time-course or treatment studies, normalize to untreated control

• Statistical analysis:

  • Perform multiple independent experiments (n≥3)

  • Apply appropriate statistical tests based on data distribution

  • Report means, standard deviations, and p-values

• Data visualization:

  • Present both representative blot images and quantification graphs

  • Include all experimental replicates in analysis

This systematic approach ensures reliable quantification and meaningful interpretation of FKBP13 expression changes .

What criteria should be used to evaluate FKBP13 antibody specificity in experimental results?

To rigorously evaluate FKBP13 antibody specificity in experimental results, researchers should apply these criteria:

• Band/signal molecular weight: Confirm detection at the expected molecular weight (approximately 13 kDa)

• Knockout/knockdown validation: Demonstration of signal reduction in FKBP13-depleted samples

• Peptide competition: Signal elimination when antibody is pre-incubated with immunizing peptide

• Cross-reactivity profile: Minimal reactivity with other FKBP family members

• Subcellular localization: Proper ER localization consistent with FKBP13 biology

• Consistent detection across methods: Similar detection patterns in different applications

• Protocol dependency: Consistency across different experimental conditions

• Comparison with multiple antibodies: Similar results using antibodies targeting different epitopes

These criteria establish a comprehensive framework for validating antibody specificity, enhancing result reliability and reproducibility .

How can researchers distinguish between FKBP13 and other FKBP family members in their results?

To distinguish FKBP13 from other FKBP family members in experimental results:

• Molecular weight discrimination:

  • FKBP13: ~13 kDa

  • FKBP12: ~12 kDa

  • FKBP25: ~25 kDa

  • FKBP51: ~51 kDa

  • FKBP52: ~52 kDa

• Subcellular localization:

  • FKBP13: Predominantly in the endoplasmic reticulum

  • FKBP12: Cytoplasmic

  • Other family members: Various localizations including cytoplasm, nucleus, mitochondria

• Expression pattern analysis:

  • Compare tissue/cell type expression profiles characteristic of each family member

• Specific antibody selection:

  • Use antibodies raised against unique, non-conserved regions

  • Verify lack of cross-reactivity against recombinant FKBP family proteins

• Functional assays:

  • Assess specific biological activities (e.g., FKBP13's role in the unfolded protein response)

• Mass spectrometry validation:

  • Confirm protein identity through peptide mass fingerprinting

These approaches help ensure that observed signals genuinely represent FKBP13 rather than related family members .