FKBP4 Antibody, FITC conjugated

What is FKBP4 and why is it important in cancer research?

FKBP4 is an HSP90-associated co-chaperone that plays a significant role in tumorigenesis. Research has demonstrated that FKBP4 is overexpressed in breast cancer tissues and cell lines at both mRNA and protein levels. This protein has been identified as a tumor-specific antigen capable of eliciting an immune response in breast cancer patients. Notably, FKBP4 expression is associated with breast cancer progression and prognosis, particularly in estrogen receptor (ER)-negative breast cancer . As a co-chaperone, FKBP4 regulates various client proteins including steroid hormone receptors such as androgen, glucocorticoid, progesterone, mineralocorticoid, and estrogen receptors, as well as nuclear factors like NF-kB and other proteins including Argonaute 2 and Tau .

What applications are suitable for FITC-conjugated FKBP4 antibodies?

FITC-conjugated FKBP4 antibodies are versatile tools suitable for multiple experimental applications:

The FITC-conjugated FKBP4 antibody (clone Hi52C) detects a protein of approximately 52 kDa, with the caveat that heavy chain migrates close to FKBP52 on SDS PAGE, which may require optimization for Western blot applications .

What species cross-reactivity does the FITC-conjugated FKBP4 antibody exhibit?

The FITC-conjugated FKBP4 monoclonal antibody (clone Hi52C) demonstrates broad cross-reactivity across several mammalian species. Based on validated testing, this antibody effectively recognizes FKBP4 in human, mouse, rat, dog, and hamster samples . This extensive cross-reactivity makes this antibody particularly valuable for comparative studies across different model organisms and translational research connecting animal models to human applications.

How does FKBP4 interact with the PI3K/Akt/mTOR pathway in cancer progression?

Recent studies utilizing protein interactomics approaches have revealed FKBP4 as a novel PI3K-Akt-mTOR proximal interacting protein. Specifically, using BirA proximity-dependent biotin identification methodology, researchers have demonstrated that FKBP4 interacts with PI3K and enhances Akt activation through PDK1 and mTORC2 . This interaction has significant implications for cancer cell growth and proliferation.

The mechanistic pathway can be summarized as:

• FKBP4 acts as a proximal interacting protein with PI3K

• This interaction facilitates PDK1 activation

• PDK1 and mTORC2 then phosphorylate Akt at distinct sites

• Activated Akt promotes cell growth and proliferation

Depletion of FKBP4 has been shown to specifically reduce cell growth and proliferation in triple-negative breast cancer cell models and xenograft tumor models, highlighting its potential as a therapeutic target .

What methodological considerations are important when using FITC-conjugated FKBP4 antibodies for detecting subcellular localization?

When investigating FKBP4 subcellular localization using FITC-conjugated antibodies, several methodological considerations are critical:

• Fixation Protocol: Use 4% paraformaldehyde for 15-20 minutes at room temperature to preserve both antigenicity and fluorescence.

• Permeabilization: For intracellular detection, use 0.1-0.5% Triton X-100 for 5-10 minutes. Over-permeabilization can lead to signal loss, while insufficient permeabilization may prevent antibody access.

• Blocking: Implement rigorous blocking (5% BSA or 10% normal serum from the host species of the secondary antibody) for at least 1 hour to reduce background fluorescence.

• Signal-to-Noise Ratio: FITC has an excitation maximum at 495nm and emission maximum at 519nm. Ensure your microscopy setup has appropriate filter sets to distinguish FITC signal from autofluorescence.

• Counterstaining: When co-staining with other fluorescent markers, select dyes with minimal spectral overlap with FITC (e.g., DAPI for nuclei, rhodamine or Cy5 for other targets).

• Photobleaching: FITC is susceptible to photobleaching; minimize exposure to light during preparation and imaging, and consider anti-fade mounting media.

• Controls: Always include a negative control (isotype control antibody, IgG2a for the Hi52C clone) to assess non-specific binding .

How can researchers validate FKBP4 antibody specificity in the context of steroid receptor research?

Validating FKBP4 antibody specificity in steroid receptor research requires multiple complementary approaches:

• FKBP4 Knockdown/Knockout Validation: Generate FKBP4-depleted cells using siRNA, shRNA, or CRISPR-Cas9 approaches. The FITC-conjugated antibody signal should diminish proportionally to knockdown efficiency.

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide (synthetic peptide corresponding to human FKBP52) before application to samples. Specific binding should be blocked by this competition.

• Western Blot Correlation: Confirm that the pattern of FKBP4 detection in immunofluorescence correlates with Western blot results, noting that FKBP4 appears at approximately 52 kDa, with potential proximity to heavy chain migration .

• Co-localization Studies: In steroid receptor research, validate functional relevance by demonstrating co-localization with known FKBP4 interaction partners like HSP90 or steroid receptors (androgen, glucocorticoid, progesterone, or estrogen receptors) .

• Cross-validation with Multiple Antibodies: Compare results with other FKBP4 antibodies targeting different epitopes, such as those recognizing AA 301-410, AA 1-459, or AA 220-459 .

What is the optimal protocol for using FITC-conjugated FKBP4 antibody in immunofluorescence studies?

Optimized Immunofluorescence Protocol for FITC-conjugated FKBP4 Antibody:

• Sample Preparation:

  • Culture cells on glass coverslips or prepare tissue cryosections (8-10 μm thickness)

  • Wash in PBS (2 × 5 minutes)

  • Fix with 4% paraformaldehyde (15 minutes, room temperature)

  • Wash in PBS (3 × 5 minutes)

• Permeabilization and Blocking:

  • Permeabilize with 0.2% Triton X-100 in PBS (10 minutes, room temperature)

  • Wash in PBS (3 × 5 minutes)

  • Block with 5% BSA in PBS (1 hour, room temperature)

• Antibody Incubation:

  • Incubate with FITC-conjugated FKBP4 antibody (clone Hi52C) at 1:100-1:200 dilution in 1% BSA/PBS (overnight, 4°C, in dark)

  • Wash in PBS (5 × 5 minutes)

• Nuclear Counterstaining and Mounting:

  • Counterstain with DAPI (1 μg/mL, 5 minutes)

  • Wash in PBS (3 × 5 minutes)

  • Mount using anti-fade mounting medium

  • Seal with nail polish and store at 4°C in the dark

• Imaging Parameters:

  • Excitation: 495 nm

  • Emission: 519 nm

  • Use appropriate filter sets to minimize bleed-through

• Controls:

  • Negative control: FITC-conjugated isotype control (mouse IgG2a)

  • Positive control: Known FKBP4-expressing cell line (e.g., breast cancer cell lines)

How should researchers optimize Western blotting conditions for FITC-conjugated FKBP4 antibody?

While FITC-conjugated antibodies are primarily designed for direct fluorescence applications, they can be used in Western blotting with specific considerations:

Optimized Western Blotting Protocol:

• Sample Preparation:

  • Extract proteins using RIPA buffer supplemented with protease inhibitors

  • Quantify protein concentration (BCA or Bradford assay)

  • Load 20-40 μg protein per lane

• Gel Electrophoresis:

  • Use 10% SDS-PAGE gels (FKBP4 is approximately 52 kDa)

  • Include molecular weight markers

• Transfer and Blocking:

  • Transfer to PVDF membrane (preferred over nitrocellulose for fluorescence)

  • Block with 5% non-fat milk or BSA in TBST (1 hour, room temperature)

• Antibody Incubation:

  • Incubate with FITC-conjugated FKBP4 antibody (1:500-1:1000) in blocking buffer (overnight, 4°C, in dark)

  • Wash with TBST (4 × 10 minutes)

• Detection:

  • Image directly using a fluorescence imager with appropriate filters (excitation ~495 nm, emission ~519 nm)

  • Alternative: if signal is weak, use an anti-FITC HRP-conjugated antibody as a secondary enhancer, followed by chemiluminescent detection

• Critical Considerations:

  • Protect membrane from light during incubation and washing steps

  • Note that heavy chain migrates close to FKBP52 on SDS-PAGE, which may complicate interpretation

  • Consider running a denaturing gel alongside a non-denaturing gel to distinguish between potential oligomeric states

How can researchers use FITC-conjugated FKBP4 antibody to study protein-protein interactions?

Studying protein-protein interactions using FITC-conjugated FKBP4 antibody can be accomplished through several approaches:

• Co-Immunoprecipitation with Fluorescence Detection:

  • Lyse cells in non-denaturing buffer to preserve protein-protein interactions

  • Perform immunoprecipitation with non-conjugated FKBP4 antibody or antibodies against suspected interaction partners

  • Detect FKBP4 in the immunoprecipitated complex using the FITC-conjugated antibody

  • Visualize directly with fluorescence imaging systems

• Proximity Ligation Assay (PLA):

  • Fix and permeabilize cells as per standard immunofluorescence protocols

  • Incubate with FITC-conjugated FKBP4 antibody and a primary antibody against the potential interaction partner

  • Use an anti-FITC antibody and species-appropriate secondary antibody conjugated to PLA probes

  • Perform ligation and amplification according to PLA manufacturer protocols

  • This allows visualization of protein interactions within 40 nm proximity

• FRET Analysis:

  • Use FITC-conjugated FKBP4 antibody as a donor fluorophore

  • Label the suspected interaction partner with an acceptor fluorophore (e.g., rhodamine)

  • Measure FRET efficiency to determine proximity-based interactions

• BioID Approach:

  • As demonstrated in research, the BioID approach can be used to identify proximal interactions

  • This involves expressing FKBP4 fused to a promiscuous biotin ligase (BirA*)

  • Proteins in close proximity become biotinylated and can be isolated using streptavidin

  • The protocol involves generating stable cell lines expressing FLAGBirA*-FKBP4, inducing expression with tetracycline, supplementing with biotin, and then analyzing biotinylated proteins

How can FITC-conjugated FKBP4 antibody be used to study its role in breast cancer progression?

FITC-conjugated FKBP4 antibodies provide valuable tools for investigating FKBP4's role in breast cancer progression through multiple experimental approaches:

• Expression Analysis in Patient Samples:

  • Use immunofluorescence with FITC-conjugated FKBP4 antibody on tissue microarrays or patient-derived samples

  • Quantify expression levels using fluorescence intensity measurements

  • Correlate with clinical parameters (tumor grade, subtype, patient outcomes)

  • Research has shown FKBP4 expression is associated with breast cancer progression and prognosis, especially in ER-negative breast cancer

• Triple-Negative Breast Cancer Models:

  • Employ the antibody to monitor FKBP4 expression in cell line models before and after genetic manipulation

  • Track changes in subcellular localization during cancer progression

  • Research has demonstrated that FKBP4 depletion specifically reduces cell growth and proliferation in triple-negative breast cancer cell models and xenograft tumor models

• PI3K/Akt/mTOR Pathway Analysis:

  • Use multicolor immunofluorescence to co-localize FITC-labeled FKBP4 with components of the PI3K/Akt/mTOR pathway

  • Combine with proximity ligation assays to confirm direct interactions

  • Research has identified FKBP4 as a novel PI3K-Akt-mTOR proximal interacting protein that enhances Akt activation through PDK1 and mTORC2

• Therapeutic Response Monitoring:

  • Monitor changes in FKBP4 expression and localization following treatment with targeted therapies

  • Assess the potential of FKBP4 as a biomarker for treatment response

What considerations are important when interpreting FKBP4 staining patterns in tumor tissues?

When interpreting FKBP4 staining patterns in tumor tissues using FITC-conjugated antibodies, researchers should consider several critical factors:

What are common issues encountered with FITC-conjugated antibodies and how can they be addressed?

Researchers working with FITC-conjugated FKBP4 antibodies may encounter several challenges. Here are common issues and their solutions:

How should researchers validate batch-to-batch consistency of FITC-conjugated FKBP4 antibodies?

Ensuring batch-to-batch consistency of FITC-conjugated FKBP4 antibodies is crucial for reproducible research. A comprehensive validation approach includes:

• Fluorophore-to-Protein Ratio Assessment:

  • Measure absorbance at 280nm (protein) and 495nm (FITC)

  • Calculate the F/P ratio to ensure consistent conjugation between batches

  • Acceptable variation should be within ±20% of the reference batch

• Positive Control Testing:

  • Use a well-characterized cell line with stable FKBP4 expression

  • Perform side-by-side comparison between batches under identical conditions

  • Quantify mean fluorescence intensity and staining pattern consistency

• Western Blot Validation:

  • Perform parallel Western blots with previous and new antibody batches

  • Compare band intensity at 52 kDa and check for consistent detection of FKBP4

  • Note that heavy chain migrates close to FKBP52 on SDS PAGE

• Cross-Reactivity Profiling:

  • Test each batch against samples from multiple species (human, mouse, rat, dog, hamster)

  • Confirm consistent cross-reactivity profile as specified in product documentation

• Functional Validation:

  • For critical applications, perform immunoprecipitation followed by mass spectrometry

  • Confirm that each batch pulls down FKBP4 and its known interacting partners

• Specificity Controls:

  • Test each batch against FKBP4-knockdown or knockout samples

  • Perform peptide competition assays using the immunizing peptide (synthetic peptide corresponding to human FKBP52)

What emerging applications may benefit from FITC-conjugated FKBP4 antibodies in cancer research?

FITC-conjugated FKBP4 antibodies are poised to contribute to several emerging areas in cancer research:

• Single-Cell Analysis: The direct fluorescence labeling enables integration into single-cell protein profiling workflows, allowing researchers to investigate FKBP4 expression heterogeneity at the individual cell level within tumors.

• Liquid Biopsy Development: As FKBP4 has been identified as a tumor-specific antigen capable of eliciting immune responses in breast cancer, FITC-conjugated antibodies could be utilized to detect circulating tumor cells or extracellular vesicles expressing FKBP4.

• Theranostic Applications: The dual role of FKBP4 in cancer progression and as a potential therapeutic target suggests applications in developing theranostic approaches, where the FITC-conjugated antibody could serve both diagnostic and therapeutic functions.

• PI3K/Akt/mTOR Pathway Modulation Monitoring: As research has demonstrated FKBP4's role in enhancing Akt activation through PDK1 and mTORC2 , FITC-conjugated antibodies could be valuable tools in monitoring pathway modulation during drug screening and development.

• Multiplexed Tissue Imaging: Integration into multiplexed immunofluorescence panels to simultaneously assess FKBP4 expression alongside other cancer biomarkers, providing comprehensive tumor profiling.

• Therapeutic Response Prediction: Development of FKBP4 expression assays as potential predictive biomarkers for response to targeted therapies, particularly for triple-negative breast cancers where treatment options remain limited .