FKBP1B Antibody, HRP conjugated

What is FKBP1B and why is it significant in research?

FKBP1B (FK506 Binding Protein 1B), also known as FKBP12.6 or calstabin 2, is a 12.6 kDa peptidyl-prolyl cis-trans isomerase that catalyzes the isomerization of proline imidic peptide bonds in oligopeptides, thereby accelerating protein folding processes. This protein has significant research importance due to its role in immunoregulation, protein folding, and cellular signaling. FKBP1B has the potential to contribute to the immunosuppressive and toxic effects of FK506 (tacrolimus) and rapamycin, making it a crucial target in transplantation and immunology research . Most notably, FKBP1B is localized in the sarcoplasmic reticulum where it associates with the Ryanodine receptor RYR2, playing a critical role in calcium release regulation in cardiac tissue. This association has profound implications for heart failure research, as male FKBP12.6 knockout mice exhibit cardiac hypertrophy and lethal exercise arrhythmias .

What are the recommended applications for FKBP1B HRP-conjugated antibodies?

FKBP1B HRP-conjugated antibodies are validated for multiple research applications, with the most common being Western blot, immunohistochemistry on paraffin-embedded sections (IHC-P), immunofluorescence (IF), immunocytochemistry (ICC), and ELISA . For Western blot applications, these antibodies typically detect FKBP1B at approximately 12-13 kDa under reducing conditions, as demonstrated in human brain tissue samples . In immunohistochemistry, they have been successfully used to visualize FKBP1B distribution in tissues such as human brain (caudate nucleus and hippocampus) . The direct HRP conjugation particularly benefits high-throughput ELISA protocols by reducing incubation steps and streamlining the detection process. When designing experiments, researchers should consider using the recommended dilution ranges for each application (typically 1:500-1:2000 for Western blot, 1:50-1:100 for IHC-P and IF/ICC) to achieve optimal signal-to-noise ratios .

How can I verify FKBP1B antibody specificity against related FKBP family proteins?

Verifying antibody specificity is crucial when studying FKBP1B due to structural similarities with other FKBP family members. To rigorously assess specificity, implement a multi-faceted approach combining complementary techniques. First, perform Western blot analysis using recombinant proteins representing various FKBP family members (FKBP12, FKBP13, FKBP25, FKBP38, FKBP52) alongside FKBP1B/FKBP12.6 as demonstrated in specificity testing . This comparative analysis reveals potential cross-reactivity patterns, with some antibodies showing approximately 15% cross-reactivity with recombinant human FKBP12, while maintaining high specificity for FKBP1B . Second, validate antibody performance using FKBP1B knockout/knockdown samples as negative controls. Third, conduct peptide competition assays using the immunizing peptide sequence (typically corresponding to amino acids 1-80 of human FKBP1B, NP_473374.1) . Finally, confirm specificity through orthogonal detection methods such as mass spectrometry validation of immunoprecipitated proteins. Document the antibody's recognition of specific FKBP1B isoforms and cross-species reactivity to ensure accurate data interpretation.

What controls are essential when using FKBP1B HRP-conjugated antibodies?

Implementing a comprehensive set of controls is essential for generating reliable and interpretable data with FKBP1B HRP-conjugated antibodies. Positive controls should include samples known to express FKBP1B, such as human brain tissue (particularly hippocampus and cortex regions), 22Rv1 cell line, mouse brain, or rat brain tissue . Tissue specificity controls are important since FKBP1B shows differential expression, with isoform 1 and isoform 2 being ubiquitously expressed but with highest levels in brain and thymus . For negative controls, include samples lacking FKBP1B expression or samples from FKBP1B knockout models. Additional essential controls include: (1) loading controls to normalize for protein quantity variations; (2) isotype controls using non-specific IgG from the same host species; (3) blocking peptide controls to demonstrate binding specificity to the immunizing peptide sequence; and (4) substrate-only controls to assess potential endogenous peroxidase activity or non-specific substrate reactions. When conducting quantitative analyses, include standard curves with purified recombinant FKBP1B protein to ensure accurate protein quantification.

How do I optimize detection protocols for FKBP1B in cardiac tissue samples?

Optimizing FKBP1B detection in cardiac tissue requires consideration of its subcellular localization and interaction with the Ryanodine receptor (RYR2). Begin with careful sample preparation by using a lysis buffer containing phosphatase inhibitors to preserve the phosphorylation state of RYR2, which affects FKBP1B association . Since FKBP1B localizes to the sarcoplasmic reticulum in cardiac tissue, consider subcellular fractionation protocols to enrich for these compartments. For Western blot detection, use reducing conditions with Immunoblot Buffer Group 2 as recommended for optimal detection of the 12.6-13 kDa FKBP1B protein . When performing immunohistochemistry on cardiac sections, implement antigen retrieval methods to expose epitopes potentially masked by formalin fixation. Consider dual immunostaining with RYR2 antibodies to investigate co-localization patterns. In research focusing on heart failure models, carefully monitor FKBP1B dissociation from hyperphosphorylated RYR2, as this association is significantly reduced in failing cardiac tissue . For quantitative analysis of FKBP1B expression or RYR2 association, densitometry analysis of Western blots should be performed with appropriate normalization to loading controls.

What sample preparation protocols yield optimal results with FKBP1B HRP-conjugated antibodies?

Optimal sample preparation significantly impacts experimental success with FKBP1B HRP-conjugated antibodies. For protein extraction, use a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with protease and phosphatase inhibitors. This formulation effectively preserves both FKBP1B integrity and its interactions with binding partners. For tissues with high endogenous peroxidase activity, include hydrogen peroxide treatment steps to quench background signal that could interfere with specific HRP detection. When preparing samples for Western blot, denature proteins using reducing conditions and use Immunoblot Buffer Group 1 or 2 as recommended for optimal resolution of the 12.6-13 kDa FKBP1B protein . For fixed tissue sections intended for immunohistochemistry, implement heat-induced epitope retrieval using citrate buffer (pH 6.0) to maximize antibody accessibility to FKBP1B epitopes. When processing samples from different species, consider potential variations in epitope conservation that might affect antibody recognition, though most FKBP1B antibodies show cross-reactivity with human, mouse, and rat samples .

How should dilution optimization be approached for different applications?

Systematic dilution optimization is crucial for achieving robust and reproducible results with FKBP1B HRP-conjugated antibodies across different applications. Begin with the manufacturer's recommended dilution ranges: 1:500-1:2000 for Western blot, 1:50-1:100 for IHC-P/IF/ICC, and starting at 1 μg/mL for ELISA . Perform initial experiments using a dilution series spanning these ranges to identify the optimal antibody concentration for your specific sample type and experimental conditions. For Western blot optimization, prepare identical blots with equal protein loading and test at least three dilutions (e.g., 1:500, 1:1000, 1:2000) to identify the concentration providing the best signal-to-noise ratio. For immunohistochemistry and immunofluorescence, optimize not only antibody dilution but also incubation time and temperature, as these parameters collectively affect specific binding and background levels. When performing ELISA, create standard curves using purified recombinant FKBP1B at each tested antibody dilution to determine both sensitivity and dynamic range. Document optimal dilutions for each application, sample type, and detection method, as these may vary depending on experimental conditions and FKBP1B expression levels in different tissues or cell lines.

What detection systems work best with FKBP1B HRP-conjugated antibodies?

The selection of an appropriate detection system is critical for maximizing signal quality when using FKBP1B HRP-conjugated antibodies. For Western blot applications, enhanced chemiluminescence (ECL) substrates provide excellent sensitivity for detecting the typically low-abundance FKBP1B protein. Enhanced ECL substrates with extended signal duration are particularly valuable when detecting FKBP1B in samples with naturally low expression levels. For colorimetric detection in immunohistochemistry, 3,3'-diaminobenzidine (DAB) substrate systems work effectively when combined with appropriate blocking of endogenous peroxidase activity. When performing ELISA, tetramethylbenzidine (TMB) substrate offers a good balance of sensitivity and stability. For multiplexed detection alongside other proteins, consider using fluorescent substrate systems compatible with HRP that emit at distinct wavelengths from other fluorophores in your experiment. The optimal exposure time for chemiluminescent detection should be empirically determined for each experiment, as FKBP1B expression varies considerably between tissues, with highest levels reported in brain and thymus . For digital imaging systems, implement consistent acquisition parameters across experimental replicates to enable accurate quantitative comparisons of FKBP1B levels.

How can I address weak or absent signals when detecting FKBP1B?

Weak or absent signals when detecting FKBP1B can stem from multiple factors requiring systematic troubleshooting. First, verify sample integrity by assessing total protein with stains like Ponceau S or by detecting abundant housekeeping proteins. If sample quality is confirmed, examine antibody handling conditions, as HRP conjugates are sensitive to repeated freeze-thaw cycles and improper storage. The manufacturer's recommendation to store at -20°C for up to 1 year and avoid repeated freeze-thaw cycles should be strictly followed . If signals remain weak, implement signal enhancement strategies: (1) increase protein loading (up to 50-100 μg for low-expressing samples); (2) adjust antibody concentration incrementally above recommended ranges; (3) extend primary antibody incubation time to overnight at 4°C; (4) use more sensitive detection substrates specifically designed for low-abundance proteins; and (5) implement tyramide signal amplification for immunohistochemistry applications. Consider sample enrichment through subcellular fractionation focusing on cytoplasmic and sarcoplasmic reticulum fractions where FKBP1B predominantly localizes . Additionally, review fixation methods for tissue samples, as overfixation can mask epitopes and impair antibody binding.

What strategies help differentiate between FKBP1B isoforms or post-translational modifications?

Differentiating between FKBP1B isoforms or post-translational modifications requires specialized experimental strategies beyond standard detection protocols. First, determine which specific FKBP1B isoform(s) your antibody recognizes by reviewing the immunogen sequence information. Many antibodies are raised against amino acids 1-80 of human FKBP1B (NP_473374.1) , which may recognize multiple isoforms. For isoform discrimination, employ high-resolution SDS-PAGE systems (15-20% acrylamide gels) capable of resolving small molecular weight differences between isoforms. Consider using 2D gel electrophoresis to separate isoforms based on both molecular weight and isoelectric point differences. For detecting post-translational modifications, complement standard Western blotting with phospho-specific antibodies if studying FKBP1B in the context of PKA-induced phosphorylation events that affect its association with RYR2 . Implement mass spectrometry analysis of immunoprecipitated FKBP1B to definitively identify isoforms and post-translational modifications present in your experimental system. When interpreting results, correlate observed molecular weights with predicted values for specific isoforms: isoform 1 should appear at approximately 12.6 kDa, while differences in migration patterns may indicate post-translational modifications or alternative splicing variants.

How can I optimize experimental conditions for studying FKBP1B-RYR2 interactions?

Optimizing conditions for studying FKBP1B-RYR2 interactions requires specialized approaches that preserve physiologically relevant protein complexes. Begin by using cardiac tissue samples or cardiomyocyte cell lines where this interaction is well-characterized. Critical to success is the lysis buffer composition; use mild non-denaturing buffers containing 1% Triton X-100 or 0.5% NP-40 with physiological salt concentrations (150 mM NaCl) to maintain protein-protein interactions. Include phosphatase inhibitors to preserve the phosphorylation state of RYR2, as PKA-induced phosphorylation causes FKBP1B dissociation . For co-immunoprecipitation experiments, use anti-FKBP1B antibodies that don't interfere with the RYR2 binding interface. Proximity ligation assays provide a powerful alternative for visualizing FKBP1B-RYR2 interactions in situ with subcellular resolution. When analyzing heart failure models, implement quantitative co-immunoprecipitation followed by Western blot to measure the FKBP1B:RYR2 ratio, as decreased association correlates with disease severity . Consider pharmacological manipulations with FK506 to compete for FKBP1B binding or PKA activators to induce RYR2 phosphorylation and subsequent FKBP1B dissociation as experimental controls. For functional studies, combine biochemical interaction analysis with calcium imaging to correlate FKBP1B-RYR2 binding status with calcium handling properties in cardiomyocytes.

How can FKBP1B HRP-conjugated antibodies be utilized in cardiac disease research?

FKBP1B HRP-conjugated antibodies serve as powerful tools in cardiac disease research due to FKBP1B's critical role in calcium handling through RYR2 regulation. These antibodies enable researchers to quantify FKBP1B expression and localization changes in various cardiac pathologies. In heart failure models, use these antibodies to document the characteristic reduction in FKBP1B-RYR2 association resulting from RYR2 hyperphosphorylation . The direct HRP conjugation facilitates efficient detection in high-throughput screening of cardiac tissue samples from patient cohorts or animal models, allowing correlation of FKBP1B levels with disease progression markers. Implement co-staining protocols in cardiac tissue sections to visualize FKBP1B redistribution relative to structural proteins or calcium handling machinery. For drug development research, these antibodies provide a means to assess whether candidate compounds restore normal FKBP1B-RYR2 binding in diseased tissue. When designing such experiments, include analysis of both total FKBP1B expression and its subcellular distribution, as redistribution away from the sarcoplasmic reticulum may occur independently of expression changes. Complementary techniques should include calcium imaging and electrophysiological measurements to establish functional correlates of observed FKBP1B alterations. Document findings using consistent quantification methods to enable meta-analysis across studies.

What are the considerations for using FKBP1B antibodies in neurological research?

FKBP1B antibodies offer valuable research applications in neuroscience due to the protein's high expression in brain tissue and emerging roles in neurological function. When designing neurological studies, consider that FKBP1B shows regional variation within the brain, with documented expression in hippocampus, cortex, and caudate nucleus . For immunohistochemical studies, optimize antigen retrieval protocols specifically for neural tissue to maximize epitope accessibility while preserving delicate neural architecture. When analyzing FKBP1B in neurodegenerative disease models, combine protein expression analysis with assessment of PPIase activity, as functional alterations may occur independently of expression changes. The HRP conjugation facilitates sensitive detection in neural tissues where FKBP1B may be present at variable levels. Consider using these antibodies in combination with markers for specific neural cell types (neurons, astrocytes, microglia) to determine cell-type-specific expression patterns. For studies involving FKBP1B's potential role in protein misfolding disorders, perform co-localization analysis with disease-associated proteins that undergo conformational changes involving proline isomerization. When quantifying FKBP1B in neural tissue extracts via Western blot, include region-specific positive controls (e.g., hippocampus) where expression is well-documented .

How can quantitative analysis of FKBP1B be standardized across different experimental systems?

Standardizing quantitative analysis of FKBP1B across different experimental systems requires implementing consistent protocols throughout the research workflow. Begin by establishing a reference standard using recombinant human FKBP1B protein to create calibration curves for absolute quantification. This approach is particularly valuable for cross-laboratory comparisons or longitudinal studies. For Western blot quantification, implement technical standardization including fixed protein loading amounts, consistent transfer conditions, standardized blocking procedures, and uniform antibody dilutions as recommended (1:500-1:2000) . Always normalize FKBP1B signals to appropriate loading controls that reflect the subcellular localization pattern of FKBP1B, preferably using cytoplasmic or sarcoplasmic reticulum markers. For immunohistochemical quantification, establish standard acquisition parameters including exposure times, gain settings, and threshold values for image analysis. Implement automated image analysis workflows using software that allows batch processing with identical parameters across experimental groups. Consider adopting absolute quantification approaches such as ELISA using purified FKBP1B standards at known concentrations. For multi-center studies, distribute aliquots of reference samples to all participating laboratories for internal calibration of detection methods. Document all methodological details comprehensively, including antibody catalog numbers, lot numbers, and detailed protocols to facilitate reproducibility and meaningful comparison of results across research groups.