MTA3 Antibody

What is MTA3 and why is it important in cancer research?

MTA3 is a component of the Metastasis-associated protein family (MTA) that functions as part of the histone deacetylase NuRD complex involved in chromatin remodeling . Unlike other MTA family members that typically promote tumor progression, MTA3 often demonstrates tumor-suppressive properties in various cancers. MTA3 plays a crucial role in maintaining normal epithelial architecture by repressing SNAI1 transcription in a histone deacetylase-dependent manner, thereby regulating E-cadherin levels . This makes MTA3 particularly significant in cancer research, as its expression is frequently downregulated in malignancies including colorectal, breast, and ovarian cancer . The molecular mechanisms through which MTA3 regulates epithelial-to-mesenchymal transition (EMT) position it as an important research target for understanding cancer progression and potential therapeutic interventions.

What applications are MTA3 antibodies suitable for?

MTA3 antibodies can be utilized across multiple experimental applications:

These applications enable researchers to detect, quantify, and localize MTA3 protein in various experimental models and clinical samples . Optimal dilutions should be determined empirically for each specific application and sample type.

How do I select the appropriate MTA3 antibody for my experiment?

When selecting an MTA3 antibody, consider the following factors:

• Target region specificity: Determine whether you need an antibody targeting the C-terminal, N-terminal, or internal region of MTA3. For instance, some antibodies specifically target the C-terminal region (amino acids 400-450) .

• Species reactivity: Verify the antibody's cross-reactivity with your model organism. Available antibodies react with various species including human, mouse, rat, and even cow, chicken, and Xenopus laevis .

• Clonality: Choose between polyclonal antibodies (broader epitope recognition, higher sensitivity) and monoclonal antibodies (greater specificity, consistency between lots) based on your experimental needs .

• Conjugation: Determine whether you need an unconjugated antibody or one conjugated to a detection molecule, based on your detection method .

• Validation data: Review available validation data for your application to ensure the antibody has been verified for your specific usage scenario .

What are the optimal conditions for Western blot detection of MTA3?

For optimal Western blot detection of MTA3:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for cell lysis

  • Include phosphatase inhibitors if studying phosphorylation status

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

• Gel electrophoresis:

  • Use 10% SDS-PAGE gels for optimal separation

  • Load appropriate positive controls (e.g., Jurkat, MCF-7, A375, or Raji cell lysates)

• Transfer and detection:

  • Transfer to PVDF membrane (preferred over nitrocellulose for MTA3)

  • Block with 5% non-fat milk or BSA in TBST for 1 hour

  • Incubate with primary antibody (1:5000-1:50000 dilution) overnight at 4°C

  • Use HRP-conjugated secondary antibody and ECL detection system

• Expected results:

  • MTA3 typically appears at 62kDa and 68kDa

  • Verify specificity using appropriate positive and negative controls

Remember that optimization may be necessary for your specific experimental system.

How can I optimize immunohistochemical staining for MTA3?

For optimal immunohistochemical detection of MTA3:

• Tissue preparation:

  • Use 10% neutral-buffered formalin fixation (12-24 hours)

  • Process and embed in paraffin following standard protocols

  • Section tissues at 3-5μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is recommended

  • Microwave or pressure cooker methods are effective

• Blocking and antibody incubation:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide

  • Block non-specific binding with 5-10% normal serum

  • Incubate with primary antibody overnight at 4°C

  • Use appropriate detection system (e.g., HRP-polymer)

• Visualization and analysis:

  • MTA3 staining is predominantly cytoplasmic in colorectal tissues

  • Include positive controls (normal colorectal epithelium shows positive MTA3 expression)

  • Negative controls should omit primary antibody

  • Use established scoring systems for consistent evaluation

What controls should I use when working with MTA3 antibodies?

Appropriate controls are essential for reliable results with MTA3 antibodies:

• Positive tissue/cell controls:

  • Normal colorectal epithelium (strong MTA3 expression)

  • MCF-7 cells (breast cancer cell line with detectable MTA3)

  • Jurkat cells, A375 cells, and Raji cells

• Negative controls:

  • Omitting primary antibody in parallel samples

  • Isotype controls to assess non-specific binding

  • Tissues known to have low MTA3 expression (certain colorectal cancer samples)

• Technical validation controls:

  • Antibody specificity validation by peptide competition

  • siRNA knockdown of MTA3 to confirm antibody specificity

  • Recombinant MTA3 protein as a positive control in Western blots

• Internal controls:

  • Housekeeping proteins (β-actin, GAPDH) for protein loading normalization in Western blots

  • Non-malignant adjacent tissue in tumor samples

How can MTA3 antibodies be used to study the NuRD complex and chromatin remodeling?

MTA3 functions as a component of the histone deacetylase NuRD complex, making MTA3 antibodies valuable tools for studying chromatin remodeling:

• Co-immunoprecipitation (Co-IP) applications:

  • Use MTA3 antibodies to precipitate the NuRD complex

  • Analyze co-precipitated proteins by mass spectrometry or Western blot

  • Target known NuRD components like HDAC1/2, Mi-2α/β, RbAp46/48

  • Optimize buffer conditions to maintain complex integrity

• Chromatin immunoprecipitation (ChIP) assays:

  • MTA3 antibodies can identify genomic regions where the NuRD complex acts

  • Focus on SNAI1 and other EMT-related gene promoters

  • Combine with sequencing (ChIP-seq) for genome-wide binding profiles

  • Use appropriate crosslinking and sonication protocols to capture chromatin-protein interactions

• Proximity ligation assays (PLA):

  • Visualize interactions between MTA3 and other NuRD complex components

  • Combine with immunofluorescence to assess cellular localization of interactions

  • Particularly useful for studying context-dependent associations in different cell types

• Sequential ChIP (Re-ChIP):

  • Use to determine if MTA3-containing NuRD complexes co-occupy genomic regions with other transcription factors such as BCL6

These approaches provide insights into how MTA3-containing NuRD complexes regulate gene expression programs involved in cancer development and progression.

What are the considerations for studying MTA3 in epithelial-to-mesenchymal transition (EMT)?

MTA3 plays a significant role in EMT regulation, particularly through SNAI1 repression and E-cadherin regulation:

• Experimental models:

  • Select appropriate cell lines with intact EMT machinery

  • Consider breast cancer (MCF-7) or colorectal cancer cell lines

  • 3D organoid culture systems provide physiologically relevant context

• Analytical approaches:

  • Use immunofluorescence to co-localize MTA3 with E-cadherin and other EMT markers

  • Implement time-course experiments during EMT induction to track MTA3 dynamics

  • Combine MTA3 detection with analysis of SNAI1 and other EMT regulators

• Functional studies:

  • MTA3 overexpression systems to assess EMT reversal

  • MTA3 knockdown to evaluate enhancement of EMT phenotypes

  • Combine with migration/invasion assays to assess functional outcomes

  • Correlate MTA3 levels with E-cadherin expression patterns

• Mechanistic investigations:

  • Analyze histone modifications at EMT gene promoters using ChIP

  • Assess MTA3 binding to SNAI1 promoter during EMT progression

  • Investigate cooperation with other transcription factors

Understanding MTA3's role in EMT could provide insights into cancer metastasis mechanisms and potential therapeutic strategies.

How can MTA3 antibodies be used in prognostic studies of colorectal and other cancers?

MTA3 expression has been associated with cancer prognosis, particularly in colorectal cancer:

These applications provide valuable insights into the clinical relevance of MTA3 in cancer progression and patient outcomes .

How can I address weak or absent MTA3 signal in Western blots?

When encountering weak or absent MTA3 signal in Western blots:

• Sample preparation optimization:

  • Ensure complete lysis using appropriate buffers (RIPA with protease inhibitors)

  • Avoid repeated freeze-thaw cycles of protein samples

  • Prepare fresh samples or add additional protease inhibitors

  • Consider subcellular fractionation as MTA3 can be primarily nuclear

• Technical adjustments:

  • Increase protein loading (50-80 μg recommended for lower abundance proteins)

  • Reduce antibody dilution (start with 1:5000 and adjust as needed)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use more sensitive detection methods (enhanced chemiluminescence substrate)

• Transfer optimization:

  • For 62-68 kDa proteins like MTA3, verify efficient transfer

  • Consider longer transfer times or semi-dry transfer systems

  • Use PVDF membranes for better protein retention

• Antibody selection:

  • Try alternative MTA3 antibodies targeting different epitopes

  • Consider antibodies with validated Western blot applications

  • Verify antibody reactivity with your species of interest

• Positive controls:

  • Include known MTA3-expressing cell lines (Jurkat, MCF-7, A375, NIH/3T3, C2C12, Raji)

  • Consider recombinant MTA3 protein as a positive control

What factors contribute to non-specific staining in MTA3 immunohistochemistry?

Non-specific staining in MTA3 immunohistochemistry can be addressed by considering:

• Antibody specificity issues:

  • Use antibodies with validated IHC applications

  • Titrate antibody concentration (perform dilution series)

  • Include peptide competition controls to confirm specificity

  • Consider monoclonal antibodies for higher specificity

• Tissue preparation factors:

  • Optimize fixation times (over-fixation can mask epitopes)

  • Ensure complete deparaffinization and rehydration

  • Test different antigen retrieval methods (citrate vs. EDTA buffers)

  • Evaluate fresh vs. archived tissues (epitope degradation over time)

• Blocking improvements:

  • Extend blocking time (60 minutes minimum)

  • Use higher concentrations of blocking serum (5-10%)

  • Add bovine serum albumin (BSA) to reduce background

  • Include avidin/biotin blocking for biotin-based detection systems

• Detection system optimization:

  • Consider polymer-based detection systems to reduce background

  • Use species-specific secondary antibodies

  • Include additional washing steps with 0.1% Tween-20

  • Dilute chromogen appropriately to minimize non-specific reaction

• Controls and verification:

  • Run controls omitting primary antibody

  • Use isotype controls to assess non-specific binding

  • Compare staining patterns with published MTA3 localization data

How can I optimize detection of both MTA3 isoforms?

MTA3 can be detected at both 62kDa and 68kDa molecular weights, representing different isoforms or post-translational modifications . To optimize detection of both forms:

• Gel separation strategies:

  • Use 8-10% acrylamide gels for better separation in the 60-70kDa range

  • Consider gradient gels (4-15%) for optimal resolution

  • Extend electrophoresis time to improve band separation

• Antibody selection:

  • Choose antibodies recognizing epitopes common to both isoforms

  • Consider antibodies raised against the C-terminal region

  • Verify expected molecular weights in antibody documentation

• Sample preparation considerations:

  • Test different lysis buffers to ensure complete extraction of both forms

  • Consider native vs. denaturing conditions

  • Evaluate the effects of phosphatase inhibitors on mobility shifts

• Cell/tissue type selection:

  • Different cell types may express isoforms at varying levels

  • Compare expression patterns across multiple cell lines

  • Consider tissue-specific expression patterns

• Analysis approaches:

  • Quantify both bands independently when assessing expression levels

  • Consider ratios between isoforms as potentially biologically relevant

  • Correlate isoform expression with functional assays

How can MTA3 antibodies be used in combination with other markers for cancer subtyping?

MTA3 can be integrated into multimarker panels for refined cancer classification:

• Multiplex immunohistochemistry/immunofluorescence:

  • Combine MTA3 detection with EMT markers (E-cadherin, vimentin, SNAI1)

  • Include cell type-specific markers for contextual analysis

  • Implement spectral unmixing for simultaneous detection of multiple markers

  • Correlate MTA3 with established molecular subtype markers

• Flow cytometry applications:

  • Develop multi-parameter flow panels including MTA3 for cell subtyping

  • Combine with markers of differentiation, stemness, or EMT

  • Use intracellular staining protocols optimized for nuclear/cytoplasmic proteins

  • Implement in cell sorting applications for functional studies

• Digital pathology approaches:

  • Use image analysis software for quantitative co-localization studies

  • Implement machine learning algorithms for pattern recognition

  • Develop automated scoring systems for multiple markers

  • Create spatial maps of marker expression in tumor microenvironments

• Clinical application considerations:

  • Test prognostic value of MTA3 in combination with established markers

  • Evaluate predictive value for therapy response

  • Develop standardized reporting frameworks for complex marker panels

These approaches can provide more nuanced classification of tumors and potentially reveal functional subtypes relevant to prognosis and treatment decisions.

What are the considerations for studying MTA3 in patient-derived xenograft (PDX) models?

Patient-derived xenograft models offer valuable systems for studying MTA3 in cancer biology:

• Antibody cross-reactivity considerations:

  • Verify that selected antibodies can distinguish human (patient-derived) MTA3 from mouse (host) proteins

  • Test antibody specificity in mixed human-mouse samples

  • Consider human-specific MTA3 antibodies for cleaner detection

• Experimental design factors:

  • Compare MTA3 expression between primary patient samples and derived PDX

  • Track MTA3 expression across PDX passages to assess stability

  • Correlate MTA3 expression with PDX growth, invasion, and metastasis

  • Implement IHC protocols optimized for xenograft tissues

• Functional investigations:

  • Test MTA3-targeting interventions on PDX growth and metastasis

  • Evaluate MTA3 as a biomarker for therapy response in PDX models

  • Combine with patient clinical data for translational relevance

  • Consider ex vivo manipulation of PDX-derived cells

• Analysis approaches:

  • Implement spatial analysis of MTA3 expression within PDX tumors

  • Correlate with stromal infiltration patterns

  • Integrate with genomic and transcriptomic profiling

  • Compare expression patterns between primary tumor and metastatic PDX models

PDX models provide systems to study MTA3 biology in contexts that maintain tumor heterogeneity and microenvironment interactions.

How can MTA3 expression analysis contribute to understanding therapy resistance mechanisms?

MTA3's role in regulating EMT and differentiation suggests potential involvement in therapy resistance:

• Expression analysis in resistant models:

  • Compare MTA3 levels between therapy-sensitive and resistant cell lines

  • Analyze MTA3 expression before and after treatment exposure

  • Correlate MTA3 with established resistance markers

  • Implement longitudinal sampling from patients during treatment course

• Mechanistic investigations:

  • Assess MTA3 regulation of drug efflux pumps or detoxification enzymes

  • Investigate connections between MTA3-regulated EMT and therapy resistance

  • Explore MTA3's role in cancer stem cell maintenance

  • Evaluate interactions with therapy-induced signaling pathways

• Manipulation approaches:

  • Test effects of MTA3 overexpression or knockdown on therapy sensitivity

  • Combine with pharmacological modulation of NuRD complex activity

  • Assess potential for MTA3-targeted interventions to overcome resistance

  • Evaluate synergistic approaches targeting MTA3-regulated pathways

• Clinical correlation strategies:

  • Analyze MTA3 expression in pre- and post-treatment patient samples

  • Correlate expression patterns with therapy response and outcomes

  • Investigate value as a predictive biomarker

  • Consider differential expression across tumor regions (core vs. invasive front)

These investigations could identify novel mechanisms of therapy resistance and potentially reveal new therapeutic strategies to overcome resistance in cancer patients.