FOXL2 Antibody, HRP conjugated

What is FOXL2 and why is it significant in research?

FOXL2 is a transcription factor belonging to the forkhead box (FOX) superfamily, characterized by the forkhead box/winged-helix DNA-binding domain . It serves as a critical regulator essential for ovary differentiation and maintenance while repressing the genetic program for somatic testis determination. Specifically, FOXL2 prevents trans-differentiation of ovary to testis through transcriptional repression of the Sertoli cell-promoting gene SOX9 . This makes it a crucial target in reproductive biology research.

In cellular processes, FOXL2 demonstrates apoptotic activity in ovarian cells, suppresses ESR1-mediated transcription of PTGS2/COX2 stimulated by tamoxifen, regulates CYP19 expression, participates in SMAD3-dependent transcription of FST via intronic SMAD-binding elements, functions as a transcriptional repressor of STAR, activates SIRT1 transcription under cellular stress conditions, and activates transcription of OSR2 . These diverse functions make FOXL2 antibodies essential tools for investigating reproductive development, ovarian pathologies, and transcriptional regulation.

How does HRP conjugation enhance FOXL2 antibody utility?

HRP conjugation directly links the Horseradish Peroxidase enzyme to the FOXL2 antibody, eliminating the need for secondary antibody incubation in detection workflows . This modification offers several methodological advantages:

• Reduced experimental time by eliminating secondary antibody incubation steps

• Decreased background signal that may arise from cross-reactivity of secondary antibodies

• Enhanced sensitivity in detection systems using chemiluminescent substrates

• Compatibility with multiple detection platforms including Western blotting and immunohistochemistry

For optimal results when using HRP-conjugated FOXL2 antibodies, researchers should use specific detection reagents optimized for peroxidase activity and consider shorter incubation times compared to unconjugated primary antibodies followed by HRP-labeled secondary antibodies .

What are the standard applications and dilutions for FOXL2-HRP antibodies?

FOXL2-HRP antibodies demonstrate versatility across multiple experimental applications. The following table summarizes recommended applications and dilutions based on compiled research data:

For immunohistochemistry applications, FOXL2 antibodies successfully detect the protein in ovarian cancer tissue samples when used at approximately 5 μg/mL for 1 hour at room temperature, followed by incubation with anti-mouse IgG HRP polymer antibody . Tissue preparation typically requires heat-induced epitope retrieval using basic antigen retrieval reagents before incubation with the primary antibody .

What are the optimal storage conditions for maintaining FOXL2-HRP antibody activity?

Proper storage is critical for maintaining antibody functionality. Based on manufacturer specifications, FOXL2-HRP antibodies require the following storage conditions:

• Store at -20°C or -80°C for long-term preservation

• Alternatively, store at 4°C in the dark for more frequently used aliquots

• Avoid repeated freeze-thaw cycles as this significantly degrades antibody performance

• When stored in 50% glycerol buffer with 0.01M PBS (pH 7.4), stability is enhanced

For laboratories engaged in extended research projects, creating multiple small-volume aliquots upon receipt is recommended to prevent degradation from repeated handling. Working dilutions should be prepared fresh before each experiment to ensure consistent results across experimental replicates .

How can researchers verify FOXL2 antibody specificity?

Validation of antibody specificity is essential for generating reliable research data. Multiple approaches should be employed:

• Positive and negative control tissues/cell lines: PANC-1 human pancreatic carcinoma cell line shows positive nuclear staining for FOXL2, while MCF-7 human breast cancer cell line serves as a negative control .

• Phosphatase treatment: For phospho-specific analyses, treating samples with alkaline phosphatase (AP) and comparing to untreated samples can confirm phosphorylation-specific recognition .

• Western blot profile: Verify antibody specificity by confirming band migration at the expected molecular weight of 39 kDa for FOXL2 .

• Immunoprecipitation validation: When immunoprecipitating with FOXL2 antibodies, subsequent immunoblotting should detect both FOXL2 and any interacting partners under investigation, such as LATS1 .

What experimental approaches are recommended for studying FOXL2 phosphorylation?

FOXL2 undergoes post-translational modification through phosphorylation, which affects its activity. To study this process:

• Co-immunoprecipitation with phospho-antibodies: Immunoprecipitate FOXL2 using anti-FOXL2 antibodies, then probe with phosphoserine antibodies to detect phosphorylated FOXL2 variants .

• In vitro kinase assays: Purify FLAG-tagged FOXL2 proteins and combine with immunoprecipitates containing potential kinases (like LATS1) in buffer containing 50 mM Tris·HCl, 150 mM sodium chloride, 2 mM ATP, 10 mM magnesium chloride, and 10 mM manganese chloride at 30°C .

• Alkaline phosphatase treatment: Treat immunoprecipitated FOXL2 with alkaline phosphatase to remove phosphate groups, then compare migration patterns with untreated samples using SDS-PAGE .

• Western blot band shift analysis: Phosphorylated FOXL2 typically appears as a higher molecular weight band compared to unphosphorylated FOXL2, allowing visual confirmation of phosphorylation status .

Research has demonstrated that FOXL2 is phosphorylated at serine residues, and this modification regulates its transcriptional activity . Specifically, when cell lysates are immunoprecipitated and probed with phosphoserine antibodies, the upper band of FOXL2 is detected, confirming serine phosphorylation .

What are common challenges in Western blotting with FOXL2-HRP antibodies?

Western blotting with FOXL2-HRP antibodies may present several technical challenges requiring specific optimization:

• Multiple bands: FOXL2 typically shows two bands in Western blot analyses—one corresponding to unphosphorylated FOXL2 and a higher molecular weight band representing phosphorylated FOXL2 . Researchers should anticipate this pattern rather than considering it non-specific binding.

• Signal intensity: For optimal chemiluminescent detection, enhanced chemiluminescence Western blotting detection reagents are recommended . Incubation time with detection reagents may need adjustment based on expression levels.

• Membrane selection: PVDF membranes have shown better results than nitrocellulose for FOXL2 detection .

• Blocking optimization: A 5% non-fat milk solution in TBS-T provides effective blocking while preserving epitope accessibility for FOXL2 antibodies.

When troubleshooting, sequential adjustment of primary antibody concentration, incubation time, and detection exposure should be performed to identify optimal conditions for specific experimental systems.

How can researchers optimize immunohistochemistry protocols for FOXL2-HRP antibodies?

For successful immunohistochemical detection of FOXL2 in tissue sections:

• Antigen retrieval: Heat-induced epitope retrieval using basic antigen retrieval reagents is critical before incubation with FOXL2 antibodies .

• Antibody concentration: For paraffin-embedded sections, 5-8 μg/mL of FOXL2 antibody with 1-hour room temperature incubation provides optimal staining .

• Detection systems: DAB (3,3'-diaminobenzidine) chromogen produces brown staining that effectively highlights nuclear localization of FOXL2 .

• Counterstaining: Hematoxylin counterstaining provides clear visualization of tissue architecture while contrasting with the FOXL2-specific nuclear staining .

• Fixation considerations: Overfixation can mask epitopes; therefore, tissue processing should be standardized and controlled to ensure consistent results.

When interpreting results, researchers should note that FOXL2 demonstrates specific localization to cell nuclei, as confirmed in multiple studies .

What controls should be included when working with FOXL2-HRP antibodies?

Rigorous experimental design requires appropriate controls:

• Positive tissue controls: Ovarian tissue sections showing nuclear FOXL2 expression should be included in each experimental run .

• Negative tissue controls: MCF-7 human breast cancer cell line has been validated as an appropriate negative control for FOXL2 expression .

• Isotype controls: Rabbit IgG at equivalent concentrations to the FOXL2 antibody should be used to identify non-specific binding .

• Phosphorylation controls: When studying phosphorylated FOXL2, samples treated with alkaline phosphatase provide essential dephosphorylated controls .

• Peptide competition: Pre-incubation of the antibody with excess immunizing peptide should eliminate specific staining in validation experiments.

How can FOXL2-HRP antibodies be utilized to study protein-protein interactions?

FOXL2 participates in numerous protein-protein interactions that regulate ovarian development and function. To study these interactions:

• Co-immunoprecipitation: Immunoprecipitate FOXL2 from cell lysates using specific antibodies, then probe for potential interacting partners like LATS1 . When cells expressing FLAG-FOXL2 are lysed and immunoprecipitated with an antibody to FLAG, both FLAG-FOXL2 and LATS1 can be identified in the immunoprecipitates, confirming their interaction .

• Proximity ligation assays: This technique allows visualization of protein-protein interactions in situ by generating fluorescent signals only when two antibody-targeted proteins are in close proximity.

• ChIP-reChIP: For studying FOXL2 interactions with other transcription factors at specific genomic loci, sequential chromatin immunoprecipitation can be employed.

• Bimolecular fluorescence complementation: By fusing complementary fragments of fluorescent proteins to FOXL2 and potential interactors, interaction-dependent fluorescence can be visualized in living cells.

Research has established that FOXL2 interacts with the tumor suppressor LATS1, which phosphorylates FOXL2 and regulates its transcriptional activity . This interaction was initially identified through yeast two-hybrid screening and subsequently confirmed by co-immunoprecipitation experiments .

What methodologies are recommended for studying FOXL2 in ovarian pathologies?

FOXL2 expression levels play a role in several ovarian pathologies, including adult granulosa cell tumors and endometriosis . To investigate these connections:

• Tissue microarray analysis: Enables high-throughput screening of FOXL2 expression across multiple patient samples using HRP-conjugated antibodies with standardized detection methods .

• Mutation analysis combined with immunohistochemistry: Particularly relevant for adult granulosa cell tumors where FOXL2 mutations correlate with protein expression patterns.

• Laser capture microdissection with immunostaining: Allows isolation of specific FOXL2-expressing cell populations from heterogeneous tissue samples for downstream molecular analysis.

• Quantitative image analysis: Digital pathology approaches can quantify nuclear FOXL2 intensity across different tissue samples, enabling correlation with clinical parameters.

Researchers should note that FOXL2 shows specific nuclear localization in immunostaining experiments of ovarian cancer tissue . This characteristic pattern provides valuable diagnostic information when evaluating ovarian pathologies.

How can FOXL2-HRP antibodies contribute to research on transcriptional regulation?

As a transcription factor, FOXL2 plays critical roles in gene regulation. To study these functions:

• Chromatin immunoprecipitation (ChIP): Using FOXL2 antibodies to identify genomic loci bound by FOXL2 in various cell types or tissues.

• Reporter gene assays: Measuring the effect of FOXL2 expression on the activity of promoter constructs, particularly those containing forkhead binding elements.

• Gene expression analysis following FOXL2 manipulation: Combining FOXL2 antibodies for validation of FOXL2 levels with transcriptome analysis.

• Sequential ChIP with transcriptional co-regulators: Identifying genomic regions where FOXL2 collaborates with other transcription factors or chromatin modifiers.

FOXL2 has been shown to suppress ESR1-mediated transcription of PTGS2/COX2, regulate CYP19 expression, participate in SMAD3-dependent transcription of FST, repress STAR transcription, activate SIRT1 transcription under cellular stress, and activate OSR2 transcription . These diverse transcriptional activities make FOXL2-HRP antibodies valuable tools for studying gene regulation in reproductive biology.

What emerging technologies are enhancing FOXL2 antibody applications?

Recent technological advances are expanding the utility of FOXL2 antibodies in research:

• Single-cell protein analysis: Combining FOXL2 antibodies with mass cytometry or microfluidic platforms to analyze protein expression at the single-cell level.

• Super-resolution microscopy: Enabling visualization of FOXL2 nuclear distribution patterns with nanometer-scale resolution.

• Multiplexed antibody imaging: Simultaneous detection of FOXL2 with multiple other proteins using cyclic immunofluorescence or spectral unmixing approaches.

• CRISPR-based genomic tagging: Creating endogenously tagged FOXL2 variants for validation of antibody specificity and live-cell imaging applications.

These technologies will continue to enhance our understanding of FOXL2 biology and its role in development and disease processes.

What considerations should guide selection of FOXL2-HRP antibodies for specific experimental goals?

When selecting FOXL2-HRP antibodies, researchers should consider:

• Clonality: Both polyclonal and monoclonal FOXL2 antibodies are available. Polyclonal antibodies may provide higher sensitivity by recognizing multiple epitopes, while monoclonal antibodies offer greater specificity and lot-to-lot consistency.

• Host species: Rabbit-derived antibodies are common for FOXL2 detection . The host species should be compatible with other antibodies used in multiplex experiments.

• Epitope location: Antibodies targeting different regions of FOXL2 may perform differently in specific applications. For example, antibodies raised against amino acids 20–33 of FOXL2 have been successfully used in Western blotting .

• Validation data: Priority should be given to antibodies with extensive validation data across multiple applications, particularly those demonstrating specificity in tissues relevant to the planned experiments .