FGFR1OP Antibody

What is FGFR1OP and why is it important in cancer research?

FGFR1OP (fibroblast growth factor receptor 1 oncogene partner) is a protein that has been identified as significantly overexpressed in various cancer types, particularly in non-small cell lung cancer (NSCLC). Gene expression profile analysis of lung carcinomas has revealed elevated expression of FGFR1OP in the majority of lung cancers, with immunohistochemical studies confirming positive staining in approximately 89.8% of NSCLC specimens . Its importance stems from its correlation with shorter tumor-specific survival times and its role in promoting cell cycle progression and cellular motility . FGFR1OP interacts with important cellular proteins such as ABL1 (Abelson murine leukemia viral oncogene homolog 1) and WRNIP1 (Werner helicase interacting protein 1), suggesting its involvement in critical cellular processes related to cancer progression . These characteristics make FGFR1OP a promising prognostic biomarker and potential therapeutic target for lung cancer.

How does FGFR1OP function at the molecular level?

At the molecular level, FGFR1OP functions through interactions with key cellular proteins involved in signaling and cell cycle regulation. Research has identified that FGFR1OP interacts with ABL1 (Abelson murine leukemia viral oncogene homolog 1) and WRNIP1 (Werner helicase interacting protein 1), which is known to be involved in cell cycle progression . FGFR1OP significantly reduces ABL1-dependent phosphorylation of WRNIP1, which results in the promotion of cell cycle progression . Additionally, induction of FGFR1OP has been shown to increase cellular motility and growth-promoting activity in mammalian cells . These molecular interactions help explain why FGFR1OP overexpression contributes to cancer cell proliferation and tumor progression, particularly in lung cancers where it is commonly overexpressed.

What are the common applications for FGFR1OP antibodies in basic research?

FGFR1OP antibodies have several validated applications in basic research settings:

• Western Blot: Used for protein expression analysis and quantification in cell lysates, typically at dilutions around 1:500 .

• Immunocytochemistry/Immunofluorescence: Employed to visualize cellular localization of FGFR1OP in cultured cells, with recommended dilutions between 1:10 and 1:500 .

• Immunohistochemistry (IHC): Applied to detect FGFR1OP in both paraffin-embedded and frozen tissue sections, allowing evaluation of expression in clinical samples .

• ELISA: Used for quantitative detection of FGFR1OP, typically at dilutions ranging from 1:100 to 1:2000 .

• Block/Neutralize: Some antibodies have been cited for use in blocking or neutralizing FGFR1OP function in experimental settings .

These applications enable researchers to investigate FGFR1OP expression patterns, subcellular localization, and functional relationships in various experimental models.

How can FGFR1OP antibodies be utilized in cancer biomarker research?

FGFR1OP antibodies can be strategically employed in cancer biomarker research through multiple advanced approaches:

Tissue Microarray Analysis: Studies have utilized tumor tissue microarrays consisting of hundreds of archived NSCLC specimens to evaluate FGFR1OP expression patterns. This high-throughput approach revealed positive staining in 89.8% (334 of 372) of NSCLC cases, establishing FGFR1OP as a prevalent biomarker in lung cancer . When conducting such analyses, researchers should use standardized scoring systems that account for both staining intensity and percentage of positive cells.

Prognostic Correlation Studies: FGFR1OP expression levels can be correlated with clinical outcomes using Kaplan-Meier survival analyses. Research has demonstrated that high levels of FGFR1OP expression are significantly associated with shorter tumor-specific survival times (p < 0.0001), indicating its value as a prognostic biomarker . For such studies, patient cohorts should be well-characterized with complete follow-up data and appropriate statistical methods.

Multi-marker Panels: FGFR1OP antibodies can be integrated into multi-marker immunohistochemical panels to improve diagnostic and prognostic accuracy. This approach requires careful optimization of staining protocols for each antibody in the panel and sophisticated image analysis methods to quantify co-expression patterns.

Circulating Tumor Cell Analysis: While not explicitly mentioned in the search results, FGFR1OP antibodies could potentially be used to detect FGFR1OP expression in circulating tumor cells, providing a less invasive method to monitor cancer progression and treatment response.

What methodological approaches can be used to investigate FGFR1OP interactions with other proteins?

Several sophisticated methodological approaches can be employed to investigate FGFR1OP interactions with other proteins:

Co-immunoprecipitation (Co-IP): This technique has been successfully used to identify FGFR1OP-interacting proteins in lung cancer cells, revealing interactions with ABL1 and WRNIP1 . For optimal results, researchers should use cell lysis conditions that preserve protein-protein interactions and include appropriate controls to confirm specificity.

Immunofluorescence Co-localization: Double-labeling approaches can visualize the spatial relationship between FGFR1OP and its binding partners. As demonstrated in the search results, FGFR1OP (labeled in green) and GT335 (cilia marker, labeled in red) have been co-visualized in ependymal whole mounts from rabbit brain tissue . Confocal microscopy with appropriate controls for spectral overlap is essential for accurate co-localization analysis.

Proximity Ligation Assay (PLA): Though not explicitly mentioned in the search results, PLA represents an advanced technique to detect protein-protein interactions in situ with high sensitivity and specificity, allowing visualization of interactions at endogenous expression levels.

Phosphorylation Studies: Given that FGFR1OP affects ABL1-dependent phosphorylation of WRNIP1 , phospho-specific antibodies can be used in combination with FGFR1OP antibodies to investigate the regulatory mechanisms of these interactions. Western blotting with phospho-specific antibodies following FGFR1OP manipulation (overexpression or knockdown) can reveal the signaling consequences of these interactions.

How can siRNA-mediated knockdown be combined with FGFR1OP antibody detection to study functional effects?

Combining siRNA-mediated knockdown with FGFR1OP antibody detection provides a powerful approach to study the functional effects of FGFR1OP in cancer cells:

Experimental Design Framework:

• Transfection Optimization: First establish optimal transfection conditions for your specific cell line using a fluorescently labeled control siRNA to assess transfection efficiency.

• Knockdown Validation: Following transfection with FGFR1OP siRNA, use Western blot with FGFR1OP antibodies at a recommended dilution of 1:500 to confirm protein reduction . Quantify the degree of knockdown compared to cells transfected with scrambled siRNA control.

• Temporal Analysis: Perform time-course experiments to determine the optimal time point for functional assays based on maximum knockdown efficiency, typically 48-72 hours post-transfection.

Research has demonstrated that treatment of lung cancer cells (which overexpress endogenous FGFR1OP) with FGFR1OP siRNA suppressed its expression and resulted in inhibition of cell growth . This experimental approach can be extended to investigate various cellular phenotypes:

• Proliferation Assays: Measure cell growth using methods such as MTT assay, BrdU incorporation, or real-time cell analysis following FGFR1OP knockdown.

• Cell Cycle Analysis: Use flow cytometry to determine if FGFR1OP knockdown affects cell cycle distribution, particularly given its role in promoting cell cycle progression through WRNIP1 regulation .

• Migration and Invasion Assays: Since FGFR1OP increases cellular motility , Transwell or wound healing assays can reveal its impact on cancer cell migration.

• Protein Interaction Studies: Examine whether knockdown of FGFR1OP affects its interactions with partners like ABL1 and WRNIP1 using co-immunoprecipitation followed by Western blotting.

What are the critical factors for optimizing immunohistochemistry protocols using FGFR1OP antibodies?

Optimizing immunohistochemistry protocols for FGFR1OP antibodies requires attention to several critical factors:

Tissue Preparation and Fixation:

• FGFR1OP antibodies have been successfully used on both paraffin-embedded and frozen tissue sections . For paraffin sections, ensure consistent fixation times (typically 24 hours in 10% neutral buffered formalin) to preserve antigenic sites.

• For frozen sections, 4% paraformaldehyde fixation has been validated for FGFR1OP detection, as demonstrated in rabbit brain tissue samples .

Antigen Retrieval Methods:

• Heat-induced epitope retrieval (HIER) is typically necessary for paraffin sections. The optimal buffer (citrate buffer pH 6.0 or EDTA buffer pH 9.0) should be determined empirically for each FGFR1OP antibody.

• For frozen sections, antigen retrieval may be less critical but should be evaluated.

Antibody Selection and Dilution:

• Both monoclonal (e.g., clone 2B1) and polyclonal FGFR1OP antibodies are available .

• Starting dilutions range from 1:10 to 1:500 for immunofluorescence and immunohistochemistry applications .

• For the polyclonal antibody described in search result , a dilution of 1:50 was used successfully for paraffin-embedded human colon and cervical cancer tissues.

Detection Systems:

• Secondary antibody selection should match the host species of the primary antibody (e.g., anti-mouse for monoclonal 2B1; anti-rabbit for polyclonal antibodies) .

• Signal amplification systems may be required for detecting low-abundance expression.

Controls:

• Positive controls should include tissues known to express FGFR1OP, such as lung cancer tissues where 89.8% of NSCLC specimens show positive staining .

• Negative controls should include omission of primary antibody and, ideally, tissues or cells with confirmed low expression.

What are the best practices for validating FGFR1OP antibody specificity?

Validating FGFR1OP antibody specificity is crucial for ensuring reliable research outcomes. Best practices include:

Genetic Modification Approaches:

• Overexpression Validation: Compare antibody staining between FGFR1OP-transfected and non-transfected cell lines. Western blot analysis has shown clear distinction between FGFR1OP transfected lysates (showing a 43.1 KDa band) and non-transfected lysates .

• Knockdown Validation: Confirm reduced antibody signal following siRNA-mediated knockdown of FGFR1OP, as demonstrated in lung cancer cell studies .

Molecular Weight Verification:

• In Western blot applications, confirm that the detected band appears at the expected molecular weight for FGFR1OP (approximately 43.1 KDa) .

• Be aware of potential post-translational modifications that may alter the apparent molecular weight.

Cross-Reactivity Assessment:

• Test antibody performance across multiple species if cross-reactivity is claimed. The monoclonal 2B1 antibody has validated reactivity with human and mouse FGFR1OP, with rabbit reactivity reported in customer reviews .

• Consider testing in tissues known to have variable FGFR1OP expression levels.

Comparative Antibody Analysis:

• When possible, compare results using multiple antibodies targeting different epitopes of FGFR1OP.

• For monoclonal antibodies like 2B1, the epitope information (full-length recombinant protein with GST tag) provides context for potential binding specificity .

Control Experiments:

• Include peptide competition assays where the antibody is pre-incubated with the immunizing peptide prior to application, which should abolish specific staining.

• For immunofluorescence applications, counterstaining with well-established markers (such as GT335 for cilia) can help confirm the expected subcellular localization pattern .

What technical challenges might researchers encounter when using FGFR1OP antibodies in Western blot applications?

Researchers using FGFR1OP antibodies in Western blot applications may encounter several technical challenges:

Sample Preparation Considerations:

• Protein Extraction Efficiency: FGFR1OP is associated with the centrosome (also known as Centrosomal protein 43 or CEP43) , which may require specialized lysis buffers to ensure complete solubilization.

• Protein Degradation: Use of fresh protease inhibitors is essential to prevent degradation of FGFR1OP during sample preparation.

Electrophoresis and Transfer Parameters:

• Molecular Weight Detection: FGFR1OP has a reported molecular weight of approximately 43.1 KDa , which should transfer efficiently under standard conditions, but optimization may be needed.

• Transfer Efficiency: Using PVDF membranes rather than nitrocellulose may improve retention of FGFR1OP during transfer for some antibodies.

Antibody Performance Variables:

• Dilution Optimization: The recommended dilution for Western blot is 1:500 for the monoclonal 2B1 antibody , but this may require adjustment based on expression levels and detection methods.

• Non-specific Binding: Background issues may occur, particularly with polyclonal antibodies. Extended blocking times (2+ hours) and thorough washing steps can help reduce background.

Detection Challenges:

• Signal Intensity: Endogenous FGFR1OP expression may vary significantly between cell types, potentially requiring signal enhancement methods for cells with lower expression.

• Specificity Confirmation: As demonstrated in validation studies, comparing FGFR1OP-transfected lysates (showing a 43.1 KDa band) with non-transfected lysates provides a critical control for specificity .

Reproducibility Issues:

• Antibody Lot Variability: Concentrations may vary between lots, requiring adjustment of dilutions. As noted in search result , "Concentrations vary lot to lot. See vial label for concentration."

• Storage Conditions: Proper aliquoting and storage at -20°C or -80°C is recommended to avoid freeze-thaw cycles that could reduce antibody performance .

How can researchers optimize double immunofluorescence staining for FGFR1OP and interacting proteins?

Optimizing double immunofluorescence staining for FGFR1OP and its interacting proteins requires careful consideration of several technical parameters:

Sequential vs. Simultaneous Staining:

• For co-detection of FGFR1OP with proteins like GT335 (as shown in ependymal whole mounts from rabbit brain) , sequential staining may reduce potential cross-reactivity between secondary antibodies.

• If using sequential staining, start with the weaker signal antibody first, followed by the stronger one.

Primary Antibody Selection:

• Choose primary antibodies from different host species (e.g., mouse anti-FGFR1OP and rabbit anti-interacting protein) to allow for simultaneous detection with species-specific secondary antibodies.

• The monoclonal mouse FGFR1OP antibody (clone 2B1) has been successfully used in immunofluorescence at dilutions between 1:10 and 1:500 .

Secondary Antibody Optimization:

• Use highly cross-adsorbed secondary antibodies to minimize cross-reactivity.

• Select fluorophores with minimal spectral overlap (e.g., Alexa Fluor 488 for green and Alexa Fluor 594 for red channels).

• Titrate secondary antibodies to determine optimal concentration that provides sufficient signal while minimizing background.

Sample Preparation Considerations:

• Fixation method significantly impacts epitope preservation. For FGFR1OP detection in frozen sections, 4% paraformaldehyde fixation has been validated .

• Permeabilization conditions should be optimized to allow antibody access to intracellular targets without disrupting protein localization.

Controls and Validation:

• Single-stain controls are essential to confirm specificity and check for bleed-through between channels.

• Include a co-localization control (known interacting proteins) to validate your protocol.

• Use Z-stack imaging to confirm true co-localization in three dimensions, particularly important for centrosomal proteins like FGFR1OP/CEP43.

Image Acquisition Parameters:

• Use sequential scanning rather than simultaneous acquisition when possible.

• Match laser power and detector settings to avoid saturation while capturing the full dynamic range of the signal.

• For quantitative co-localization studies, maintain consistent imaging parameters across all samples.

What strategies can address inconsistent results in detecting FGFR1OP expression across different tissue types?

Addressing inconsistent results in detecting FGFR1OP expression across different tissue types requires a systematic troubleshooting approach:

Tissue-Specific Optimization Table:

Fixation and Processing Variables:

• Fixation time significantly impacts antibody performance. Standardize to 24 hours for formalin-fixed tissues.

• For tissues with high adipose content, extend fixation and processing times to ensure complete fixation.

• Consider using tissue-specific fixatives for challenging tissues (e.g., Bouin's for testicular tissue).

Epitope Accessibility Strategies:

• Different tissues may require different antigen retrieval methods. Systematically compare heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0).

• For tissues with high endogenous peroxidase activity, include additional blocking steps.

• Consider enzymatic digestion (e.g., proteinase K, trypsin) as an alternative to heat-based retrieval for certain tissues.

Detection System Adjustments:

• Amplification systems (e.g., tyramide signal amplification) may be necessary for tissues with low FGFR1OP expression.

• For tissues with high background, use polymer-based detection systems rather than avidin-biotin methods.

• Optimize incubation times based on tissue density and antibody penetration requirements.

Expression Level Verification:

• When possible, verify immunohistochemistry results with complementary methods such as Western blot or qRT-PCR from the same tissue samples.

• Consider microdissection of specific tissue components followed by Western blot for more precise quantification.

How can researchers effectively use FGFR1OP antibodies to investigate its role in cancer cell migration and invasion?

Researchers can effectively use FGFR1OP antibodies to investigate its role in cancer cell migration and invasion through several advanced experimental approaches:

Functional Assay Integration:

• Wound Healing Assays: Following manipulation of FGFR1OP expression (overexpression or siRNA knockdown), monitor cell migration into the wound area over time. Use FGFR1OP antibodies (1:10-1:500 dilution for immunofluorescence) to confirm expression levels in migrating cells at wound edges.

• Transwell Migration/Invasion Assays: Quantify the number of cells passing through uncoated (migration) or Matrigel-coated (invasion) membranes after FGFR1OP manipulation. Collect cells from both sides of the membrane for Western blot analysis (1:500 dilution) to correlate migration/invasion capacity with FGFR1OP expression levels.

• 3D Spheroid Invasion Assays: Embed cancer cell spheroids in extracellular matrix and monitor invasion patterns. Use immunofluorescence with FGFR1OP antibodies to visualize protein localization in invasive projections.

Molecular Mechanism Investigation:

• Co-immunoprecipitation (Co-IP): Use FGFR1OP antibodies to pull down protein complexes from migrating versus stationary cancer cells to identify differential binding partners involved in migration.

• Phosphorylation Status Analysis: Since FGFR1OP affects ABL1-dependent phosphorylation of WRNIP1 , investigate whether this signaling axis is modulated during migration by blotting for phosphorylated targets in migrating versus non-migrating cells.

• Cytoskeletal Interaction Studies: Perform double immunofluorescence for FGFR1OP and cytoskeletal components (actin, tubulin) in migrating cells to identify potential structural roles during migration.

Live Cell Imaging Applications:

• Antibody Fragment Labeling: For live cell imaging, consider using fluorescently labeled FGFR1OP antibody fragments (Fab) to track protein dynamics during migration without interfering with function.

• Correlation with Migration Markers: Combine FGFR1OP immunostaining with markers of migration (e.g., focal adhesion proteins) to establish spatial and temporal relationships during the migration process.

Data Analysis Approaches:

• Quantitative Image Analysis: Develop algorithms to quantify FGFR1OP expression levels, subcellular localization, and co-localization with migration-related proteins at different stages of migration.

• Correlation Analysis: Statistically correlate FGFR1OP expression or localization patterns with quantitative migration parameters (velocity, directionality, persistence).

This experimental framework leverages various antibody applications (Western blot, immunofluorescence, Co-IP) to comprehensively investigate FGFR1OP's functional role in cancer cell migration and invasion, building upon the established finding that FGFR1OP increases cellular motility in mammalian cells .