FKBP1B Antibody

What is FKBP1B and what cellular functions does it regulate?

FKBP1B (FK506 binding protein 1B, 12.6 kDa) is a protein that functions as a negative regulator of ryanodine receptor Ca2+ release, playing a crucial role in calcium homeostasis. The protein has a calculated molecular weight of 12 kDa, which matches its observed molecular weight in experimental settings . FKBP1B is particularly important in neuronal and cardiac tissues, where it regulates calcium signaling pathways. Research has shown that FKBP1B overexpression can reverse aging-induced memory impairment and neuronal Ca2+ dysregulation, suggesting its critical role in maintaining normal neuronal function . The protein appears to protect downstream transcriptional networks from aging-induced dysregulation, affecting approximately 37% of genes that show altered expression with aging .

What are the key specifications of the FKBP1B antibody (15114-1-AP)?

The FKBP1B antibody (15114-1-AP) is a rabbit polyclonal antibody produced using FKBP1B fusion protein Ag7153 as the immunogen . It has been validated for several experimental applications with specific reactivity profiles:

This antibody has been experimentally verified to detect endogenous FKBP1B in multiple tissue types, including brain and heart tissues, making it suitable for comparative studies across these tissues .

What are the validated applications for FKBP1B antibody and their recommended dilutions?

The FKBP1B antibody (15114-1-AP) has been validated for multiple experimental applications with specific recommended dilutions for optimal results:

For immunohistochemistry applications, optimal antigen retrieval conditions have been established: TE buffer pH 9.0 is suggested, with citrate buffer pH 6.0 as an alternative option . Researchers should note that the antibody may require titration in each testing system to obtain optimal results, as performance can be sample-dependent .

What is the recommended protocol for FKBP1B Western blot analysis?

For Western blot analysis of FKBP1B, researchers should follow these methodological steps:

• Sample Preparation: Prepare protein lysates from tissues (brain or heart recommended) or cell lines (U-937 or Jurkat cells have been validated) .

• Dilution Optimization: Start with a 1:500-1:2000 dilution range of the antibody. The exact dilution should be optimized for each experimental system .

• Expected Results: The antibody should detect a band at approximately 12 kDa, which corresponds to the observed molecular weight of FKBP1B .

• Controls: Include positive controls such as mouse brain tissue, which has been validated to express FKBP1B detectable by this antibody .

• Storage Considerations: Store the antibody at -20°C, where it remains stable for one year after shipment. For the 20μl size, note that it contains 0.1% BSA .

How should immunohistochemistry for FKBP1B be performed?

For immunohistochemical detection of FKBP1B, researchers should implement the following procedure:

• Section Preparation: Cut coronal sections (30 μm recommended based on published protocols) .

• Antigen Retrieval: Use TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0 as an alternative .

• Primary Antibody Incubation: Dilute the FKBP1B antibody 1:50-1:500 (optimize for your tissue) and incubate overnight .

• Secondary Antibody Selection: Use an appropriate biotinylated secondary antibody followed by ExtrAvidin incubation (2 hours each) .

• Visualization: Develop with Ni-enhanced DAB solution (approximately 3 minutes) .

• Standardization: To ensure comparable staining intensity, process all sections simultaneously in the same staining tray .

The antibody's staining pattern should match established topography for FKBP1B in the target tissue. For brain tissue, the pattern has been well-characterized in previous studies, particularly in the CA1 region of the hippocampus . To validate specificity, include negative controls by omitting the primary antibody and using only secondary antibody on adjacent sections from the same subjects .

How can FKBP1B antibody be used to study aging-related processes?

FKBP1B plays a significant role in aging-related processes, particularly in the brain. Researchers can use the FKBP1B antibody for several advanced applications:

• Expression Analysis: Compare FKBP1B expression levels between young and aged subjects using Western blot or immunohistochemistry. Note that region-specific analysis (e.g., focused on CA1) may be necessary to detect aging-related changes that might be obscured in whole-hippocampus analyses .

• Intervention Studies: Examine the effects of FKBP1B overexpression on age-related phenotypes. Previous research has shown that both short-term and long-term overexpression of FKBP1B can improve memory performance in aged rats .

• Transcriptional Network Analysis: Investigate how FKBP1b modulates aging-related gene expression. Research has identified that FKBP1b overexpression can restore expression of approximately 99.5% of aging-affected genes in the direction opposite to the aging effect .

• Structural Analysis: Study the impact of FKBP1b on cellular structures. Immunohistochemical analysis has confirmed that FKBP1b overexpression can restore neuronal microtubular structure that undergoes rarefaction with aging .

When designing these experiments, researchers should consider using region-specific analyses (particularly CA1 of the hippocampus) to maximize detection sensitivity for age-related changes in FKBP1B expression.

What transcriptional networks are affected by FKBP1B and how can they be studied?

Research has identified distinct transcriptional networks affected by FKBP1B, which can be studied using a combination of FKBP1B antibody techniques and transcriptional profiling:

• Genes Restored by FKBP1B: Of 2342 genes with expression altered by aging, 876 (37%) showed expression changes with FKBP1B treatment, with 99.5% of these being restored in the direction opposite to the aging effect . These genes predominantly associated with brain structural categories including:

  • Cytoskeleton

  • Membrane channels

  • Extracellular region

• Genes Not Restored by FKBP1B: Genes upregulated with aging but not affected by FKBP1B were primarily associated with:

  • Glial-neuroinflammatory processes

  • Ribosomal pathways

  • Lysosomal categories

To study these networks, researchers can implement a combined approach:

• Viral Vector Overexpression: Use AAV-FKBP1b injection to overexpress FKBP1b, followed by verification of overexpression using qRT-PCR and immunohistochemistry with the FKBP1B antibody .

• Transcriptional Profiling: Perform microarray or RNA-seq analysis to identify differentially expressed genes.

• Functional Validation: Confirm expression changes of key genes at the protein level using western blot or immunohistochemistry.

• Pathway Analysis: Use tools like DAVID to identify overrepresented functional categories among differentially expressed genes .

This integrated approach can reveal the complex interplay between FKBP1B, calcium regulation, and aging-related transcriptional networks.

How can researchers address potential cross-reactivity or specificity issues with FKBP1B antibody?

When using FKBP1B antibody, researchers should implement several validation steps to ensure specificity and address potential cross-reactivity:

• Negative Controls: Always include negative controls by omitting the primary antibody and using only secondary antibody on adjacent sections from the same subjects .

• Knockdown Validation: Consider validating antibody specificity through knockdown experiments, such as using short hairpin RNA targeting FKBP1b, as demonstrated in previous studies .

• Overexpression Controls: Include FKBP1b-overexpressing samples as positive controls, which should show increased immunoreactivity .

• Western Blot Validation: Confirm the molecular weight of detected bands (expected at 12 kDa for FKBP1B) .

• Cross-Species Verification: When working across species, verify that the staining pattern is consistent with known species-specific expression patterns of FKBP1B.

The FKBP1B antibody (15114-1-AP) has been validated in previous studies through these approaches, showing consistent topography of FKBP1B immunostaining and successful detection of experimental manipulations and aging-related differences in FKBP1B expression in the CA1 region .

What are the recommended approaches for quantifying FKBP1B expression in tissue samples?

For accurate quantification of FKBP1B expression in tissue samples, researchers should consider these methodological approaches:

• Optical Densitometry for IHC:

  • Digitize immunostained sections using standardized camera settings (e.g., using an Olympus DP73 camera)

  • Analyze using software such as ImageJ

  • For hippocampal analyses, measure at least two sections of the apical dendritic layer (stratum radiatum) of CA1 pyramidal neurons per animal

  • Ensure investigators are blinded to animal number and condition

• Western Blot Quantification:

  • Use appropriate housekeeping protein controls (e.g., GAPDH) for normalization

  • Implement densitometric analysis of band intensity

  • Ensure equal protein loading across samples

• Gene Expression Analysis:

  • For qRT-PCR quantification, normalize FKBP1b expression to stable reference genes (e.g., GAPDH)

  • Verify RNA quality (RNA integrity number ≥ 9.0 recommended based on previous studies)

  • For microarray studies, an n of 5-10 per group has been determined sufficient to identify distinct hippocampal transcriptome signatures

When comparing young and aged subjects, researchers should be aware that whole dorsal hippocampus samples might not show age-related differences in FKBP1B expression that would be detectable in region-specific analyses (particularly in CA1) .

How should researchers interpret changes in FKBP1B expression in relation to functional outcomes?

When interpreting FKBP1B expression changes in relation to functional outcomes, researchers should consider several important factors:

• Correlation with Behavioral Measures: Previous research has shown that FKBP1B overexpression can substantially improve memory performance in aged rats in tests such as the Morris water maze . When analyzing FKBP1B expression data, correlate expression levels with behavioral performance measures.

• Regional Specificity: Changes in FKBP1B expression may be region-specific, particularly in the brain. The CA1 region of the hippocampus has shown more pronounced age-related changes in FKBP1B expression compared to whole hippocampus .

• Downstream Pathway Analysis: Consider analyzing markers of downstream pathways affected by FKBP1B, such as:

  • Cytoskeletal components (e.g., MAP2) which can reflect FKBP1B's effects on neuronal structure

  • Calcium regulatory pathways, given FKBP1B's role as a negative regulator of ryanodine receptor Ca2+ release

• Expression Restoration Patterns: When using interventions like FKBP1B overexpression, examine whether expression patterns of age-affected genes return to levels similar to young controls. Prior research indicates that FKBP1B restoration moves gene expression in the direction opposite to aging effects .

• Timing Considerations: Both short-term (ST) and long-term (LT) FKBP1B overexpression have shown beneficial effects, but LT treatment (initiated at 13 months of age in rats) has demonstrated more intense FKBP1B upregulation compared to ST treatment (initiated at 19 months) .

What are common challenges when using FKBP1B antibody and how can they be addressed?

Researchers may encounter several challenges when working with FKBP1B antibody that can be addressed through methodological adjustments:

• Variable Signal Intensity:

  • Challenge: Inconsistent staining intensity between samples

  • Solution: Process all sections simultaneously in the same staining tray and standardize all steps of the protocol, including incubation times and temperatures

• Background Staining:

  • Challenge: High background obscuring specific signal

  • Solution: Optimize blocking conditions, increase washing steps, and titrate antibody dilution within the recommended range (1:50-1:500 for IHC, 1:500-1:2000 for WB)

• Antigen Retrieval Issues:

  • Challenge: Poor or inconsistent staining in fixed tissues

  • Solution: Compare recommended TE buffer pH 9.0 with alternative citrate buffer pH 6.0 to determine optimal conditions for your specific tissue samples

• Detection in Whole Tissue vs. Specific Regions:

  • Challenge: Failure to detect age-related changes in FKBP1B expression in whole tissue samples

  • Solution: Focus analysis on specific regions known to show stronger age-related changes (e.g., CA1 region of hippocampus rather than whole hippocampus)

• Storage-Related Antibody Performance Decline:

  • Challenge: Reduced antibody performance over time

  • Solution: Store at -20°C as recommended, where the antibody remains stable for one year after shipment. For 20μl sizes containing 0.1% BSA, avoid repeated freeze-thaw cycles

How can I optimize FKBP1B antibody protocols for different experimental models?

Optimizing FKBP1B antibody protocols for different experimental models requires systematic adjustment of several parameters:

• Species-Specific Optimization:

  • The antibody has confirmed reactivity with human, mouse, and rat samples

  • When working with new species, validate antibody performance using positive controls from validated species alongside your experimental samples

  • Consider cross-validation with alternative FKBP1B antibodies if available

• Application-Specific Dilution Optimization:

  • Western Blot: Begin with 1:1000 dilution and adjust within the 1:500-1:2000 range

  • IHC: Start with 1:100 dilution and optimize within the 1:50-1:500 range

  • For each new cell line or tissue type, perform a dilution series to determine optimal concentration

• Tissue-Specific Antigen Retrieval:

  • For formalin-fixed tissues, compare the recommended TE buffer pH 9.0 with citrate buffer pH 6.0

  • Optimize retrieval times based on fixation duration and tissue type

  • For heart tissue, which has been validated for this antibody, follow the specific protocol recommendations provided by the manufacturer

• Transgenic or Manipulated Models:

  • For FKBP1B overexpression studies, verify increased expression using both qRT-PCR and immunohistochemistry

  • For knockdown studies, confirm reduced expression similarly through dual methods

  • When using viral vector delivery (e.g., AAV-FKBP1b), confirm successful transduction through immunohistochemistry

Testing these parameters systematically will help establish optimal conditions for your specific experimental model while ensuring reliable and reproducible results.