FKBP10 Antibody

What is FKBP10 and what cellular functions does it perform?

FKBP10, also known as FKBP65 (65 kDa FK506-binding protein), is an endoplasmic reticulum (ER) resident protein with four tandem peptidyl-prolyl cis/trans isomerase (PPIase) domains. It functions as a molecular chaperone that modulates the folding and trafficking of secretory proteins . FKBP10 plays a particularly important role in collagen folding and secretion, with mutations in this protein being associated with osteogenesis imperfecta . The protein is typically expressed in developing tissues and re-expressed in adult tissues following injury . Beyond its structural roles, FKBP10 has been implicated in cell adhesion processes and may function through the integrin/AKT signaling pathway .

What applications are FKBP10 antibodies commonly used for?

FKBP10 antibodies are validated for multiple research applications including:

• Western Blotting (WB): Typically used at dilutions of 1:1000-1:16000 depending on the specific antibody

• Immunohistochemistry (IHC): Used at dilutions of 1:50-1:500, often with TE buffer pH 9.0 for antigen retrieval

• Immunoprecipitation (IP): Approximately 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• Immunofluorescence (IF): Validated in multiple published studies

• ELISA: Validated for human and mouse samples

These applications allow researchers to detect endogenous FKBP10 in various cell lines including HEK-293, HeLa, A375, and NIH/3T3 cells, as well as in human tissues such as brain, placenta, kidney, and ovarian cancer samples .

How should FKBP10 antibodies be stored for optimal performance?

FKBP10 antibodies should be stored at -20°C for long-term stability. Most commercial preparations come in PBS buffer with 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, the antibodies remain stable for at least one year after shipment. For the 20 μl size that contains 0.1% BSA, aliquoting is unnecessary for -20°C storage . It's important to avoid repeated freeze-thaw cycles to maintain antibody activity and specificity. Do not aliquot certain commercial antibody preparations as specified by the manufacturer .

What are the optimal antigen retrieval methods for FKBP10 immunohistochemistry?

For optimal FKBP10 immunohistochemistry staining, two antigen retrieval methods have shown consistent results:

• Primary recommendation: TE buffer at pH 9.0

• Alternative method: Sodium citrate buffer at pH 6.0

For sodium citrate buffer method, tissue sections should be boiled at 100-120°C for 5 minutes . Following antigen retrieval, endogenous peroxidase activity should be blocked using 3% hydrogen peroxide at room temperature for 10 minutes . The sections should then be incubated with the primary FKBP10 antibody (dilutions ranging from 1:50 to 1:700 depending on the specific antibody) overnight at 4°C, followed by incubation with an appropriate conjugated secondary antibody at room temperature for 30 minutes . Visualization can be performed using 3',3'-diaminobenzidene staining at room temperature for approximately 5 minutes .

How should researchers evaluate FKBP10 expression in immunohistochemistry samples?

When evaluating FKBP10 expression in immunohistochemistry, a systematic scoring approach is recommended:

• Select five random fields under a light microscope (magnification ×200)

• Calculate an immunoreaction score (IRS) based on:

  • Intensity of staining: 0 (negative), 1 (weak), 2 (moderate), 3 (strong)

  • Percentage of positive cells: <5% (0), 5-25% (1), 26-50% (2), 51-75% (3), >75% (4)

This standardized scoring system allows for consistent quantification of FKBP10 expression across different samples and studies, facilitating reliable comparison of results. For prognostic studies, correlation of these scores with clinical outcomes can help establish the value of FKBP10 as a biomarker.

What controls should be included when using FKBP10 antibodies in Western blotting?

For rigorous Western blot experiments with FKBP10 antibodies, researchers should include:

• Positive controls: HEK-293 cells, HeLa cells, and A375 cells have been validated to express detectable levels of endogenous FKBP10

• Negative controls: Include a lane with the secondary antibody only (no primary antibody)

• Knockdown/knockout controls: siRNA-mediated knockdown of FKBP10 (as described in several publications) provides a critical specificity control

• Loading controls: β-actin is commonly used as a loading control in FKBP10 studies

The expected molecular weight for FKBP10 is approximately 64-70 kDa , although the calculated molecular weight based on amino acid sequence is 64 kDa . This slight discrepancy may be due to post-translational modifications. Researchers should ensure their gel system can resolve proteins in this range and adjust antibody dilutions based on expression levels in their specific samples.

How can FKBP10 knockdown experiments be designed to investigate its functional role in cancer?

For investigating FKBP10 function in cancer through knockdown experiments:

• Cell line selection: Based on published research, gastric cancer cell lines HGC-27 and MKN-7 show high endogenous expression of FKBP10, making them suitable models . Other cancer cell lines with verified FKBP10 expression include HEK-293, HeLa, and A375 .

• Knockdown strategy:

  • siRNA transfection: Use sequence-specific siRNA targeting FKBP10 (siFKBP10) with appropriate negative controls (siNC)

  • Validation of knockdown efficiency: Confirm reduced expression at both mRNA (RT-PCR) and protein levels (Western blot)

• Functional assays:

  • Adhesion assay: FKBP10 knockdown significantly suppresses cell adhesion in gastric cancer cells

  • Integrin pathway analysis: Measure changes in expression of integrin family proteins, particularly integrin αV and α6, which are reduced following FKBP10 knockdown

  • PI3K-AKT signaling assessment: Examine phosphorylated AKT levels (both P-AKT 473 and P-AKT 308), which decrease after FKBP10 knockdown while total AKT remains unchanged

• Metastasis-related phenotypes: Since FKBP10 expression correlates with lymph node metastasis in gastric cancer, assess cell migration and invasion capacities before and after knockdown .

This experimental design enables comprehensive evaluation of FKBP10's role in cancer progression, particularly in adhesion-mediated processes that may contribute to metastasis.

What bioinformatic approaches can be used to identify FKBP10-associated pathways in cancer?

Several sophisticated bioinformatic approaches have successfully identified FKBP10-associated pathways in cancer:

• Differential expression gene (DEG) screening:

  • Analyze multiple independent databases (e.g., GSE27342, GSE29272, GSE54129, TCGA-STAD for gastric cancer)

  • Identify consistently upregulated genes across databases

  • Perform pathway analysis on these DEGs to reveal functional associations (e.g., "Focal adhesion" pathway)

• Prognostic value assessment:

  • Test candidate genes in multiple independent cohorts (e.g., GSE15459, GSE62254, TCGA-STAD)

  • Perform Kaplan-Meier survival analysis to identify genes with stable prognostic value

  • Further stratify analysis based on clinical parameters such as lymph node metastasis status

• Gene Set Enrichment Analysis (GSEA):

  • Apply GSEA to identify biological processes and pathways associated with FKBP10 expression

  • Research has shown FKBP10 is primarily involved in cell adhesion processes and PI3K-AKT signaling

• Integrative multi-omics analysis:

  • Correlate FKBP10 expression with other molecular features (mutations, copy number alterations, methylation)

  • Map FKBP10 into protein-protein interaction networks to identify functional modules

These approaches provide a comprehensive understanding of FKBP10's role in cancer biology and help identify potential therapeutic implications.

How can FKBP10 antibodies be utilized in investigating disease mechanisms in osteogenesis imperfecta?

FKBP10 antibodies can be instrumental in investigating osteogenesis imperfecta (OI) mechanisms through several specialized approaches:

• Mutation-specific analyses:

  • Compare wild-type and mutant FKBP10 protein localization using immunofluorescence

  • Assess protein stability and degradation rates of mutant forms using pulse-chase experiments with Western blot detection

  • Examine potential dominant-negative effects of mutant proteins on collagen folding

• Collagen interaction studies:

  • Co-immunoprecipitation of FKBP10 with type I collagen to assess binding efficiency in normal versus OI models

  • Proximity ligation assays to visualize and quantify FKBP10-collagen interactions in situ

  • Analysis of collagen post-translational modifications in FKBP10-deficient models

• ER stress response evaluation:

  • Assess unfolded protein response (UPR) activation in cells with FKBP10 mutations

  • Examine co-localization of FKBP10 with other ER chaperones using dual immunofluorescence

  • Investigate calcium homeostasis alterations, as FKBP10 contains calcium-binding EF-hand motifs

• Therapeutic targeting:

  • Screen for compounds that might stabilize mutant FKBP10 or enhance remaining PPIase activity

  • Evaluate the effects of chemical chaperones on restoring proper collagen folding in FKBP10-deficient cells

These approaches capitalize on the specificity of FKBP10 antibodies to illuminate the molecular mechanisms underlying OI and potentially identify therapeutic interventions.

How can researchers address non-specific binding issues with FKBP10 antibodies?

When encountering non-specific binding with FKBP10 antibodies, researchers should implement the following strategies:

• Antibody validation and selection:

  • Use recombinant antibodies (e.g., rabbit recombinant antibodies) which often show higher specificity than conventional polyclonal antibodies

  • Verify antibody specificity through FKBP10 knockdown/knockout controls

  • Consider antibodies purified by antigen affinity methods rather than protein A purification alone

• Protocol optimization:

  • Adjust antibody concentration: Test a dilution series (e.g., 1:2000-1:16000 for Western blot, 1:50-1:500 for IHC)

  • Increase blocking stringency: Use 5% non-fat milk or BSA in TBST for Western blot

  • Add 0.1-0.3% Triton X-100 in blocking buffer for IHC to reduce background

  • Optimize incubation times and temperatures

• Sample-specific considerations:

  • For IHC, compare both recommended antigen retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Use fresh tissue samples where possible, as fixation artifacts can increase non-specific binding

  • For cell lines, ensure endogenous expression levels are sufficient (HEK-293, HeLa, A375, and MKN-7 cells show strong expression)

• Confirmatory approaches:

  • Use two different FKBP10 antibodies targeting distinct epitopes to confirm specificity

  • Include comprehensive controls (positive, negative, and knockdown) in each experiment

Implementing these approaches systematically will help resolve specificity issues and ensure reliable FKBP10 detection.

What are the common pitfalls in quantifying FKBP10 expression in clinical samples?

When quantifying FKBP10 expression in clinical samples, researchers should be aware of several common pitfalls:

• Sample heterogeneity challenges:

  • Tumor samples often contain varying amounts of stromal cells and infiltrating immune cells

  • Solution: Use laser capture microdissection to isolate specific cell populations or employ dual staining with cell-type specific markers

• Standardization issues:

  • Variability in fixation protocols and processing times between samples affects antigen preservation

  • Solution: Standardize pre-analytical variables and include standardized control samples in each batch

• Scoring system limitations:

  • Subjective interpretation of staining intensity can introduce investigator bias

  • Solution: Implement the systematic immunoreaction score (IRS) system combining intensity and percentage of positive cells

  • Consider using digital image analysis software for objective quantification

• Reference gene selection for RT-PCR:

  • Inappropriate housekeeping genes may themselves be regulated in disease states

  • Solution: Validate multiple reference genes for stability in your specific tissue type before quantitative analysis

• Prognostic interpretation challenges:

  • Correlating FKBP10 expression with clinical outcomes requires appropriate statistical approaches

  • Solution: Use multivariate analysis to account for confounding factors and validate findings in independent cohorts, as demonstrated in gastric cancer studies

How should researchers interpret discrepancies between FKBP10 mRNA and protein expression data?

When encountering discrepancies between FKBP10 mRNA and protein expression data, researchers should consider several biological and technical factors:

• Post-transcriptional regulation mechanisms:

  • miRNA-mediated regulation may target FKBP10 mRNA without affecting transcription

  • RNA-binding proteins may alter mRNA stability or translation efficiency

  • Systematically investigate potential regulatory elements in the FKBP10 3'UTR

• Post-translational modifications and protein stability:

  • FKBP10 may undergo proteolytic processing or degradation in certain contexts

  • The observed molecular weight of FKBP10 (65-70 kDa) compared to the calculated weight (64 kDa) suggests post-translational modifications

  • Examine proteasome or autophagy inhibition effects on FKBP10 protein levels

• Technical considerations:

  • Antibody specificity: Validate antibody performance with positive and negative controls

  • PCR primer design: Ensure primers detect all relevant FKBP10 transcripts

  • Sample processing: Protein degradation during extraction can affect detection while preserving RNA

• Experimental validation approaches:

  • Pulse-chase experiments to determine protein half-life

  • Use of proteasome inhibitors to assess degradation pathways

  • Analysis of polysome fractions to evaluate translation efficiency

  • Comparison of multiple independent detection methods

• Biological significance assessment:

  • In gastric cancer research, both mRNA and protein levels were assessed to confirm FKBP10 upregulation

  • Consider that discrepancies may themselves reveal important regulatory mechanisms in disease contexts

Understanding these factors will help researchers interpret discrepancies meaningfully and may lead to discoveries about FKBP10 regulation in normal and disease states.

How might FKBP10 serve as a therapeutic target in cancer and fibrotic diseases?

FKBP10's emerging roles in multiple pathological processes suggest several promising therapeutic targeting strategies:

• Cancer therapeutic approaches:

  • Targeted inhibition: Develop specific small molecule inhibitors against FKBP10's PPIase domains

  • Disruption of integrin interactions: As FKBP10 knockdown reduces integrin αV and α6 and impairs cell adhesion, targeting this interaction could inhibit metastatic potential

  • PI3K-AKT pathway modulation: FKBP10's involvement in AKT phosphorylation suggests combination therapies with existing PI3K/AKT inhibitors might be synergistic

  • Biomarker-guided therapy: High FKBP10 expression correlates with poor prognosis and lymph node metastasis in gastric cancer, potentially identifying patients who would benefit from more aggressive treatment regimens

• Fibrotic disease interventions:

  • Anti-fibrotic applications: FKBP10 is considered a therapeutic target for idiopathic pulmonary fibrosis (IPF)

  • Collagen deposition modulation: Given FKBP10's role in collagen folding and secretion, inhibitors could potentially reduce excessive collagen deposition in fibrotic conditions

  • Conditional targeting: Develop strategies that selectively inhibit FKBP10 in activated fibroblasts while sparing normal tissue functions

• Technical challenges and solutions:

  • Specificity concerns: Design inhibitors that distinguish between different FKBP family members

  • Delivery strategies: Develop targeted delivery systems to reach specific tissue microenvironments

  • Combination approaches: Identify synergistic combinations with existing therapies

• Clinical development considerations:

  • Patient stratification: Use FKBP10 expression as a biomarker to select patients most likely to respond to targeted therapies

  • Monitoring efficacy: Employ FKBP10 antibodies in pharmacodynamic studies to assess target engagement

These multifaceted approaches highlight FKBP10's potential as both a biomarker and therapeutic target across multiple diseases.

What novel applications of FKBP10 antibodies are emerging in translational research?

Several innovative applications of FKBP10 antibodies are emerging in translational research:

• Liquid biopsy development:

  • Detection of circulating FKBP10 as a potential non-invasive biomarker for gastric cancer progression and lymph node metastasis

  • Combined assessment with other circulating proteins to develop multi-marker panels with improved sensitivity and specificity

• Theranostic applications:

  • Antibody-drug conjugates targeting FKBP10 in cancers with high expression

  • Imaging applications using labeled FKBP10 antibodies to visualize tumor margins or metastatic spread

  • Monitoring treatment response through serial measurement of FKBP10 levels

• Tissue microenvironment analysis:

  • Multiplex immunohistochemistry combining FKBP10 with integrin family proteins and phospho-AKT to map signaling networks in tumor samples

  • Spatial transcriptomics combined with FKBP10 protein detection to correlate expression patterns with specific microenvironmental features

• Disease heterogeneity characterization:

  • FKBP10 expression patterns across cancer subtypes to identify distinct biological behaviors

  • Correlation with treatment resistance phenotypes in various cancer types

  • Integration with genomic profiling to identify synthetic lethality opportunities

• Organoid and 3D culture applications:

  • Using FKBP10 antibodies to assess protein expression and localization in patient-derived organoids

  • Functional studies in 3D cultures to better recapitulate in vivo collagen interactions

These emerging applications demonstrate how FKBP10 antibodies are expanding beyond basic research tools to become valuable assets in translational medicine and personalized healthcare approaches.

How might multi-omics approaches integrate FKBP10 antibody-based proteomics with other data types?

Advanced multi-omics integration strategies involving FKBP10 antibody-based proteomics include:

• Integrated protein-transcriptome analysis:

  • Correlation of FKBP10 protein levels (detected via antibody-based methods) with mRNA expression

  • Single-cell proteogenomics to identify cell populations with discordant FKBP10 protein and mRNA levels

  • Analysis of alternative splicing events affecting FKBP10 function through combined RNA-seq and protein isoform detection

• Epigenetic-proteomic integration:

  • Correlation of FKBP10 protein expression with promoter methylation status

  • Chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify transcription factors regulating FKBP10

  • Investigation of histone modifications at the FKBP10 locus and their correlation with protein expression

• Protein interaction networks:

  • Immunoprecipitation with FKBP10 antibodies followed by mass spectrometry to identify protein interaction partners

  • Proximity-dependent biotin identification (BioID) to map the FKBP10 protein interaction neighborhood

  • Integration of these data with public protein-protein interaction databases to place FKBP10 in functional networks

• Metabolomic correlations:

  • Association of FKBP10 expression levels with metabolic profiles

  • Investigation of collagen metabolism alterations in relation to FKBP10 expression

  • Integration with lipid profiles, given the potential role of FKBP10 in ER function

• Clinical data integration for precision medicine:

  • Correlation of FKBP10 protein levels with treatment responses

  • Development of integrated biomarker panels combining FKBP10 with other molecular features

  • Machine learning approaches to identify patient subgroups based on integrated multi-omics profiles including FKBP10 expression

These integrative approaches leverage the specificity of FKBP10 antibodies while providing a more comprehensive understanding of FKBP10's role in complex biological systems and disease processes.