FKBP7 Antibody

What is FKBP7 and why is it important in research?

FKBP7, also known as FKBP23, belongs to the immunophilin class of proteins. It contains an N-terminal hydrophobic signal sequence, a PPIase motif, 2 EF-hand domains, and 2 putative N-glycosylation sites. The calculated molecular mass is about 25 kDa, but becomes 33 kDa following cleavage of the signal sequence . FKBP7 is primarily known for its role in protein folding during protein synthesis, as PPIases (peptidyl-prolyl cis-trans isomerases) accelerate this process . Research interest in FKBP7 has grown significantly due to its implications in cancer, particularly regarding chemoresistance in prostate cancer . Its ability to bind to BiP in the endoplasmic reticulum in a process regulated by calcium concentration makes it relevant to cellular stress responses and protein quality control pathways .

What are the main structural and functional characteristics of FKBP7?

FKBP7 has several key structural and functional characteristics:

• Contains an N-terminal hydrophobic signal sequence

• Features a PPIase motif essential for its isomerase activity

• Contains 2 EF-hand domains, which are calcium-binding motifs

• Has 2 putative N-glycosylation sites, with confirmed N45-glycosylation

• Primarily localizes to the endoplasmic reticulum but can also be found in the cytosol

• Exists in 3 isoforms produced by alternative splicing

• Calculated molecular weight is 25 kDa, but observed at 30-33 kDa after signal sequence cleavage

• Functions in protein folding acceleration during synthesis

• Binds to BiP in the ER in a calcium-dependent manner

• Interacts with the translation initiation complex eIF4F

How do I choose the appropriate FKBP7 antibody for my research?

When selecting an FKBP7 antibody, consider these factors:

• Target species reactivity: Determine whether you need antibodies that react with human, mouse, rat, or other species. Many commercially available antibodies show reactivity across human, mouse, and rat FKBP7 .

• Application compatibility: Different antibodies are optimized for specific applications:

  • Western Blot (WB): Generally use dilutions of 1:500-1:2400 or 1:1000-1:10000

  • Immunohistochemistry (IHC): Typically use dilutions of 1:50-1:500 or 5-20 μg/mL

  • ELISA, FACS, ICC, IP: Check specific validated applications for each antibody

• Clonality: Choose between:

  • Polyclonal antibodies: Recognize multiple epitopes, often providing stronger signals

  • Monoclonal antibodies: Recognize a single epitope, offering higher specificity

• Host: Most commonly available in rabbit or mouse hosts

• Epitope specificity: Some antibodies target specific regions like the C-terminus (AA 195-224) or center region

• Validation data: Review available validation data in applications that match your experiments

What are the optimal conditions for using FKBP7 antibodies in Western blotting?

For optimal Western blotting with FKBP7 antibodies:

• Sample preparation: FKBP7 has been successfully detected in human lung tissue, human heart tissue, and recombinant FKBP7 protein .

• Expected molecular weight: Look for bands at approximately 30 kDa, which corresponds to the observed molecular weight of FKBP7 .

• Dilution optimization:

  • Start with manufacturer-recommended dilutions, typically 1:500-1:2400 or 1:1000-1:10000

  • It's recommended that this reagent should be titrated in each testing system to obtain optimal results

• Blocking conditions: Use standard blocking buffers compatible with the secondary antibody system.

• Positive controls: Human lung tissue and human heart tissue have been validated as positive controls for FKBP7 expression in Western blot applications .

• Cross-reactivity considerations: Many FKBP7 antibodies show reactivity across human, mouse, and rat samples, which can be beneficial for comparative studies but requires validation in each species .

How should I optimize FKBP7 antibody use in immunohistochemistry?

For optimal immunohistochemistry with FKBP7 antibodies:

• Tissue preparation and antigen retrieval:

  • Use TE buffer pH 9.0 for antigen retrieval

  • Alternatively, citrate buffer pH 6.0 can be used

• Antibody dilution:

  • Start with recommended dilutions: 1:50-1:500 or 5-20 μg/mL

  • Optimize based on signal-to-noise ratio in your specific tissue samples

• Validated tissue samples:

  • Positive reactions have been documented in human liver cancer tissue and human lung cancer tissue

  • Additional validation has been shown in human skin cancer, kidney, and stomach tissues

• Detection systems:

  • For example, use 2 μg/mL HRP-Linked Caprine Anti-Rabbit IgG Polyclonal Antibody as a secondary antibody with DAB staining

• Controls:

  • Include positive controls (tissues known to express FKBP7)

  • Include negative controls (primary antibody omitted or non-specific IgG substituted)

What are the proper storage and handling conditions for FKBP7 antibodies?

To maintain FKBP7 antibody stability and functionality:

How can I use FKBP7 antibodies to investigate its role in cancer chemoresistance mechanisms?

FKBP7 has emerged as a potential therapeutic target in cancer, particularly in chemoresistance:

• Expression analysis in resistant cell lines:

  • Use Western blotting to compare FKBP7 expression in parental versus chemoresistant cell lines

  • FKBP7 is overexpressed in taxane-resistant prostate cancer cells

  • Monitor FKBP7 expression changes upon treatment with various chemotherapies using validated Western blot protocols

• Mechanistic studies:

  • Investigate FKBP7's interaction with the translation initiation complex eIF4F using co-immunoprecipitation

  • Examine subcellular localization changes using fractionation techniques (e.g., digitonin-based fractionation)

  • Study glycosylation status differences between ER-resident and cytosolic FKBP7 pools

• Clinical correlation:

  • Correlate FKBP7 expression with clinical prognosis using TCGA data

  • TCGA data has shown that FKBP7 impacts survival in multiple cancer types

• Functional validation:

  • Utilize FKBP7 overexpression or knockdown models to directly assess its impact on cell proliferation and chemotherapy efficacy

  • Research has shown FKBP7 impacts both cell proliferation and docetaxel efficacy in chemoresistant models

• Potential therapeutic targeting:

  • Investigate FKBP7's interaction with known FKBP ligands using structural studies

  • Utilize purified recombinant catalytic domain of FKBP7 for binding studies

What is the relationship between FKBP7 and tumor microenvironment, and how can I study it?

The relationship between FKBP7 and the tumor microenvironment can be studied using several approaches:

• Correlation with stromal markers:

  • Multi-omics analysis has shown that FKBP7, along with FKBP9 and FKBP10, is positively correlated with stromal cells

  • This contrasts with FKBP3 and FKBP4, which showed negative correlation with stromal components

• Immunohistochemical co-localization:

  • Use FKBP7 antibodies in conjunction with stromal markers to study co-localization in tumor tissues

  • Recommended FKBP7 antibody dilution for IHC: 1:50-1:500 or 5-20 μg/mL

• Cancer stemness correlation:

  • FKBP7 expression has been associated with decreased stemness, in contrast to FKBP3 and FKBP4 which contribute to stemness

  • Analyze stemness indices in relation to FKBP7 expression levels

• Gene expression correlation analysis:

  • FKBP7 shows significant positive correlation with FKBP1A (r = 0.51, p < 0.05) and FKBP14 (r = 0.57, p < 0.05)

  • These correlations may provide insights into functional networks in the tumor microenvironment

How can I validate FKBP7 antibody specificity for my particular experimental system?

Thorough validation of FKBP7 antibody specificity is crucial for experimental reliability:

• Multiple antibody comparison:

  • Use at least two antibodies targeting different epitopes of FKBP7

  • Compare antibodies recognizing the C-terminus (AA 195-224) versus center region epitopes

• Positive and negative controls:

  • Use tissues or cell lines with confirmed FKBP7 expression as positive controls

  • Human lung tissue, heart tissue, liver cancer tissue, and lung cancer tissue have been validated for FKBP7 expression

  • Use FKBP7 knockout or knockdown samples as negative controls

• Cross-reactivity assessment:

  • Test the antibody against recombinant FKBP7 protein

  • Compare observed molecular weight (30 kDa) with predicted molecular weight

  • Assess potential cross-reactivity with other FKBP family members, particularly those with high sequence homology

• Application-specific validation:

  • For WB: Include positive control lysates and blocking peptides

  • For IHC: Perform peptide competition assays and compare staining patterns with literature

  • For IP: Confirm pulled-down protein by mass spectrometry

• Orthogonal validation:

  • Correlate protein detection with mRNA expression

  • Use primers for RT-PCR validation: design specific to FKBP7 to avoid amplification of other FKBP family members

How do I troubleshoot non-specific banding or high background in FKBP7 Western blots?

When encountering issues with FKBP7 Western blots:

• Non-specific bands:

  • Verify the expected molecular weight: FKBP7 should appear at approximately 30 kDa

  • Consider isoforms: FKBP7 has 3 isoforms produced by alternative splicing

  • Post-translational modifications: FKBP7 is N45-glycosylated, which can affect migration

  • Increase antibody dilution: Try more dilute solutions, starting from manufacturer recommendations (1:500-1:2400 or 1:1000-1:10000)

• High background:

  • Optimize blocking: Increase blocking time or use alternative blocking reagents

  • Increase washing steps: More thorough or additional washing steps

  • Reduce antibody concentration: Dilute primary and/or secondary antibodies further

  • Check secondary antibody compatibility: Ensure proper host species matching

• Weak or no signal:

  • Confirm expression in your sample: FKBP7 is highly expressed in certain tissues like human lung and heart

  • Increase protein loading: Load more total protein

  • Decrease antibody dilution: Use more concentrated antibody solution

  • Optimize antigen retrieval/sample preparation: Different lysis buffers may improve detection

  • Extend exposure time: Longer exposure may be needed for low abundance protein

• Sample-specific considerations:

  • Different sample types may require optimization of the recommended dilution range

  • It's recommended to titrate the reagent in each testing system to obtain optimal results

What are the critical considerations when designing multi-color immunofluorescence experiments that include FKBP7?

For multi-color immunofluorescence including FKBP7:

• Antibody compatibility:

  • Choose FKBP7 antibodies raised in different host species than other primary antibodies

  • Consider using directly conjugated antibodies to avoid secondary antibody cross-reactivity

  • Rabbit-hosted FKBP7 antibodies are commonly available and can be paired with mouse antibodies for co-labeling

• Cellular localization considerations:

  • FKBP7 is primarily localized to the endoplasmic reticulum but can also be found in the cytosol

  • Choose appropriate co-markers for ER (e.g., calnexin, BiP) or cytosolic compartments

  • Consider the glycosylation status, as cytosolic FKBP7 appears mainly glycosylated

• Signal intensity balancing:

  • Titrate each antibody individually before combining them

  • For FKBP7 ICC, start with recommended dilutions of 5-20 μg/mL

  • Balance exposure times/laser powers to compensate for different signal intensities

• Spectral overlap minimization:

  • Choose fluorophores with minimal spectral overlap

  • Perform proper controls: single-stained samples for compensation settings

  • Include unstained and secondary-only controls

• Sequential staining consideration:

  • If antibody host species overlap is unavoidable, consider sequential staining protocols

  • Use Fab fragments to block/mask primary antibodies between staining rounds

How can I quantitatively assess FKBP7 expression in relation to cancer progression or treatment response?

For quantitative assessment of FKBP7 expression in cancer contexts:

• Western blot quantification:

  • Use appropriate housekeeping proteins for normalization

  • Employ densitometry software for quantification

  • Ensure you're working within the linear range of detection

  • Compare expression levels across treatment conditions or disease stages

  • Recommended dilutions: 1:500-1:2400 or 1:1000-1:10000

• Immunohistochemistry scoring:

  • Develop a standardized scoring system (e.g., H-score, Allred score)

  • Consider both percentage of positive cells and staining intensity

  • Use digital pathology software for automated quantification

  • Compare tumor tissue with matched normal tissue

  • Recommended dilutions: 1:50-1:500 or 5-20 μg/mL

• qRT-PCR correlation:

  • Correlate protein expression with mRNA levels

  • Use validated FKBP7-specific primers

  • Calculate relative expression using the 2^-ΔΔCt method

  • Compare results with protein expression to identify post-transcriptional regulation

• Clinical correlation analysis:

  • Stratify patients based on FKBP7 expression levels

  • Correlate with clinical parameters: survival, treatment response, progression

  • Consider multi-omics analysis as FKBP7 has been studied in relation to stemness indices and stromal cell correlation

• Treatment response monitoring:

  • Measure FKBP7 expression changes before and after treatment

  • FKBP7 expression increases upon treatment with several cytotoxic chemotherapies in prostate cancer cell lines

  • Track correlation between expression changes and clinical outcomes

What are the emerging roles of FKBP7 beyond protein folding, and how can antibodies help investigate them?

Recent research has uncovered several novel roles for FKBP7 beyond its classical protein folding function:

• Translation regulation:

  • FKBP7 affects translation by binding to the translation initiation complex eIF4F

  • Use co-immunoprecipitation with FKBP7 antibodies to identify new binding partners in the translation machinery

  • Investigate polysome profiles in FKBP7-manipulated cells

• Chemoresistance mechanisms:

  • FKBP7 is overexpressed in taxane-resistant prostate cancer cells

  • It impacts both cell proliferation and docetaxel efficacy in chemoresistant models

  • Use FKBP7 antibodies to track expression changes during resistance development

• Stromal interactions:

  • Multi-omics analysis showed FKBP7 positively correlates with stromal cells

  • Use dual immunolabeling with FKBP7 and stromal markers to investigate this relationship in tissue sections

• ER-cytosol trafficking:

  • FKBP7 has been detected in both ER and cytosolic fractions

  • The cytosolic FKBP7 appears mainly glycosylated, suggesting ER-resident FKBP7 could be refluxed to the cytosol

  • Use subcellular fractionation and FKBP7 antibodies to track this trafficking

• Calcium signaling connections:

  • FKBP7 contains EF-hand domains (calcium-binding motifs)

  • Its binding to BiP is regulated by calcium concentration

  • Investigate calcium-dependent interactions using FKBP7 antibodies in different calcium conditions

How do methylation patterns and epigenetic modifications affect FKBP7 expression in different cancer types?

Understanding epigenetic regulation of FKBP7 in cancer:

• DNA methylation analysis:

  • FKBP7 methylation patterns have been studied in relation to stemness indices

  • Compare methylation profiles in tumor vs. normal tissues

  • Correlate methylation status with protein expression levels using FKBP7 antibodies

• Stemness correlation:

  • The expression and DNA methylation data of FKBP7 have been used to calculate stemness indices

  • FKBP7 appears to have opposite effects compared to FKBP3 and FKBP4, which contribute to stemness

  • Use FKBP7 antibodies to study expression in cancer stem cell populations

• Transcriptional regulation:

  • In taxane-resistant models, FKBP7 overexpression results from transcriptional regulation rather than protein stability

  • Investigate promoter activity and transcription factor binding using complementary approaches to antibody detection

• Correlation with other FKBP family members:

  • FKBP7 shows significant positive correlation with FKBP1A (r = 0.51, p < 0.05) and FKBP14 (r = 0.57, p < 0.05)

  • Use multiplexed antibody detection to study co-expression patterns

• Multi-cancer analysis:

  • TCGA data analysis has shown FKBP7 impacts survival in multiple cancer types beyond prostate cancer

  • Compare FKBP7 expression and methylation patterns across cancer types using tissue microarrays and FKBP7 antibodies

What are the latest structural insights into FKBP7, and how might they inform therapeutic targeting?

Recent structural and functional studies of FKBP7 provide new perspectives for therapeutic development:

• Domain analysis and function:

  • FKBP7 contains a PPIase motif essential for peptidyl-prolyl isomerase activity

  • It has 2 EF-hand domains (calcium-binding motifs)

  • Contains 2 putative N-glycosylation sites, with confirmed N45-glycosylation

  • Use domain-specific antibodies to study the accessibility and function of different domains

• Recombinant protein studies:

  • The recombinant catalytic domain of FKBP7 has been produced and purified

  • High-quality 15N HSQC NMR spectra have been collected

  • Interactions with known FKBP ligands have been examined

  • Use antibodies that recognize specific conformational states to study structural dynamics

• Post-translational modifications:

  • Glycosylation affects FKBP7 localization and potentially function

  • The calculated molecular mass is about 25 kDa, but becomes 33 kDa following signal sequence cleavage

  • Cytosolic FKBP7 appears mainly glycosylated

  • Use glycosylation-sensitive antibodies or deglycosylation experiments with standard FKBP7 antibodies

• Binding partner interactions:

  • FKBP7 binds to BiP in the ER in a calcium-dependent manner

  • It also interacts with the translation initiation complex eIF4F

  • Use co-immunoprecipitation with FKBP7 antibodies to identify and characterize these interactions

• Therapeutic targeting potential:

  • FKBP7's increased expression in chemoresistant cancer cells makes it a potential therapeutic target

  • Structural insights can guide the development of specific inhibitors

  • Use competitive binding assays with FKBP7 antibodies to screen potential therapeutic compounds