FOXQ1 Antibody, HRP conjugated

What is FOXQ1 and why is it significant in cancer research?

FOXQ1 (also known as HFH1) is a member of the FOX gene family characterized by a conserved 110-amino acid DNA-binding motif called the forkhead or winged helix domain. This transcription factor plays crucial roles in embryonic development, cell cycle regulation, tissue-specific gene expression, cell signaling, and tumorigenesis .

Research has shown that FOXQ1 is frequently overexpressed in colorectal cancer (CRC) and correlates with poor prognosis. It promotes invasion and metastasis through the HB-EGF/EGFR pathway, activating downstream genes including AKT, RAF, and KRAS . Studies have demonstrated that knockdown of FOXQ1 suppresses cell proliferation, migration, and invasion in CRC cells .

What are the key characteristics of FOXQ1 protein for antibody detection?

When designing experiments using FOXQ1 antibodies, researchers should note these key characteristics:

For optimal detection, researchers should be aware that the actual observed molecular weight may sometimes differ from the calculated value due to post-translational modifications or other factors affecting mobility during electrophoresis .

How should I optimize Western blot protocols when using HRP-conjugated FOXQ1 antibodies?

For optimal Western blot results with HRP-conjugated FOXQ1 antibodies:

• Sample preparation: Extract total protein using RIPA lysis buffer. Load approximately 30μg protein per sample on 10% polyacrylamide-SDS gels .

• Blocking conditions: Block membranes in 5% fat-free milk to reduce non-specific binding .

• Antibody dilution: For HRP-conjugated FOXQ1 antibodies, the recommended dilution ranges from 1:500-1:2000, though this should be optimized for each specific antibody . For example, Proteintech's FOXQ1 antibody (23718-1-AP) is recommended at 1:500-1:1000 dilution for Western blot .

• Detection system: When using HRP-conjugated antibodies, no secondary antibody is required, which simplifies the protocol and may reduce background signal.

• Signal development: Use enhanced chemiluminescence (ECL) substrate for visualization. Note that HRP-conjugated antibodies in light-protected vials or covered with aluminum foil should be used to maintain enzymatic activity .

• Expected band: Look for a band at approximately 42 kDa, though the observed molecular weight may differ slightly depending on cell/tissue type and possible post-translational modifications .

What are the recommended protocols for immunohistochemistry (IHC) using HRP-conjugated FOXQ1 antibodies?

For effective IHC using HRP-conjugated FOXQ1 antibodies:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) tissue sections.

• Antigen retrieval: Use TE buffer at pH 9.0 for optimal results. Alternatively, citrate buffer at pH 6.0 may be used, though this may yield different sensitivity .

• Antibody dilution: The recommended dilution range is 1:400-1:1600 for the Proteintech antibody (23718-1-AP) . For Elabscience's E-AB-19882, a more concentrated dilution of 1:25-1:50 is recommended . Always optimize for your specific sample and antibody.

• Incubation conditions: Incubate with primary antibody overnight at 4°C for best results .

• Detection: Being HRP-conjugated, no secondary antibody is needed. Develop with DAB (3,3′-diaminobenzidine) substrate.

• Counterstaining: Use hematoxylin for nuclear counterstaining.

• Positive controls: Human colorectal cancer and ovarian cancer tissues have been verified as positive samples for IHC with FOXQ1 antibodies . Human stomach cancer tissue has also been validated .

How can FOXQ1 antibodies be used to investigate its role in cancer signaling pathways?

FOXQ1 antibodies can be instrumental in elucidating cancer signaling pathways through multiple approaches:

• Pathway analysis: Studies using FOXQ1 antibodies have revealed that FOXQ1 activates the EGFR pathway through HB-EGF. Western blotting after FOXQ1 knockdown showed decreased expression of EGFR downstream genes including AKT, RAF, and KRAS . This experimental approach involves:

  • Establishing knockdown models (using siRNA or shRNA)

  • Confirming knockdown efficiency using FOXQ1 antibodies

  • Assessing downstream pathway components

• EMT regulation: FOXQ1 influences epithelial-mesenchymal transition (EMT), which can be monitored by examining EMT markers:

  • E-cadherin

  • N-cadherin

  • Vimentin

  These can be assessed by Western blotting following FOXQ1 manipulation .

• Co-IP experiments: HRP-conjugated FOXQ1 antibodies can be used in immunoprecipitation followed by blotting for potential interaction partners to map signaling networks. Research indicates FOXQ1 may interact with multiple signaling pathways .

• ChIP assays: To identify direct transcriptional targets of FOXQ1, which helps establish its position in signaling hierarchies.

How can FOXQ1 expression be effectively used as a prognostic biomarker in cancer studies?

Based on multiple studies, FOXQ1 has emerged as a promising prognostic biomarker in several cancers. When designing studies to evaluate FOXQ1 as a biomarker:

• Expression analysis: Use IHC with carefully standardized protocols to quantify FOXQ1 expression in tumor samples. Studies have divided patients into "high" and "low" FOXQ1 expression groups .

• Scoring systems: Develop consistent scoring methods for FOXQ1 immunoreactivity:

  • Based on staining intensity (0, 1+, 2+, 3+)

  • Percentage of positive cells

  • Combined scores

• Correlation analysis: FOXQ1 expression should be correlated with:

  • Clinicopathological features (tumor size, lymph node metastasis, TNM stage)

  • Other biomarkers (e.g., CEA in NSCLC studies)

  • Survival outcomes (DFS, OS)

• Statistical analysis: Use appropriate statistical methods:

  • Kaplan-Meier survival analysis for DFS and OS

  • Cox proportional hazards regression for multivariate analysis

  • Statistical significance defined as P < 0.05

Example results from a NSCLC study :

Multivariate analysis confirmed FOXQ1 as an independent prognostic factor for both DFS (HR=1.379, 95% CI: 1.011-1.882, P=0.043) and OS (HR=1.498, 95% CI: 1.064-2.108, P=0.021) .

What are common technical issues when using HRP-conjugated FOXQ1 antibodies and how can they be resolved?

How should HRP-conjugated FOXQ1 antibodies be stored and handled to maintain optimal activity?

To preserve the activity of HRP-conjugated FOXQ1 antibodies:

• Storage temperature: Store at -20°C for long-term storage. Antibodies are typically stable for 12 months when properly stored .

• Light protection: HRP conjugates should be stored in light-protected vials or covered with aluminum foil to prevent photo-degradation of the enzyme .

• Aliquoting: For antibodies without glycerol, aliquot upon receipt to avoid repeated freeze-thaw cycles. For antibodies in 50% glycerol, aliquoting is typically unnecessary for -20°C storage .

• Shipping and receipt: Products are typically shipped with ice packs. Upon receipt, store immediately at the recommended temperature .

• Buffer composition: HRP-conjugated antibodies are typically provided in PBS with preservatives such as sodium azide (0.02%) and 50% glycerol at pH 7.3-7.6 .

• Working solution preparation: Dilute only the amount needed for immediate use. Prepare fresh working dilutions on the day of the experiment.

• Handling during experiments: Keep on ice when in use and return to -20°C promptly after use. Avoid prolonged exposure to room temperature.

How can FOXQ1 antibodies be utilized in developing targeted cancer therapies?

FOXQ1 antibodies play a crucial role in developing targeted cancer therapies through several methodological approaches:

What are the considerations for using FOXQ1 antibodies in multiplex staining protocols?

When incorporating FOXQ1 antibodies into multiplex immunostaining protocols:

• Antibody compatibility:

  • Ensure all primary antibodies are raised in different host species to avoid cross-reactivity

  • If using multiple rabbit antibodies, consider sequential staining with stripping between rounds

  • Test for cross-reactivity between all antibodies in the panel

• HRP-conjugate considerations:

  • For multiplex fluorescence, direct HRP conjugates can be used with tyramide signal amplification (TSA)

  • After each HRP detection round, perform complete HRP inactivation (e.g., with hydrogen peroxide)

  • Validate complete inactivation before proceeding to next marker

• Antigen retrieval optimization:

  • FOXQ1 detection often requires TE buffer at pH 9.0 for optimal retrieval

  • Ensure compatible retrieval conditions for all target antigens

  • Consider a compromise retrieval method that works acceptably for all targets

• Recommended marker combinations:

  • FOXQ1 with EMT markers (E-cadherin, N-cadherin, vimentin)

  • FOXQ1 with EGFR pathway components (EGFR, HB-EGF, phospho-AKT, phospho-MAPK)

  • FOXQ1 with proliferation markers (Ki-67) and apoptosis markers

• Controls:

  • Single-stained controls for each antibody

  • Isotype controls

  • Known positive tissues (colorectal cancer, ovarian cancer, stomach cancer)

  • Blocking peptide controls to confirm specificity

• Order of antibody application:

  • Begin with the least abundant target or the one requiring most sensitivity

  • Consider nuclear antigens (like FOXQ1) early in the sequence

How should researchers design experiments to evaluate FOXQ1's role in cancer progression models?

A comprehensive experimental design to investigate FOXQ1's role in cancer progression should include:

• Expression profiling:

  • Compare FOXQ1 expression between tumor and adjacent normal tissues using Western blot and IHC

  • Correlate with clinical parameters using statistical analysis

  • Use databases like GEPIA (Gene Expression Profiling Interactive Analysis) to analyze expression patterns across large patient cohorts

• Functional studies:

  • Create stable cell lines with FOXQ1 knockdown using validated siRNAs/shRNAs

  • Confirm knockdown efficiency via qRT-PCR and Western blotting

  • Perform complementary overexpression studies

  • Assess effects on:

    • Proliferation (MTT/CCK-8 assays, colony formation)

    • Migration (transwell assays, scratch tests)

    • Invasion (Matrigel invasion assays)

    • EMT marker expression

    • Apoptosis and cell cycle distribution

• Mechanistic investigation:

  • Perform pathway analysis after FOXQ1 modulation

  • Use rescue experiments (e.g., adding recombinant HB-EGF to FOXQ1 knockdown cells)

  • Employ ChIP assays to identify direct transcriptional targets

  • RNA-seq to identify global transcriptome changes

  • Co-IP to identify protein interaction partners

• In vivo validation:

  • Xenograft models with FOXQ1-modulated cells

  • Metastasis models (tail vein injection, orthotopic implantation)

  • Analysis of tumor growth, metastasis formation, and survival

• Translational relevance:

  • Correlate experimental findings with patient data

  • Develop scoring systems for FOXQ1 expression in patient samples

  • Validate findings across multiple cancer types

What considerations should be made when interpreting contradictory FOXQ1 expression data from different detection methods?

When faced with contradictory FOXQ1 expression data across different detection methods:

• Methodological differences:

  • Each detection method has specific sensitivity and specificity profiles

  • Western blot provides molecular weight confirmation but limited spatial information

  • IHC provides cellular/tissue localization but potential epitope masking

  • qRT-PCR measures mRNA but not protein levels

• Antibody epitope considerations:

  • Different antibodies target different regions of FOXQ1:

    • N-terminal antibodies (e.g., orb2126822)

    • Antibodies targeting specific amino acid regions (AA 110-219, AA 29-78, etc.)

  • Post-translational modifications might affect epitope accessibility

  • Denaturation differences between methods may expose different epitopes

• Normalization approaches:

  • For Western blots, choice of loading control (β-actin, GAPDH)

  • For IHC, scoring methods and threshold definitions

  • For qRT-PCR, reference gene selection

• Sample preparation variables:

  • Tissue fixation method and duration for IHC

  • Protein extraction methods for Western blotting

  • RNA quality for qRT-PCR

• Resolution of contradictions:

  • Use multiple antibodies targeting different epitopes

  • Employ orthogonal validation methods

  • Include appropriate positive and negative controls

  • Consider cell/tissue-specific context

  • Validate findings using genetic approaches (siRNA/shRNA with rescue)

• Reporting guidelines:

  • Document detailed methodological information

  • Report antibody validation data

  • Provide all raw data and analysis methods

  • Acknowledge limitations of each approach