SSX4 Antibody

What is SSX4 and what is its biological significance?

SSX4 (Synovial Sarcoma, X Breakpoint 4) belongs to the family of highly homologous synovial sarcoma X (SSX) breakpoint proteins. These proteins function primarily as transcriptional repressors and have significant biological relevance in cancer research. SSX genes are expressed in various human tumors, including melanomas, but are notably absent in adult somatic tissues, making them cancer/testis antigens . Their restricted expression pattern has made them particularly valuable in cancer research as potential biomarkers and immunotherapy targets. The SSX4 protein is predominantly localized in the nucleus and has a calculated molecular weight of approximately 22kDa, though it is often observed at 27kDa in experimental conditions . The unique expression profile of SSX4 in tumor cells versus normal tissues has positioned it as a promising target for developing cancer diagnostics and therapies.

What are the typical applications of SSX4 antibodies in research?

SSX4 antibodies serve multiple research applications, with the most common being:

• Western Blot (WB): For detection and quantification of SSX4 protein in cell and tissue lysates. Typically used at dilutions of 1:500-1:2000 .

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of SSX4 antigens in solution. Recommended dilutions range from 1:2000-1:5000 .

• Immunohistochemistry (IHC): For visualizing SSX4 expression patterns in tissue sections. Optimal dilutions are typically 1:25-1:100 for IHC applications .

• Immunofluorescence (IF): For cellular localization studies of SSX4 protein, particularly in cancer cell lines .

• Immunogenicity studies: For investigating immune responses directed against SSX4 in cancer patients, particularly in melanoma .

These applications are essential for studying SSX4's role in tumorigenesis, evaluating its potential as a diagnostic marker, and investigating its utility in immunotherapeutic approaches.

How do I select the appropriate SSX4 antibody for my research?

When selecting an SSX4 antibody, researchers should consider several critical parameters based on their specific experimental requirements:

For studies focusing on specific functional domains, select antibodies targeting relevant epitopes. For instance, research on the Krüppel-associated box domain would benefit from antibodies targeting the N-terminal region, which has shown high immunogenic potential in melanoma patients . For cross-reactivity studies between SSX family members, carefully evaluate the epitope sequence to ensure specificity for SSX4 versus other SSX proteins.

What is known about SSX4's role in cancer immunology?

SSX4, like other SSX family members, has emerged as a significant cancer/testis antigen with important implications for tumor immunology. Research has demonstrated that CD4+ T cell responses can be directed against SSX4 in melanoma patients bearing antigen-expressing tumors . Multiple distinct antigenic sequences derived from SSX4 have been recognized by these T cells, with the majority located within the Krüppel-associated box domain in the N-terminal region of the protein . This indicates a high immunogenic potential for this region.

The immune recognition of SSX4 involves both humoral and cellular responses:

• CD4+ T cell responses: SSX-4-specific CD4+ T cells recognizing several distinct antigenic sequences have been detected and isolated from melanoma patients .

• HLA restriction: Most identified SSX4 antigenic sequences are restricted by frequently expressed HLA class II alleles, suggesting broad potential for immune recognition across diverse patient populations .

• Spontaneous immune responses: SSX proteins can elicit spontaneous humoral and cellular immune responses in cancer patients without prior immunization .

These findings collectively support SSX4's potential utility in cancer vaccine-based immunotherapy approaches. The presence of circulating SSX4-specific T cells in melanoma patients suggests that these immune responses might naturally occur during cancer development and could potentially be enhanced through immunotherapeutic interventions .

What methodological approaches can be used to study SSX4 expression in tumor samples?

Studying SSX4 expression in tumor samples requires a multi-faceted approach that combines molecular and immunological techniques:

Immunohistochemical Detection

• Use SSX4-specific antibodies at optimized dilutions (typically 1:25-1:100)

• Include appropriate positive controls (e.g., mouse kidney, mouse lung as reported positive samples)

• Employ antigen retrieval techniques to enhance detection sensitivity

• Systematically evaluate staining patterns (nuclear localization expected)

Molecular Analysis

• RT-PCR for mRNA expression analysis

• RNA-Seq for comprehensive expression profiling

• In situ hybridization for spatial expression analysis within tissue architecture

Protein Analysis

• Western blotting using validated antibodies (1:500-1:2000 dilution)

• Expect a molecular weight of approximately 27kDa (observed) compared to calculated 22kDa

• Consider protein extraction methods optimized for nuclear proteins

When analyzing tumor samples, researchers should systematically compare SSX4 expression between tumor and adjacent normal tissues, as SSX4 expression is expected to be cancer-specific. Additionally, correlation with clinicopathological parameters can provide insights into the potential prognostic significance of SSX4 expression in different cancer types.

How can SSX4 antibodies be used to investigate potential therapeutics targeting cancer/testis antigens?

SSX4 antibodies serve as crucial tools in developing and evaluating therapeutics targeting cancer/testis antigens through several strategic approaches:

• Target Validation: Confirm SSX4 expression in tumor samples but not in normal tissues using immunohistochemistry (1:25-1:100 dilution) . This validates the cancer specificity essential for therapeutic targeting.

• Therapeutic Antibody Development:

  • Epitope mapping using various domain-specific antibodies (e.g., targeting AA 91-188, AA 63-91)

  • Screening antibody candidates for target binding affinity

  • Evaluating tumor-specific recognition

• Immunotherapy Monitoring:

  • Assess patient immune responses to SSX4-based vaccines

  • Monitor anti-SSX4 antibody development in patient sera

  • Evaluate T-cell responses against SSX4 epitopes

• Mechanistic Studies:

  • Investigate SSX4's role as a transcriptional repressor using ChIP assays

  • Study effects of SSX4 knockdown/inhibition on cancer cell phenotypes

  • Explore protein-protein interactions with SSX4 using co-immunoprecipitation

• Combination Therapy Assessment:

  • Evaluate changes in SSX4 expression following conventional therapies

  • Study synergistic effects between SSX4-targeting approaches and other treatments

The methodological approach should incorporate both in vitro studies using cancer cell lines and in vivo studies using appropriate animal models where human tumor xenografts are evaluated for SSX4 expression and therapeutic response. These approaches collectively leverage SSX4 antibodies as tools for target validation, therapeutic development, and response monitoring.

What are the optimal protocols for using SSX4 antibodies in Western blot applications?

The following protocol represents an optimized method for Western blot detection of SSX4 protein:

Sample Preparation:

• Extract total protein from cells/tissues using RIPA buffer supplemented with protease inhibitors

• Include positive controls such as mouse kidney or lung tissue lysates

• Determine protein concentration using Bradford or BCA assay

• Prepare 20-30μg of protein per lane in Laemmli buffer with reducing agent

Gel Electrophoresis and Transfer:

• Separate proteins on 10-12% SDS-PAGE gels (optimal for 22-27kDa proteins)

• Transfer to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer

• Verify transfer efficiency with Ponceau S staining

Immunoblotting:

• Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Incubate with primary SSX4 antibody at 1:500-1:2000 dilution in blocking buffer overnight at 4°C

• Wash membrane 3-4 times with TBST, 5 minutes each

• Incubate with appropriate HRP-conjugated secondary antibody (anti-rabbit or anti-mouse depending on primary antibody host) at 1:5000 dilution for 1 hour at room temperature

• Wash membrane 3-4 times with TBST, 5 minutes each

• Develop using enhanced chemiluminescence (ECL) substrate

• Image using a digital imaging system

Expected Results:

• Target band at approximately 27kDa (observed MW), though the calculated MW is 22kDa

• Possible secondary bands may represent post-translational modifications or isoforms

Troubleshooting Notes:

• If background is high, increase washing steps or reduce antibody concentration

• If signal is weak, increase antibody concentration or protein loading

• For improved specificity, consider using 3% BSA instead of milk for blocking and antibody dilution

What controls should be implemented when working with SSX4 antibodies?

Implementing appropriate controls is crucial for ensuring experimental rigor and result validity when working with SSX4 antibodies:

Positive Controls:

• Tissue/Cell Samples:

  • Mouse kidney and lung tissues have been confirmed as positive samples for SSX4 expression

  • Melanoma cell lines or tissues with confirmed SSX4 expression

• Recombinant Protein:

  • Purified recombinant SSX4 protein or fusion proteins containing SSX4 epitopes

  • Serial dilutions can serve as quantitative reference standards

Negative Controls:

• Biological:

  • Adult somatic normal tissues that should not express SSX4

  • Cell lines with confirmed absence of SSX4 expression

• Technical:

  • Primary antibody omission control

  • Isotype control using non-specific IgG from the same host species

Validation Controls:

• Peptide Competition:

  • Pre-incubation of antibody with immunizing peptide should abolish specific signal

  • Particularly useful for validating antibody specificity

• Knockdown/Knockout Validation:

  • Samples with siRNA-mediated SSX4 knockdown

  • CRISPR/Cas9-generated SSX4 knockout cells

• Cross-reactivity Assessment:

  • Testing against other SSX family members (SSX1, SSX2, SSX3, SSX5) to confirm specificity

  • Particularly important due to high sequence homology among SSX family proteins

Application-Specific Controls:

• For IHC/IF:

  • Serial sections with primary antibody omission

  • Autofluorescence controls in fluorescence applications

• For ELISA:

  • Blank wells (no antigen) to assess non-specific binding

  • Standard curves using recombinant SSX4 protein

Implementing these controls systematically ensures that observed signals are specific to SSX4 and not due to technical artifacts or cross-reactivity with related proteins.

How can researchers validate the specificity of SSX4 antibodies against other SSX family members?

Validating SSX4 antibody specificity against other SSX family members is crucial due to their high sequence homology. Here's a comprehensive validation strategy:

1. Sequence Analysis and Epitope Mapping:

• Compare amino acid sequences of all SSX family members (SSX1-9)

• Identify unique regions in SSX4 that differ from other SSX proteins

• Select or design antibodies targeting SSX4-specific epitopes

• Antibodies targeting amino acids 70-188 of human SSX4 (NP_005627.1) may offer good specificity

2. Recombinant Protein Panel Testing:

• Express recombinant proteins for all SSX family members

• Perform Western blot or ELISA with the SSX4 antibody against all family members

• Quantify cross-reactivity percentages

3. Peptide Competition Assays:

• Pre-incubate SSX4 antibody with:

  • SSX4-specific immunizing peptide

  • Corresponding peptides from other SSX family members

• Measure signal reduction in Western blot or immunostaining

• Complete signal abolishment should occur only with the SSX4-specific peptide

4. Immunoprecipitation-Mass Spectrometry:

• Perform immunoprecipitation with the SSX4 antibody

• Analyze precipitated proteins by mass spectrometry

• Confirm presence of SSX4 and absence of other SSX proteins

5. Cell-Based Validation:

• Use cells overexpressing individual SSX family members

• Perform parallel detection with the SSX4 antibody

• Include cells with siRNA knockdown of specific SSX members

Validation Data Interpretation:
A highly specific SSX4 antibody should show:

• Strong reactivity with SSX4 protein/cells

• Minimal to no cross-reactivity with other SSX family members

• Signal abolishment when pre-incubated with SSX4-specific peptide

• No significant signal reduction when pre-incubated with other SSX peptides

Researchers should document all validation results thoroughly and include this information when reporting experimental findings using SSX4 antibodies.

What are the key methodological considerations for studying SSX4-specific immune responses in cancer patients?

Investigating SSX4-specific immune responses in cancer patients requires careful methodological considerations spanning sample collection, processing, and analysis:

Sample Collection and Processing:

• Peripheral Blood Collection:

  • Collect peripheral blood mononuclear cells (PBMCs) using standardized protocols

  • Process fresh samples within 4-6 hours of collection for optimal viability

  • Consider cryopreservation in liquid nitrogen for long-term storage

• Tumor Tissue Processing:

  • Process fresh tumor samples to isolate tumor-infiltrating lymphocytes (TILs)

  • Perform parallel analysis of SSX4 expression in the tumor specimen

T Cell Response Analysis:

• In Vitro Stimulation:

  • Stimulate PBMCs with pools of long peptides spanning the SSX4 protein sequence

  • Focus particularly on peptides from the Krüppel-associated box domain, which has shown high immunogenicity

  • Include appropriate positive controls (e.g., viral peptides) and negative controls

• Detection Methods:

  • ELISPOT assays for quantifying cytokine-producing cells

  • Intracellular cytokine staining followed by flow cytometry

  • HLA-peptide tetramer staining for antigen-specific T cells

  • Proliferation assays (CFSE dilution) to assess T cell expansion

• T Cell Isolation and Characterization:

  • Isolate SSX4-specific T cell clones through limiting dilution or cell sorting

  • Characterize TCR sequences and functional properties

  • Determine HLA restriction of identified T cell responses

Humoral Response Analysis:

• Serum Collection:

  • Process serum samples uniformly and store at -80°C

  • Avoid repeated freeze-thaw cycles

• Detection Methods:

  • ELISA using recombinant SSX4 protein

  • Western blot analysis against recombinant SSX4

  • Multiplex assays for simultaneous analysis of antibodies against multiple cancer/testis antigens

Data Analysis and Interpretation:

• Correlation Analysis:

  • Compare immune responses with clinical parameters (tumor stage, treatment response)

  • Correlate with SSX4 expression levels in patient tumors

  • Track longitudinal changes in immune responses during disease progression or treatment

• Statistical Considerations:

  • Define clear positivity thresholds based on healthy donor responses

  • Account for HLA distribution in study populations

  • Use appropriate statistical methods for comparing patient subgroups

These methodological approaches build upon established protocols in cancer immunology research, including those used to identify CD4+ T cell responses to SSX4 in melanoma patients .

What are common technical challenges when using SSX4 antibodies and how can they be addressed?

Researchers working with SSX4 antibodies frequently encounter several technical challenges. Here are the most common issues and their solutions:

Additional Optimization Strategies:

• For enhanced detection in low-expressing samples:

  • Consider tyramide signal amplification for IHC/IF

  • Use ultra-sensitive Western blot detection systems

  • Perform immunoprecipitation before Western blot

• For improved specificity:

  • Use lower antibody concentrations with longer incubation times

  • Optimize washing steps (increase number and duration)

  • Consider using 3% BSA instead of milk for blocking

• For reproducibility:

  • Maintain detailed records of antibody lot numbers and dilutions

  • Create aliquots of antibodies to avoid freeze-thaw cycles

  • Validate each new antibody lot before experimental use

How can researchers optimize immunohistochemistry protocols for SSX4 detection in tumor samples?

Optimizing immunohistochemistry (IHC) protocols for SSX4 detection requires systematic adjustment of multiple parameters:

Tissue Preparation and Antigen Retrieval:

• Fixation:

  • Use 10% neutral buffered formalin for 24-48 hours

  • Avoid overfixation which can mask epitopes

• Antigen Retrieval Methods (test and compare):

  • Heat-induced epitope retrieval (HIER):

    • Citrate buffer (pH 6.0) at 95-98°C for 20 minutes

    • EDTA buffer (pH 9.0) at 95-98°C for 20 minutes

  • Enzymatic retrieval:

    • Proteinase K (10-20 μg/mL) for 10-15 minutes at room temperature

Blocking and Antibody Incubation:

• Blocking:

  • 5-10% normal serum (from secondary antibody host species)

  • Add 0.1-0.3% Triton X-100 for improved penetration

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

• Primary Antibody:

  • Test dilution series from 1:25 to 1:100 (recommended range)

  • Optimize incubation time and temperature:

    • 1 hour at room temperature

    • Overnight at 4°C (often yields better results for nuclear antigens)

• Secondary Detection:

  • Use polymer-based detection systems for enhanced sensitivity

  • Consider biotin-free systems to eliminate endogenous biotin interference

Signal Development and Counterstaining:

• Chromogen Selection:

  • DAB (3,3'-diaminobenzidine) for brown signal

  • AEC (3-amino-9-ethylcarbazole) for red signal (better for tissues with endogenous brown pigments)

• Counterstaining:

  • Light hematoxylin counterstain to avoid obscuring nuclear SSX4 staining

  • Control counterstaining time (2-3 minutes typically sufficient)

Protocol Optimization Strategy:

• Initial Matrix Experiment:

  • Test two antigen retrieval methods

  • Test three antibody dilutions (e.g., 1:25, 1:50, 1:100)

  • Include positive control tissue (mouse kidney or lung)

• Fine-tuning:

  • Select best conditions from initial matrix

  • Adjust incubation times and blocking conditions

  • Optimize counterstaining intensity

• Validation:

  • Confirm specificity with peptide competition

  • Compare with other detection methods (e.g., RNAscope for mRNA)

  • Validate across multiple tumor samples

The optimized protocol should yield clear nuclear staining of SSX4 in positive tumor cells with minimal background in surrounding tissues. Document all optimization steps for reproducibility and include representative images showing optimal staining patterns in publications.

What are the emerging applications of SSX4 antibodies in cancer research and diagnostics?

SSX4 antibodies are finding increasing utility in several cutting-edge research and diagnostic applications:

1. Liquid Biopsy Development:

• Detection of circulating tumor cells (CTCs) expressing SSX4

• Evaluation of SSX4 as a circulating tumor marker in patient plasma

• Potential for early cancer detection and monitoring disease progression

2. Precision Medicine Applications:

• Stratification of patients based on SSX4 expression profiles

• Prediction of response to immunotherapy based on SSX4-specific immune responses

• Development of companion diagnostics for SSX4-targeted therapies

3. Advanced Imaging Applications:

• Fluorescently-labeled SSX4 antibodies for intraoperative tumor visualization

• Antibody-based PET imaging to detect SSX4-expressing tumors

• Multiplex immunofluorescence to study SSX4 in the tumor microenvironment

4. Therapeutic Development:

• SSX4 antibody-drug conjugates (ADCs) for targeted therapy

• Bispecific antibodies linking T cells to SSX4-expressing tumor cells

• CAR-T cell therapy targeting SSX4 epitopes

5. Fundamental Biology Research:

• Investigation of SSX4's role in transcriptional repression mechanisms

• Study of SSX4 in cancer stem cell biology

• Exploration of interactions between SSX4 and other cancer-related proteins

These emerging applications leverage the cancer-specific expression pattern of SSX4 and the availability of specific antibodies targeting different epitopes of the protein . As research progresses, SSX4 antibodies are likely to become increasingly valuable tools in both basic cancer research and clinical applications, particularly in the rapidly evolving field of cancer immunotherapy.

How is research on SSX4 contributing to our understanding of cancer immunotherapy?

Research on SSX4 is making significant contributions to cancer immunotherapy through several key mechanisms:

1. Identification of Immunogenic Epitopes:

• Studies have revealed multiple immunogenic sequences in SSX4, particularly within the Krüppel-associated box domain

• These epitopes are recognized by CD4+ T cells from melanoma patients

• The identification of naturally processed and presented SSX4 epitopes provides targets for vaccine development

2. Understanding Natural Immune Responses:

• Research has documented spontaneous CD4+ T cell responses against SSX4 in melanoma patients

• These findings indicate that SSX4 is naturally immunogenic in the context of cancer

• The presence of circulating SSX4-specific T cells suggests potential for immune response amplification

3. HLA Restriction Patterns:

• Studies have characterized the HLA class II restriction of SSX4-specific T cell responses

• Many SSX4 epitopes are restricted by frequently expressed HLA alleles, suggesting broad population coverage

• This information is crucial for designing widely applicable immunotherapeutic approaches

4. Cancer/Testis Antigen Biology:

• Research on SSX4 contributes to our understanding of cancer/testis antigens as a class

• The cancer-specific expression pattern makes SSX4 an ideal target for immunotherapy with minimal off-target effects

• SSX4's role as a transcriptional repressor may provide insights into functional consequences of targeting this protein

5. Translation to Clinical Applications:

• SSX4 research supports the development of:

  • Peptide-based cancer vaccines

  • T cell receptor (TCR)-engineered T cells

  • Immune monitoring strategies for cancer patients

The collective research on SSX4 reinforces its potential as an immunotherapeutic target while providing crucial insights into the fundamental biology of cancer/testis antigens and their interactions with the immune system. As immunotherapy continues to evolve as a pillar of cancer treatment, SSX4-focused research contributes valuable knowledge to guide the development of more effective and targeted approaches.

What new methodologies are being developed for studying SSX4 in cancer research?

The field of SSX4 research is witnessing the development of several innovative methodologies that are expanding our understanding of this cancer/testis antigen:

1. Single-Cell Analysis Technologies:

• Single-cell RNA sequencing to identify SSX4-expressing subpopulations within heterogeneous tumors

• Mass cytometry (CyTOF) with SSX4 antibodies for high-dimensional protein profiling

• Spatial transcriptomics to map SSX4 expression within the tumor microenvironment

2. Advanced Immune Monitoring Approaches:

• High-throughput T cell epitope mapping using peptide libraries and multiplexed assays

• TCR sequencing of SSX4-specific T cell clones to track clonal expansion

• Multiparameter flow cytometry to characterize functional properties of SSX4-specific T cells

3. CRISPR-Based Functional Genomics:

• CRISPR/Cas9 knockout of SSX4 to assess its functional role in tumorigenesis

• CRISPR activation/inhibition systems to modulate SSX4 expression

• CRISPR screens to identify genes interacting with SSX4 in cancer cells

4. Advanced Imaging Techniques:

• Super-resolution microscopy to study SSX4 protein localization and interactions

• Intravital microscopy to visualize SSX4-expressing cells in vivo

• Multiplexed ion beam imaging (MIBI) for simultaneous detection of multiple markers alongside SSX4

5. Computational and AI Approaches:

• Machine learning algorithms to predict immunogenic SSX4 epitopes

• Network analysis to understand SSX4's position in cancer-related pathways

• Structural biology and molecular modeling of SSX4 for rational drug design

6. Organoid and 3D Culture Systems:

• Patient-derived organoids to study SSX4 expression in more physiologically relevant models

• Co-culture systems combining SSX4-expressing cancer cells with immune components

• Microfluidic "organ-on-a-chip" platforms for studying SSX4 in complex tissue environments

These emerging methodologies are significantly enhancing our ability to study SSX4 at unprecedented resolution and in increasingly complex biological contexts. As these techniques continue to evolve and become more widely adopted, they promise to accelerate our understanding of SSX4's role in cancer biology and its potential as a therapeutic target.

What are the key considerations for researchers designing experiments involving SSX4 antibodies?

Researchers designing experiments with SSX4 antibodies should consider several critical factors to ensure robust and reproducible results:

• Antibody Selection and Validation:

  • Choose antibodies with validated specificity for SSX4 versus other SSX family members

  • Consider the target epitope in relation to experimental goals (e.g., N-terminal antibodies for studying the immunogenic Krüppel-associated box domain)

  • Validate each new antibody lot through appropriate controls before experimental use

• Experimental Design:

  • Include comprehensive positive and negative controls

  • Design experiments with appropriate biological replicates

  • Consider potential confounding factors such as SSX4 expression heterogeneity in tumors

• Technical Optimization:

  • Optimize protocols specifically for SSX4 detection in relevant applications

  • Consider the expected molecular weight (observed 27kDa vs. calculated 22kDa)

  • Anticipate technical challenges based on SSX4's biological properties (nuclear localization, expression levels)

• Data Interpretation:

  • Account for the cancer/testis antigen nature of SSX4 when interpreting expression patterns

  • Consider the biological context of SSX4 as a potential transcriptional repressor

  • Integrate findings with existing knowledge on SSX family proteins

• Translational Considerations:

  • Assess how findings may contribute to potential diagnostic or therapeutic applications

  • Consider implications for cancer immunotherapy when studying immune responses to SSX4

  • Evaluate clinical relevance in the context of patient samples and outcomes

By systematically addressing these considerations, researchers can design more effective experiments, avoid common pitfalls, and generate more meaningful and translatable data in their investigations of SSX4 biology and its applications in cancer research.

How should researchers interpret and report SSX4 antibody data in their publications?

Proper interpretation and reporting of SSX4 antibody data is essential for research transparency and reproducibility:

1. Comprehensive Methods Documentation:

• Report complete antibody information:

  • Manufacturer and catalog number

  • Clone designation for monoclonal antibodies

  • Host species and clonality

  • Target epitope (e.g., amino acids 70-188)

• Provide detailed protocols including:

  • Antibody dilutions used (e.g., 1:500-1:2000 for WB, 1:25-1:100 for IHC)

  • Incubation conditions (time, temperature)

  • Detection systems employed

2. Controls and Validation:

• Describe all controls implemented:

  • Positive controls (e.g., mouse kidney, lung tissues)

  • Negative controls (antibody omission, isotype controls)

  • Specificity controls (peptide competition, knockdown validation)

• Report cross-reactivity assessment:

  • Testing against other SSX family members

  • Confirmation of specificity for SSX4

3. Data Presentation:

• Include representative images showing:

  • Full Western blot membranes with molecular weight markers

  • Both positive and negative control tissues in IHC

  • Appropriate magnification and scale bars

• Present quantitative data with:

  • Appropriate statistical analysis

  • Clearly defined scoring systems for IHC

  • Multiple biological replicates

4. Result Interpretation:

• Discuss observed versus expected molecular weight (27kDa vs. 22kDa)

• Address any discrepancies or unexpected findings

• Consider alternative explanations for observed phenomena

• Contextualize findings within current knowledge of SSX4 biology

5. Limitations and Caveats:

• Acknowledge technical limitations

• Discuss potential for cross-reactivity with other SSX proteins

• Address sample size and representativeness limitations

• Consider biological variability in SSX4 expression

6. Data Availability:

• Consider sharing raw image data in repositories

• Make detailed protocols publicly available

• Include supplementary information on antibody validation