ITM2A Antibody

What is ITM2A and why is it of interest to researchers?

ITM2A (Integral Membrane Protein 2A) is a type II transmembrane protein that belongs to the ITM2 family. It is also known as BRICD2A, E25A, and BRICHOS domain containing 2A. The protein has a molecular weight of approximately 29.7 kilodaltons in its native form, though it may appear larger (~39-45 kDa) in some experimental conditions due to post-translational modifications .

ITM2A is of significant research interest because it is expressed in several important cell types, including chondrocytes involved in endochondrial ossification, skeletal muscle, adipose tissue-derived stem cells, and CD3-activated CD4+ and CD8+ T cells . Recent studies have also identified ITM2A as a potential tumor suppressor in breast cancer, with high expression correlating with better patient outcomes .

What species reactivity can be expected with commercially available ITM2A antibodies?

Most commercially available ITM2A antibodies are designed to detect human ITM2A, but many also cross-react with orthologs in other species. Based on gene homology, reactivity has been reported with mouse, rat, porcine, and monkey ITM2A .

When selecting an antibody for your research, it's important to verify the specific species reactivity. For example, some antibodies like the rabbit polyclonal antibody ab203620 have been specifically validated for rat samples in applications such as IHC-P and ICC/IF . Other antibodies may show broader reactivity across human, mouse, and rat samples .

Always review the technical specifications and validation data for each antibody to ensure compatibility with your experimental model.

What are the optimal fixation and antigen retrieval methods for ITM2A immunohistochemistry?

Based on validated protocols, the following methods have shown good results for ITM2A immunohistochemistry:

For formalin-fixed, paraffin-embedded (FFPE) tissues:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin following standard protocols

• Section tissues at 4-6 μm thickness

• Perform heat-induced epitope retrieval (HIER) using either:

  • Citrate buffer (pH 6.0) for 20 minutes at 95-100°C

  • EDTA buffer (pH 9.0) for 20 minutes at 95-100°C

• Cool sections to room temperature gradually

• Block endogenous peroxidase activity with 3% H₂O₂

• Apply ITM2A antibody at optimized dilution (e.g., 1:200 for ab203620)

• Incubate overnight at 4°C for best results

For frozen sections or cultured cells:

• Fix with 4% paraformaldehyde (PFA) for 10-15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

• Apply ITM2A antibody at appropriate dilution (e.g., 1:100 for immunofluorescence)

These protocols have been successfully used to detect ITM2A in rat brain tissue and rat brain vascular endothelial cells (RBE4) .

How should ITM2A antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of ITM2A antibodies is crucial for maintaining their activity and specificity. Based on manufacturer recommendations:

For lyophilized antibodies:

• Store unopened vials at -20 to -70°C for up to 12 months from the date of receipt

• Reconstitute following manufacturer's instructions, typically using sterile buffer

• After reconstitution, aliquot to avoid repeated freeze-thaw cycles

• Store reconstituted antibodies at:

  • 2-8°C for up to 1 month for short-term use

  • -20 to -70°C for up to 6 months for long-term storage

For liquid antibodies:

• Store at 2-8°C for short-term (1-2 weeks)

• For long-term storage, aliquot and freeze at -20°C

• Avoid more than 2-3 freeze-thaw cycles as this can degrade antibody performance

• When thawing, allow the antibody to equilibrate to room temperature before opening the vial

• Centrifuge the vial briefly before use to collect all material

General handling recommendations:

• Use sterile technique when handling antibodies

• Return antibodies to appropriate storage conditions immediately after use

• Work with antibodies on ice when possible

• Avoid contamination with microorganisms

What controls should be included when performing experiments with ITM2A antibodies?

Including appropriate controls is essential for validating results obtained with ITM2A antibodies:

Positive Controls:

• Cell lines known to express ITM2A, such as:

  • A-673 human Ewing's sarcoma cell line

  • Chondrocytes involved in endochondrial ossification

  • CD3-activated CD4+ and CD8+ T cells

  • Skeletal muscle tissues

• Tissues with confirmed ITM2A expression, such as rat brain tissue

Negative Controls:

• Primary antibody omission control (substitute antibody diluent for primary antibody)

• Isotype control (use non-specific antibody of the same isotype and concentration)

• Cell lines with confirmed low/no ITM2A expression

• ITM2A-knockout or knockdown samples (if available)

Additional Technical Controls:

• Secondary antibody only control (to assess non-specific binding)

• Blocking peptide competition (pre-incubate antibody with immunizing peptide)

• Parallel detection with alternative antibody clones targeting different epitopes of ITM2A

• Validation by orthogonal methods (e.g., confirm protein detection with RNA expression data)

These controls help ensure specificity of detection and rule out technical artifacts or non-specific binding.

How does ITM2A expression correlate with cancer progression and patient outcomes?

Recent research has identified ITM2A as a potential tumor suppressor in breast cancer. Analysis of gene expression profiles from multiple datasets (GSE29413, GSE61304, and TCGA) has demonstrated that ITM2A is frequently downregulated in breast cancer compared to normal tissues .

The relationship between ITM2A expression and patient outcomes includes:

These findings suggest that ITM2A may function as a tumor suppressor in breast cancer, potentially through modulation of immune responses in the tumor microenvironment. Further research is needed to determine if similar relationships exist in other cancer types.

What experimental approaches can be used to study ITM2A's role in cellular differentiation?

To investigate ITM2A's role in cellular differentiation, researchers can employ several complementary experimental approaches:

1. Expression Analysis During Differentiation:

• qRT-PCR to quantify ITM2A mRNA expression at different time points during differentiation

• Western blot with ITM2A antibodies to track protein expression changes

• Immunofluorescence to visualize subcellular localization changes during differentiation

• Flow cytometry to quantify ITM2A expression at the single-cell level

2. Gain-of-Function Studies:

• Stable or transient overexpression of ITM2A using plasmid vectors

• Inducible expression systems (e.g., Tet-On) to control timing of ITM2A expression

• Analysis of differentiation markers before and after ITM2A induction

3. Loss-of-Function Studies:

• siRNA or shRNA-mediated knockdown of ITM2A

• CRISPR-Cas9 gene editing to create ITM2A knockout cell lines

• Analysis of differentiation potential in ITM2A-depleted cells

4. Protein Interaction Studies:

• Co-immunoprecipitation with ITM2A antibodies to identify interaction partners

• Proximity labeling approaches (BioID, APEX) to identify proteins in close proximity to ITM2A

• Mass spectrometry analysis of ITM2A-containing protein complexes

5. Functional Assays:

• Cell proliferation assays in ITM2A-manipulated cells

• Migration and invasion assays to assess cellular behavior

• Lineage-specific differentiation assays (osteogenic, chondrogenic, myogenic, etc.)

• Analysis of signaling pathway activation using phospho-specific antibodies

6. In vivo Models:

• Conditional knockout mouse models to study tissue-specific roles of ITM2A

• Xenograft studies with ITM2A-overexpressing or -depleted cells

• Analysis of developmental phenotypes in model organisms

These approaches can be combined to build a comprehensive understanding of ITM2A's functional role in cellular differentiation across different cellular contexts.

What is the relationship between ITM2A expression and immune responses in cancer?

The relationship between ITM2A expression and immune responses in cancer, particularly breast cancer, involves several key aspects:

1. PD-L1 Expression Regulation:
ITM2A has been found to facilitate the expression of PD-L1 (Programmed Death-Ligand 1) in breast cancer cells. This relationship was verified through both qRT-PCR assays and public database analysis. PD-L1 is a critical immune checkpoint molecule that can inhibit T cell function, but its expression in certain contexts can also be associated with active immune responses .

2. Association with Tumor-Infiltrating Lymphocytes (TILs):
Breast cancers with higher ITM2A expression were found to have more tumor-infiltrating lymphocytes. This correlation suggests that ITM2A may play a role in modulating the immune microenvironment of tumors .

3. Differential Gene Expression:
RNA-sequencing analysis of breast cancer cells overexpressing ITM2A revealed that differentially expressed genes were associated with immunity responses, further supporting ITM2A's role in immune modulation .

4. Prognostic Implications:
Interestingly, PD-L1 expression associated with tumor-infiltrating lymphocytes has been found to be a positive prognostic feature in breast cancer. Given ITM2A's relationship with both PD-L1 expression and TILs, this could explain in part why high ITM2A expression correlates with better patient outcomes .

This emerging understanding of ITM2A's immunomodulatory roles presents new research directions, including:

• Investigating whether ITM2A could serve as a biomarker for immunotherapy response

• Exploring whether modulating ITM2A expression could enhance anti-tumor immune responses

• Determining the molecular mechanisms by which ITM2A regulates PD-L1 expression

• Examining how ITM2A affects different immune cell populations within the tumor microenvironment

What are common issues when detecting ITM2A by Western blot and how can they be resolved?

When detecting ITM2A by Western blot, researchers may encounter several challenges. Here are common issues and their solutions:

Issue 1: Multiple bands or unexpected molecular weight

• Expected observation: ITM2A should appear as a band at approximately 39 kDa , though the theoretical molecular weight is 29.7 kDa .

• Potential causes and solutions:

  • Post-translational modifications: ITM2A may undergo glycosylation or other modifications

    • Solution: Treat samples with deglycosylation enzymes (PNGase F) to confirm glycosylation

  • Incomplete protein denaturation

    • Solution: Increase SDS concentration or boiling time in sample buffer

  • Protein degradation

    • Solution: Use fresh samples and add protease inhibitors during extraction

  • Antibody cross-reactivity

    • Solution: Test multiple antibodies targeting different epitopes of ITM2A

Issue 2: Weak or no signal

• Potential causes and solutions:

  • Low protein expression

    • Solution: Load more protein (50-100 μg) or use enrichment techniques

  • Inefficient transfer

    • Solution: Optimize transfer conditions (time, voltage, buffer composition)

  • Suboptimal antibody concentration

    • Solution: Perform antibody titration; try 2 μg/ml as validated for some ITM2A antibodies

  • Incompatible blocking agent

    • Solution: Test different blocking agents (BSA vs. non-fat milk)

Issue 3: High background

• Potential causes and solutions:

  • Excessive antibody concentration

    • Solution: Dilute primary and secondary antibodies

  • Insufficient blocking

    • Solution: Increase blocking time or concentration

  • Inadequate washing

    • Solution: Add additional washing steps with 0.1% Tween-20 in TBS

  • Secondary antibody cross-reactivity

    • Solution: Use highly cross-adsorbed secondary antibodies

Technical Tips for Optimal ITM2A Detection:

• Use PVDF membranes, which have shown good results for ITM2A detection

• Perform electrophoresis under reducing conditions for optimal ITM2A resolution

• Consider using specialized Western blot buffer systems, such as Western Blot Buffer Group 1, which has been validated for ITM2A detection

• Include positive control lysates, such as A-673 human Ewing's sarcoma cell line

How can researchers validate the specificity of ITM2A antibodies in their experimental systems?

Validating antibody specificity is crucial for obtaining reliable results. For ITM2A antibodies, consider these comprehensive validation approaches:

1. Genetic Validation:

• siRNA knockdown: Demonstrate reduction in signal intensity following ITM2A knockdown

• CRISPR-Cas9 knockout: Show complete absence of signal in ITM2A knockout cells

• Overexpression: Show increased signal in cells transfected with ITM2A expression vectors

• These genetic validations should be performed in relevant cell types that express detectable levels of endogenous ITM2A

2. Orthogonal Methods Validation:

• Correlation with mRNA levels: Compare protein detection with qRT-PCR results across multiple samples

• Mass spectrometry validation: Confirm the identity of the detected protein band

• In situ hybridization: Compare antibody staining patterns with mRNA localization

• Verify results with multiple antibodies: Use antibodies targeting different epitopes of ITM2A

3. Technical Validation:

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to demonstrate signal reduction

• Isotype controls: Use matched isotype control antibodies to evaluate non-specific binding

• Signal titration: Show concentration-dependent signal with increasing amounts of target protein

• Cell type specificity: Confirm expected expression patterns across cell types known to express or lack ITM2A

4. Application-Specific Validation:

• For IHC/ICC: Compare staining patterns across multiple fixation and antigen retrieval methods

• For flow cytometry: Compare surface vs. intracellular staining protocols

• For IP experiments: Confirm pulldown of correctly sized protein by Western blot

• For functional assays: Demonstrate that antibody binding affects expected biological functions

5. Cross-reactivity Testing:

• Test the antibody against related family members (e.g., ITM2B, ITM2C)

• Evaluate species cross-reactivity using samples from different organisms

• Examine antibody performance in tissues with complex protein mixtures

Implementing multiple validation strategies provides strong evidence for antibody specificity and increases confidence in experimental results.

How should contradictory findings regarding ITM2A function be reconciled in research?

When facing contradictory findings regarding ITM2A function, researchers should employ a systematic approach to reconcile these discrepancies:

Consider Context-Dependent Effects

• Cell type specificity: ITM2A may function differently in various cell types (chondrocytes vs. T cells vs. cancer cells)

• Developmental stage: Function may vary across developmental or differentiation stages

• Pathological state: Normal vs. disease state may alter protein function

• Experimental methodology: Different techniques may reveal distinct aspects of function

Evaluate Experimental Approaches

• Create a comparison table of contradictory studies, analyzing:

  • Cell/tissue systems used

  • Expression levels (physiological vs. overexpression)

  • Knockdown/knockout strategies

  • Readout methods

  • Time points examined

Examine Protein Interactions and Modifications

• ITM2A function may depend on:

  • Specific protein-protein interactions that vary by context

  • Post-translational modifications affecting function

  • Subcellular localization differences

  • Cleavage or processing events

Design Reconciliation Experiments

• Direct comparison experiments using:

  • Multiple cell types in parallel

  • Range of expression levels

  • Both gain- and loss-of-function approaches

  • Multiple functional readouts

  • Time-course studies

5. Example Reconciliation Framework for ITM2A:
Recent studies suggest ITM2A functions as a tumor suppressor in breast cancer , but earlier work implicated it in other processes like chondrogenesis. To reconcile these findings:

• Investigate whether ITM2A's tumor suppressive effects are mediated through:

  • Differentiation pathways shared with developmental contexts

  • Novel cancer-specific mechanisms

  • Immune modulatory functions

• Examine whether ITM2A's relationship with PD-L1 expression exists across different cellular contexts

  • Is this relationship cancer-specific?

  • Does it depend on the immune microenvironment?

  • Is it influenced by other signaling pathways?

• Determine whether ITM2A's effects on cell proliferation, invasion, and migration are universal or context-dependent

By systematically analyzing contradictory findings through these approaches, researchers can develop more nuanced models of ITM2A function that accommodate seemingly disparate results within a coherent framework.

What emerging applications of ITM2A antibodies show promise for advancing our understanding of disease mechanisms?

Several emerging applications of ITM2A antibodies show significant promise for deepening our understanding of disease mechanisms:

1. Cancer Prognostic Biomarker Development:

• Using ITM2A antibodies for tissue microarray analysis to correlate expression with patient outcomes across cancer types

• Developing standardized immunohistochemical scoring systems for ITM2A expression in tumors

• Exploring whether ITM2A expression patterns can predict response to immunotherapies, given its relationship with PD-L1 and TILs

2. Single-Cell Analysis Applications:

• Combining ITM2A antibodies with other markers for multi-parameter flow cytometry to identify specific cell populations

• Applying CyTOF (mass cytometry) with metal-conjugated ITM2A antibodies for high-dimensional phenotyping of tumor and immune cells

• Using ITM2A antibodies in single-cell Western blot techniques to examine protein heterogeneity

3. Spatial Biology and Tissue Architecture:

• Employing ITM2A antibodies in multiplex immunofluorescence to map spatial relationships with immune cells in the tumor microenvironment

• Using imaging mass cytometry with ITM2A antibodies to preserve spatial context while examining multiple markers

• Correlating ITM2A expression patterns with tissue architecture in normal development and disease

4. Functional Screening Applications:

• Using ITM2A antibodies in high-content screening to identify modulators of ITM2A expression or localization

• Developing ITM2A-targeted proximity labeling approaches to identify context-specific interaction partners

• Employing antibody-based techniques to modulate ITM2A function in cellular models

5. Translational Medicine Applications:

• Investigating whether ITM2A antibodies could be used therapeutically to modulate immune responses in cancer

• Developing companion diagnostic approaches using ITM2A antibodies to stratify patients for targeted therapies

• Creating circulating tumor cell detection methods incorporating ITM2A antibodies

These emerging applications leverage the specificity of ITM2A antibodies to advance both basic understanding of ITM2A biology and its potential clinical applications, particularly in cancer research and immunology.

How might the study of ITM2A contribute to the development of novel therapeutic approaches?

The study of ITM2A offers several promising avenues for novel therapeutic development:

1. Cancer Immunotherapy Enhancement:
Given ITM2A's association with PD-L1 expression and tumor-infiltrating lymphocytes , therapeutic approaches could include:

• Modulating ITM2A expression to enhance response to existing immune checkpoint inhibitors

• Developing combination therapies targeting both ITM2A signaling and other immune pathways

• Using ITM2A expression as a biomarker to identify patients likely to respond to immunotherapies

2. Targeted Cancer Therapies:

• Restoring ITM2A expression in cancers where it is downregulated could potentially suppress tumor growth and invasiveness

• Developing small molecules or biologics that mimic ITM2A's tumor-suppressive functions

• Creating synthetic biology approaches that link ITM2A expression to therapeutic effectors

3. Regenerative Medicine Applications:
Based on ITM2A's role in cellular differentiation:

• Modulating ITM2A to enhance chondrogenic differentiation for cartilage regeneration

• Controlling ITM2A expression to direct stem cell fate in tissue engineering

• Developing tissue-specific delivery systems for ITM2A-targeting therapeutics

4. Diagnostic and Prognostic Tools:

• Creating standardized ITM2A immunohistochemistry protocols for cancer prognosis

• Developing circulating biomarker assays that include ITM2A assessment

• Integrating ITM2A status into multi-parameter prognostic models

5. Drug Development Strategies:

• Screening for compounds that modulate ITM2A expression or activity

• Identifying the molecular mechanisms of ITM2A-mediated tumor suppression to reveal novel drug targets

• Developing antibody-drug conjugates targeting cells with specific ITM2A expression patterns

6. Experimental Therapeutic Approaches:

• Engineered T cells with enhanced ITM2A signaling for adoptive cell therapy

• mRNA or gene therapy approaches to restore ITM2A expression

• Nanotechnology-based delivery of ITM2A modulators to specific tissues

As research continues to clarify ITM2A's functions in normal physiology and disease, these therapeutic directions will likely expand and become more precisely defined. The connection between ITM2A and immune regulation is particularly promising for near-term therapeutic development.

What are the best practices for designing experiments using ITM2A antibodies?

Based on collected evidence and technical considerations, here are best practices for designing experiments with ITM2A antibodies:

1. Antibody Selection:

• Choose antibodies validated for your specific application (WB, IHC, ICC/IF, etc.)

• Consider using monoclonal antibodies for higher specificity in complex applications

• Verify species reactivity matches your experimental model

• Review literature to identify antibody clones with proven performance in similar experiments

• When possible, use antibodies targeting different epitopes to confirm findings

2. Experimental Controls:

• Always include positive controls (e.g., A-673 human Ewing's sarcoma cell line)

• Include appropriate negative controls (antibody omission, isotype controls)

• Consider using genetic approaches (knockdown/knockout) to validate specificity

• Include loading controls for Western blot experiments

• Use blocking peptides when available to confirm specificity

3. Protocol Optimization:

• Determine optimal antibody concentration through titration experiments

• For Western blot, use PVDF membrane and reducing conditions as validated for ITM2A detection

• For IHC, optimize fixation and antigen retrieval methods (4% PFA fixation for IF, formalin fixation with heat-induced epitope retrieval for IHC-P)

• Adjust incubation times and temperatures based on signal strength and background

4. Data Analysis and Interpretation:

• Quantify results using appropriate software and statistical methods

• Consider ITM2A's molecular weight variation (theoretical 29.7 kDa vs. observed ~39-45 kDa) when interpreting Western blot results

• Correlate protein expression with mRNA data when possible

• Report detailed methods including antibody catalog numbers, dilutions, and incubation conditions

5. Documentation and Reporting:

• Document all experimental conditions thoroughly

• Include representative images with scale bars

• Report both positive and negative results

• Provide detailed materials and methods to enable reproducibility

• Clearly state limitations of the antibodies used

By following these best practices, researchers can ensure reliable and reproducible results when using ITM2A antibodies, contributing to our collective understanding of this protein's functions in normal physiology and disease.

What key considerations should researchers keep in mind when interpreting ITM2A expression data across different experimental systems?

When interpreting ITM2A expression data across different experimental systems, researchers should consider several important factors:

1. Expression Level Variations:

• Baseline expression levels vary significantly across tissue and cell types

• Expression may be highly context-dependent (differentiation state, activation status)

• Quantitative comparison across different systems requires standardization

• Consider both absolute and relative expression changes when interpreting results

2. Technical Considerations:

• Different detection methods (antibody-based vs. RNA-based) may not correlate perfectly

• Various antibody clones may recognize different isoforms or post-translationally modified forms

• Detection sensitivity varies across platforms (Western blot vs. IHC vs. flow cytometry)

• Image acquisition settings significantly impact quantitative comparisons in microscopy

3. Biological Context Integration:

• Interpret ITM2A expression in relation to known functions in specific tissue/cell types

• Consider the developmental or disease stage when comparing expression patterns

• Evaluate expression in the context of relevant signaling pathways

• Assess whether expression changes are cause or consequence of observed phenotypes

4. Cross-Species Considerations:

• ITM2A function may be conserved but regulation can differ across species

• Antibody cross-reactivity may vary between human, mouse, rat, and other species

• Expression patterns during development or disease progression may not be identical across species

• Validate findings in multiple species when possible

5. Disease-Specific Interpretation:

• In cancer research, consider tumor heterogeneity when interpreting ITM2A expression

• Correlate ITM2A expression with clinical parameters and outcomes when available

• Integrate findings with immune cell infiltration and activation data

• Consider microenvironmental factors that may influence expression

6. Integration Framework:
When confronted with seemingly contradictory ITM2A expression data, consider this stepwise approach:

• Verify technical aspects (antibody specificity, assay conditions)

• Compare experimental conditions (cell types, treatments, time points)

• Examine broader biological context (signaling pathways, cellular state)

• Develop testable hypotheses to explain discrepancies

• Design reconciliation experiments that directly compare systems