SOX11 Antibody, FITC conjugated

What is SOX11 and why is it an important research target?

SOX11 is a transcription factor that acts as a transcriptional activator in multiple cellular contexts. It binds cooperatively with POU3F2/BRN2 or POU3F1/OCT6 to gene promoters, enhancing transcriptional activation. It plays critical roles in embryonic neurogenesis and has been implicated in tissue modeling during development .

SOX11's significance in research stems from its differential expression pattern - it shows low expression in normal adult tissues but is overexpressed in several malignancies, particularly glioblastoma (GBM), making it a promising tumor-associated antigen for targeted therapies . Additionally, SOX11 works redundantly with SOX4 and SOX12 in promoting cell survival in developing tissues including the neural tube, branchial arches, and somites, contributing significantly to organogenesis .

What are the available formats of SOX11 antibodies and their applications?

SOX11 antibodies are available in multiple formats including:

• Unconjugated primary antibodies:

  • Polyclonal antibodies targeting various epitopes (e.g., aa 50-200)

  • Monoclonal antibodies with higher specificity

• Fluorophore-conjugated variants:

  • FITC-conjugated antibodies targeting specific regions (e.g., aa 241-341)

  • Alexa Fluor 488-conjugated antibodies for enhanced brightness and photostability

Each format has specific applications:

• Unconjugated antibodies are versatile for Western blotting (WB) and immunohistochemistry on paraffin-embedded tissues (IHC-P)

• Fluorophore-conjugated antibodies are optimal for flow cytometry and direct immunofluorescence microscopy without requiring secondary antibodies

The choice between formats depends on the experimental question, detection method, and whether multiplexing is required.

What are the optimal sample preparation methods for SOX11 detection?

Successful SOX11 detection requires proper sample preparation, which varies by application:

For immunohistochemistry:

• Heat-mediated antigen retrieval with citrate buffer pH 6 is recommended prior to staining protocols

• Paraffin-embedded tissue sections have been successfully used, particularly for lymphoma tissues and tumors with SOX11 overexpression

For flow cytometry:

• Fixation with 80% methanol (5 minutes) followed by permeabilization with 0.1% PBS-Tween for 20 minutes has proven effective

• Blocking with 1x PBS/10% normal goat serum/0.3M glycine helps reduce non-specific binding

Optimization may be required for specific sample types, and researchers should validate these conditions for their particular experimental system.

How can the specificity and sensitivity of SOX11 antibodies be validated?

Validation of SOX11 antibodies requires multiple complementary approaches:

Positive and negative controls:

• Use cell lines with known SOX11 expression (positive control, such as SH-SY5Y cells used in flow cytometry)

• Include samples known to lack SOX11 expression as negative controls

• Compare staining patterns with literature-documented SOX11 localization (nuclear for SOX11)

Cross-reactivity testing:

• Western blot analysis to confirm band size (approximately 60 kDa for SOX11)

• Testing against recombinant SOX11 protein and related SOX family members (especially SOX4 and SOX12 which share functional redundancy)

• Peptide competition assays using the immunogen peptide

Validation across multiple applications:

• Confirm consistent results across different applications (IHC, WB, flow cytometry)

• Compare multiple antibodies targeting different epitopes of SOX11 (e.g., antibodies to aa 50-200 vs. aa 241-341)

What are the technical considerations for using FITC-conjugated SOX11 antibodies in flow cytometry?

When implementing FITC-conjugated SOX11 antibodies in flow cytometry, several technical factors should be considered:

Protocol optimization:

• Fixation and permeabilization are critical since SOX11 is a nuclear transcription factor

• Methanol fixation (80%) for 5 minutes followed by permeabilization with 0.1% PBS-Tween for 20 minutes has proven effective

• Adequate blocking (using 1x PBS/10% normal goat serum/0.3M glycine) is essential to minimize background

Fluorophore considerations:

• FITC has excitation/emission peaks of approximately 495/519 nm

• Consider spectral overlap when designing multicolor panels

• When greater sensitivity is required, Alexa Fluor 488-conjugated alternatives may provide superior brightness and photostability compared to FITC

Controls and gating strategy:

• Include an isotype control conjugated to the same fluorophore

• Use single-stained controls for compensation when conducting multicolor experiments

• Implement proper gating strategies based on forward/side scatter to exclude dead cells and debris

How do polyclonal and monoclonal SOX11 antibodies compare in research applications?

The choice between polyclonal and monoclonal SOX11 antibodies significantly impacts experimental outcomes:

Polyclonal SOX11 antibodies:

• Generally recognize multiple epitopes within the target region (e.g., aa 50-200 or aa 241-341)

• Potentially higher sensitivity due to binding to multiple epitopes

• Greater batch-to-batch variation requiring more rigorous validation between lots

• May show higher background in some applications

Monoclonal SOX11 antibodies:

• Recognize a single epitope with high specificity

• Recombinant monoclonal antibodies (e.g., EPR8191(2)) offer consistent performance with minimal batch variation

• May have lower sensitivity in detecting conformationally altered SOX11

• Typically provide cleaner results in applications like flow cytometry

Selection should be based on the specific research application, with monoclonal antibodies generally preferred for diagnostic applications requiring high specificity, while polyclonal antibodies may offer advantages in detection of low-abundance targets.

How are SOX11 antibodies used in cancer research, particularly in lymphoma and glioblastoma studies?

SOX11 antibodies serve as vital tools in cancer research, with particular relevance in two key malignancies:

Mantle Cell Lymphoma (MCL):

• SOX11 is a diagnostic marker for MCL, with nuclear expression in tumor cells

• Immunohistochemical analysis using SOX11 antibodies helps distinguish MCL from other lymphoma subtypes

• The nuclear staining pattern is critical for accurate diagnosis

Glioblastoma (GBM):

• SOX11 is overexpressed in GBM while showing low expression in normal tissues

• Antibodies help characterize SOX11 expression patterns across tumor samples and subtypes

• Flow cytometry with fluorophore-conjugated SOX11 antibodies enables analysis of SOX11 expression in patient-derived samples and cell lines

Research applications include:

• Tumor classification and subtyping

• Correlation of SOX11 expression with clinical outcomes

• Identification of SOX11-positive cells for functional studies

• Development and monitoring of SOX11-targeted therapies

What is the relationship between SOX11 detection and potential immunotherapy applications?

SOX11's properties make it a promising target for immunotherapy, particularly in glioblastoma:

SOX11 as a tumor-associated antigen:

• SOX11 shows tumor-specific overexpression with limited expression in normal tissues, reducing the risk of off-target effects

• The peptide FMACSPVAL derived from SOX11 demonstrated strong binding affinity to HLA-A*0201 molecules and effectively generated SOX11-specific CD8+ T cells

• IFN-γ ELISPOT assays showed robust T-cell responses to SOX11-derived peptides

Detection methods supporting immunotherapy development:

• FITC-conjugated SOX11 antibodies enable researchers to:

  • Monitor SOX11 expression in patient samples to identify candidates for SOX11-targeted therapy

  • Evaluate SOX11 expression in cell lines used for preclinical studies

  • Track changes in SOX11 expression during disease progression or treatment

Translational applications:

• SOX11-specific T cell generation for adoptive cell therapy represents a potential approach for treating GBM patients

• Nine out of thirty-two healthy donors showed positive responses to SOX11 in ELISPOT assays, indicating the feasibility of generating SOX11-specific immune responses

How can SOX11 antibodies be used to investigate developmental processes?

SOX11 plays critical roles in embryonic development, and antibodies enable detailed investigation of these processes:

Developmental functions of SOX11:

• SOX11 is a transcriptional factor involved in embryonic neurogenesis

• It contributes to tissue modeling during development

• SOX11 works with SOX4 and SOX12 to support cell survival in developing tissues including the neural tube, branchial arches, and somites

Research applications:

• Developmental timing studies using embryonic tissue sections

• Co-localization with other developmental markers

• Lineage tracing of SOX11-expressing cells

• Investigation of SOX11's role in neuronal differentiation

• Assessment of its contribution to organogenesis through conditional knockout models

What are common problems encountered when using SOX11 antibodies and how can they be resolved?

Researchers often encounter several challenges when working with SOX11 antibodies:

High background signal:

• Problem: Non-specific binding resulting in high background

• Solutions:

  • Optimize blocking conditions (1x PBS/10% normal goat serum/0.3M glycine is recommended)

  • Increase washing steps and duration

  • Titrate antibody concentration to find optimal signal-to-noise ratio

  • Use more specific monoclonal antibodies when available

Weak or absent signal:

• Problem: Insufficient detection of SOX11

• Solutions:

  • Ensure proper antigen retrieval (heat-mediated with citrate buffer pH 6)

  • Optimize fixation and permeabilization for nuclear proteins

  • Consider alternative epitopes (antibodies targeting different regions)

  • Increase antibody concentration or incubation time

  • Verify SOX11 expression in your sample type

Inconsistent results:

• Problem: Variable staining patterns between experiments

• Solutions:

  • Standardize protocols with precise timing and temperatures

  • Use recombinant monoclonal antibodies for greater consistency

  • Maintain consistent lot numbers when possible

  • Include positive controls in each experiment

How should multiplexing with SOX11 antibodies be designed and optimized?

Multiplexing allows simultaneous detection of SOX11 with other markers, but requires careful consideration:

Panel design considerations:

• Spectral compatibility: FITC/Alexa Fluor 488-conjugated SOX11 antibodies emit green fluorescence, so pair with spectrally distinct fluorophores

• Antigen abundance matching: Pair bright fluorophores with low-expression targets and vice versa

• Host species compatibility: When using unconjugated primary antibodies, choose antibodies raised in different species to avoid cross-reactivity

Optimization approaches:

• Single-stain controls: Test each antibody individually before combining

• Fluorescence minus one (FMO) controls: Include all fluorophores except one to assess spectral spillover

• Titration: Determine optimal concentration for each antibody independently

Technical recommendations:

• For fluorescence microscopy: Use sequential scanning to minimize bleed-through

• For flow cytometry: Perform proper compensation using single-stained controls

• Consider tyramide signal amplification for detecting low-abundance targets alongside SOX11

What are the best practices for quantitative analysis of SOX11 expression?

Accurate quantification of SOX11 expression requires standardized approaches:

In flow cytometry:

• Convert fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF) using calibration beads

• Use median fluorescence intensity (MFI) rather than mean for more robust measurements

• Report fold-change relative to appropriate negative controls

• Document instrument settings and maintain consistent PMT voltages between experiments

In image-based analyses:

• Standardize image acquisition parameters (exposure time, gain, etc.)

• Measure nuclear intensity since SOX11 is a nuclear protein

• Use automated algorithms to reduce subjective bias

• Include internal reference standards across different imaging sessions

Controls for quantitative analysis:

• Use cell lines with defined SOX11 expression levels as reference standards

• Include samples with known SOX11 status (positive and negative)

• Perform replicate measurements to establish technical variability

• Consider orthogonal methods (e.g., qPCR, Western blot) to validate antibody-based quantification

How might SOX11 antibodies contribute to personalized medicine approaches?

SOX11 antibodies have significant potential in advancing personalized medicine:

Patient stratification:

• SOX11 expression analysis could identify patient subgroups most likely to benefit from SOX11-targeted therapies

• Flow cytometry with FITC-conjugated SOX11 antibodies enables rapid assessment of SOX11 status

• Integration with other biomarkers may improve prognostic classification

Therapeutic monitoring:

• Serial sampling to track changes in SOX11 expression during treatment

• Assessment of immune responses to SOX11-derived epitopes

• Monitoring for emergence of SOX11-negative subclones during therapy

Companion diagnostics:

• Development of standardized SOX11 detection assays to accompany SOX11-targeted therapies

• Implementation in clinical trials evaluating SOX11-based immunotherapies

• Potential integration into diagnostic algorithms for lymphomas and gliomas

What novel technical approaches are emerging for SOX11 detection?

The field of SOX11 detection continues to evolve with several emerging technologies:

Single-cell technologies:

• Single-cell RNA sequencing paired with protein detection (CITE-seq)

• Mass cytometry (CyTOF) using metal-conjugated SOX11 antibodies for high-parameter analysis

• Imaging mass cytometry for spatial context of SOX11 expression

In vivo imaging approaches:

• Near-infrared fluorophore-conjugated SOX11 antibodies for preclinical imaging

• Development of smaller antibody fragments (nanobodies) with improved tissue penetration

• Radiolabeled SOX11 antibodies for PET imaging in experimental models

Digital pathology integration:

• Automated image analysis algorithms for SOX11 quantification

• Machine learning approaches to correlate SOX11 expression patterns with outcomes

• Whole slide imaging with multiplex immunofluorescence including SOX11

How can SOX11 peptide epitopes advance T cell-based immunotherapies?

Recent advances in SOX11 epitope identification open new possibilities for immunotherapy:

SOX11-derived peptides for immunotherapy:

• The peptide FMACSPVAL has demonstrated superior characteristics based on:

  • Highest score in in silico prediction (6.02 nM by NetMHC-4.0)

  • Strong binding affinity to HLA-A*0201 molecules

  • High efficiency in generating SOX11-specific CD8+ T cells in IFN-γ ELISPOT assays

Clinical application potential:

• Development of peptide vaccines using SOX11-derived epitopes

• Engineering of T cells with T cell receptors (TCRs) specific for SOX11 peptides

• Creation of chimeric antigen receptor (CAR) T cells targeting SOX11

• Use of SOX11 peptides for ex vivo expansion of tumor-reactive T cells

Monitoring therapeutic responses:

• FITC-conjugated SOX11 antibodies can monitor changes in SOX11 expression following immunotherapy

• Flow cytometry to assess tumor-infiltrating lymphocytes specific for SOX11 epitopes

• ELISPOT assays to measure T cell responses to SOX11 peptides during treatment

The identification of the novel peptide FMACSPVAL represents a significant advance, potentially enabling adoptive transfer of in vitro elicited SOX11-specific CD8+ T cells as a therapeutic approach for GBM patients .