FOXQ1 Antibody, FITC conjugated

What is FOXQ1 and what cellular functions does it regulate?

FOXQ1 (also known as HFH1) belongs to the FOX gene family characterized by a conserved 110-amino acid DNA-binding motif called the forkhead or winged helix domain. It functions primarily as a nuclear transcription factor that regulates multiple cellular processes. FOXQ1 is known to repress the promoter activity of smooth muscle-specific genes, such as telokin and SM22α . Recent research has identified FOXQ1 as a novel regulator of autophagy in breast cancer by upregulating expression of several autophagy-related proteins including ATG4B and CXCR4 . Additionally, FOXQ1 plays important roles in embryonic development, cell cycle regulation, tissue-specific gene expression, cell signaling, and tumorigenesis . In normal physiology, FOXQ1 participates in hair follicle differentiation .

What are the optimal applications for FOXQ1 antibody, FITC conjugated?

FITC-conjugated FOXQ1 antibodies are particularly well-suited for flow cytometry, immunofluorescence microscopy, and high-content imaging applications where direct fluorescent detection is advantageous. While unconjugated FOXQ1 antibodies are validated for Western blot (WB) and immunohistochemistry (IHC) , the FITC-conjugated versions excel in applications requiring fluorescence detection without secondary antibody steps. For multicolor immunofluorescence experiments, researchers should consider the spectral properties of FITC (excitation ~495nm, emission ~520nm) when designing panels with other fluorophores to avoid spectral overlap. When using FITC-conjugated antibodies for intracellular targets like FOXQ1, proper cell permeabilization protocols are essential to allow antibody access to the nuclear-localized FOXQ1 protein .

What sample types and tissues have been validated for FOXQ1 antibody research?

Based on validation data, FOXQ1 antibodies have been successfully tested in several sample types. For Western blot applications, mouse and rat kidney tissues have shown positive detection . For immunohistochemistry, human stomach cancer tissue , human colorectal cancer, and human ovarian cancer tissues have been verified as suitable samples . When working with FITC-conjugated FOXQ1 antibodies in cancer research, these validated tissue types provide a starting point for experimental design. Additionally, FOXQ1 antibodies have been successfully used with HeLa S3 nuclear extracts in Western blot applications . When investigating FOXQ1 in novel tissue types or cell lines, researchers should include these validated samples as positive controls to confirm antibody functionality.

What are the recommended protocols for fixation and permeabilization when using FITC-conjugated FOXQ1 antibodies?

Since FOXQ1 is primarily localized in the nucleus , proper cell permeabilization is critical for antibody access to the target. For immunofluorescence applications with FITC-conjugated FOXQ1 antibodies, a recommended protocol includes:

• Fix cells with 4% paraformaldehyde in PBS for 15 minutes at room temperature

• Wash three times with PBS (5 minutes each)

• Permeabilize with 0.5% Triton X-100 in PBS for 10 minutes at room temperature

• Block with 5% normal serum (from the same species as the secondary antibody would be, if used) in PBS with 0.1% Triton X-100 for 1 hour

• Incubate with FITC-conjugated FOXQ1 antibody at an optimized dilution (starting with 1:25-1:50 for unconjugated versions in IHC ) in blocking buffer overnight at 4°C

• Wash three times with PBS containing 0.1% Triton X-100

• Counterstain nuclei with DAPI if desired

• Mount with anti-fade mounting medium designed for fluorescence preservation

When working with tissue sections, antigen retrieval may be necessary, with recommended methods including TE buffer pH 9.0 or citrate buffer pH 6.0 as validated for unconjugated FOXQ1 antibodies .

How does FOXQ1 regulate autophagy in cancer models?

Recent research has established FOXQ1 as a novel regulator of autophagy in breast cancer models. FOXQ1 upregulates the expression of several autophagy-related proteins, including ATG4B and CXCR4 . Mechanistically, this regulation occurs at the transcriptional level, where FOXQ1 has been shown to bind to the promoter region of ATG4B, as demonstrated through ChIP assays. Researchers identified specific binding sites in the ATG4B promoter that can be amplified using region-specific primers:

• Site 1 and Site 2, forward: 5'-ACCAGCGCAGGAAGATACTG-3' and reverse: 5'-CTCCCAAAGTGCTGGGATTA-3'

• Site 3, forward: 5'-CCTAGGGAGAGGAGGACTGG-3' and reverse: 5'-GCAGCTGTCACTACCATCCA-3'

When investigating FOXQ1's role in autophagy regulation, these primers can be valuable for ChIP-qPCR experiments to confirm direct binding. Additionally, RNA-seq analysis of FoxQ1 overexpression has shown that it affects pathways associated with cell cycle checkpoints, M phase, and cellular response to stress/external stimuli . For researchers studying FOXQ1 in cancer models, it's important to consider its complex regulatory network, including its influence on interleukin (IL)-1α, IL-8, vascular endothelial growth factor, and electron transport chain complex I subunits in breast cancer models .

What considerations should be made when designing ChIP-seq experiments with FOXQ1 antibodies?

When designing ChIP-seq experiments to investigate FOXQ1 binding sites genome-wide, several specific considerations should be addressed:

• Antibody selection: Use ChIP-validated antibodies. While the search results don't specifically mention FITC-conjugated antibodies for ChIP, unconjugated FOXQ1 antibodies have been used successfully in ChIP assays . FITC conjugation may interfere with chromatin binding, so unconjugated antibodies are typically preferred for ChIP applications.

• Controls: Include both negative controls (normal IgG) and positive controls (known FOXQ1 binding sites) in your experimental design. The ATG4B promoter regions mentioned above can serve as positive control regions .

• Chromatin preparation: Since FOXQ1 is a transcription factor with potential binding to condensed chromatin, optimization of chromatin shearing conditions is critical to generate fragments of appropriate size (typically 200-500 bp).

• Enrichment validation: Before proceeding to sequencing, validate the enrichment of known targets using qPCR. The fold enrichment should be normalized to the input as described in the ChIP protocol from search result #2 .

• Data analysis pipeline: For transcription factors like FOXQ1, peak-calling algorithms that account for the typically narrow binding patterns of transcription factors should be employed in the bioinformatic analysis.

When comparing ChIP-seq data with transcriptomic profiling, researchers should be aware that FOXQ1 can act as both an activator and repressor of transcription, as evidenced by its role in repressing smooth muscle-specific genes while potentially activating autophagy-related genes .

How can researchers address the challenge of distinguishing specific FOXQ1 isoforms or post-translationally modified variants?

Distinguishing between FOXQ1 isoforms or post-translationally modified variants requires careful experimental design:

• Antibody epitope selection: When selecting FOXQ1 antibodies, consider the epitope location relative to known modification sites or isoform-specific regions. Some available antibodies are generated against specific regions, such as amino acids 100-250 or synthetic peptides , which may detect different forms of the protein.

• Validation with multiple antibodies: Use multiple antibodies targeting different epitopes of FOXQ1 to confirm results and potentially distinguish between isoforms.

• Two-dimensional gel electrophoresis: For detailed analysis of post-translational modifications, combine isoelectric focusing with SDS-PAGE to separate FOXQ1 variants based on both charge and molecular weight.

• Mass spectrometry analysis: Following immunoprecipitation with FOXQ1 antibodies, mass spectrometry can identify specific modifications and distinguish between isoforms.

• Recombinant protein controls: Include recombinant FOXQ1 variants as controls in Western blots to establish migration patterns of specific isoforms.

• Phosphatase treatment: To determine if observed multiple bands are due to phosphorylation, treat samples with phosphatase before Western blot analysis.

Researchers should be aware that the observed molecular weight in Western blots may not always match the calculated molecular weight due to post-translational modifications and other factors affecting protein mobility in gel electrophoresis .

What strategies can minimize photobleaching of FITC-conjugated FOXQ1 antibodies in long-term imaging experiments?

Photobleaching is a significant challenge when working with FITC-conjugated antibodies in extended imaging sessions. To minimize this issue:

• Anti-fade mounting media: Use specialized anti-fade mounting media formulated for fluorescein preservation. These media often contain anti-oxidants and radical scavengers that reduce photobleaching rates.

• Oxygen scavenging systems: For live-cell imaging, consider incorporating oxygen scavenging systems (e.g., glucose oxidase/catalase) into imaging buffers to reduce reactive oxygen species that contribute to fluorophore degradation.

• Image acquisition parameters:

  • Reduce excitation light intensity to the minimum required for adequate signal

  • Minimize exposure times

  • Increase camera gain (within reasonable noise limits) to compensate for reduced excitation

  • Use confocal pinhole settings that balance resolution with signal strength

• Computational approaches:

  • Implement image acquisition strategies like time-gating or selective plane illumination

  • Use deconvolution algorithms to extract maximum information from lower-intensity images

  • Apply photobleaching correction algorithms during post-processing

• Sample preparation: Ensure complete blocking of non-specific binding sites and thorough washing to remove unbound antibody, as excess antibody can contribute to background photobleaching and reduced signal-to-noise ratio.

• Storage conditions: For prepared slides, store at 4°C in the dark and image within 1-2 weeks for optimal fluorescence preservation. The stability of FOXQ1 antibodies (unconjugated) is reported as one year when stored at -20°C .

How can researchers troubleshoot inconsistent FOXQ1 staining patterns across different cell lines or tissue samples?

Inconsistent staining patterns with FOXQ1 antibodies may result from several factors:

• Fixation and antigen retrieval optimization:

  • Different tissue types may require specific fixation protocols

  • For FOXQ1 IHC, both TE buffer pH 9.0 and citrate buffer pH 6.0 have been validated for antigen retrieval

  • Systematic comparison of multiple fixation and retrieval methods may be necessary for new sample types

• Expression level variations:

  • FOXQ1 expression varies significantly across tissues and cell types

  • Consider using positive controls like mouse kidney tissue or human cancer tissues (stomach, colorectal, ovarian) where FOXQ1 expression has been verified

  • Quantitative PCR can verify transcript levels before attempting protein detection

• Antibody dilution optimization:

  • The recommended dilutions for unconjugated antibodies (1:25-1:50 for IHC or 1:400-1:1600 ) serve as starting points

  • Perform systematic titration experiments with FITC-conjugated versions to determine optimal concentration

  • Create dilution series across multiple samples to identify optimal signal-to-noise ratios

• Cellular localization considerations:

  • FOXQ1 is primarily localized to the nucleus

  • Ensure permeabilization protocols adequately expose nuclear antigens

  • Consider counterstaining with nuclear markers to confirm localization patterns

• Validation controls:

  • Include antibody validation by FOXQ1 knockdown/knockout samples where available

  • Western blot validation alongside immunofluorescence can confirm specificity

When troubleshooting, it's important to note that the actual band observed in Western blot "may not be consistent with the expectation" due to factors affecting protein mobility , which could also influence antibody recognition in other applications.

What are the most effective blocking protocols to reduce background when using FOXQ1, FITC-conjugated antibodies in immunofluorescence?

Effective blocking is critical for maximizing signal-to-noise ratio when using FITC-conjugated FOXQ1 antibodies. The following optimized blocking protocol can significantly reduce background fluorescence:

• Multi-component blocking solution:

  • 5% normal serum (from a species different from the antibody host)

  • 1% BSA (high-quality, IgG-free)

  • 0.3% Triton X-100 for permeabilization

  • 0.05% Tween-20 to reduce non-specific interactions

  • Optional: 0.1% cold fish skin gelatin for additional blocking power

• Blocking duration and conditions:

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

  • Use gentle agitation to ensure complete coverage of sample

• Pre-adsorption treatment:

  • For tissues known to cause high background, pre-adsorb the FITC-conjugated antibody with acetone powder prepared from relevant negative control tissues

  • Mix antibody with acetone powder for 1 hour at 4°C, then centrifuge to remove powder before use

• Autofluorescence reduction:

  • Treat sections with 0.1% Sudan Black B in 70% ethanol for 20 minutes to reduce tissue autofluorescence

  • For formalin-fixed tissues, treatment with sodium borohydride (1mg/ml in PBS) for 10 minutes can reduce fixative-induced autofluorescence

• Sequential blocking:

  • Apply protein block first, followed by a separate Fc receptor block if working with tissues rich in Fc receptors

  • Commercial Fc receptor blocking reagents can be particularly effective for lymphoid tissues

• Washing optimization:

  • Increase the number and duration of washes after antibody incubation

  • Include 0.05% Tween-20 in wash buffers to reduce non-specific binding

When troubleshooting high background, researchers should remember that FOXQ1 shows nuclear localization , so cytoplasmic staining may represent background or non-specific binding requiring further optimization of blocking protocols.