MAP2K3 Antibody

What is the biological function of MAP2K3 and why is it studied?

MAP2K3 is a member of the dual specificity kinase group that plays a key role in cell differentiation, motility, division, and death. It primarily functions by phosphorylating and activating the p38 MAPK signaling pathway in response to cytokines and environmental stress stimuli. MAP2K3 can be phosphorylated at sites Ser189 and Thr193 by MKKK proteins (MEKK 1-4) . Recent research highlights MAP2K3's potential as a therapeutic target, particularly in colorectal cancer where targeting MAP2K3 may represent an effective strategy to selectively kill cancer cells .

What applications are commonly supported by commercial MAP2K3 antibodies?

Commercial MAP2K3 antibodies support various applications, with Western Blot (WB) being the most validated. For instance, the 13898-1-AP antibody has been extensively validated for Western Blot with recommended dilutions of 1:1000-1:4000. This antibody shows reactivity with human, mouse, and rat samples, making it suitable for comparative studies across these species . Other applications may include ELISA, immunohistochemistry, and immunofluorescence depending on the specific antibody formulation and validation status.

What should researchers consider when selecting a MAP2K3 antibody?

When selecting a MAP2K3 antibody, researchers should consider:

• Experimental application: Ensure the antibody is validated for your specific application (WB, IHC, IF, etc.)

• Species reactivity: Verify compatibility with your experimental model (human, mouse, rat)

• Antibody type: Consider whether polyclonal or monoclonal antibodies better suit your research needs

• Epitope recognition: Determine if you need an antibody that recognizes specific phosphorylated forms or total MAP2K3

• Molecular weight detection: Confirm the antibody detects the expected molecular weight (MAP2K3 is typically observed at 36-40 kDa)

How should MAP2K3 antibodies be stored and handled to maintain their activity?

For optimal preservation of antibody activity, MAP2K3 antibodies should be stored at -20°C in appropriate buffer conditions. For example, the 13898-1-AP antibody is stable for one year after shipment when stored at -20°C in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) . Importantly, for this particular formulation, aliquoting is unnecessary for -20°C storage. Researchers should avoid repeated freeze-thaw cycles and follow manufacturer-specific guidelines, as storage conditions may vary between products.

How can MAP2K3 antibodies be used to investigate the role of MAP2K3 in cancer progression?

MAP2K3 antibodies can be employed to elucidate the role of this kinase in cancer through several methodological approaches:

• Expression analysis in tumor vs. normal tissue: Immunohistochemistry with anti-MAP2K3 antibodies can reveal differential expression patterns. Research has shown aberrantly high MAP2K3 expression in various tumor tissues, including gliomas, correlating with poor clinicopathological characteristics and outcomes .

• Signaling pathway investigation: Western blot analysis using phospho-specific and total MAP2K3 antibodies can assess activation of the p38 MAPK pathway in response to various stimuli or therapeutic interventions.

• Correlation with immune markers: MAP2K3 expression analysis combined with immune checkpoint markers can help understand the relationship between MAP2K3 and immune infiltration in tumors. Research has demonstrated a significant relationship between MAP2K3 expression and immunological checkpoints, immune-related genes, and immune infiltration in glioma .

• Functional studies: Using MAP2K3 antibodies in conjunction with knockdown or overexpression systems enables researchers to study the functional consequences of altered MAP2K3 expression or activity.

What are the methodological considerations when studying MAP2K3 in the context of immune cell infiltration?

When investigating MAP2K3 in relation to immune cell infiltration, researchers should consider:

• Dual immunostaining approaches: Use MAP2K3 antibodies alongside immune cell markers to assess co-localization or expression patterns within the tumor microenvironment.

• Computational analysis integration: Combine antibody-based detection methods with computational approaches such as CIBERSORT and ssGSEA algorithms to correlate MAP2K3 expression with immune cell infiltration patterns .

• ESTIMATE scoring implementation: Apply the "ESTIMATE" R package to determine immune, stromal, and ESTIMATE scores based on gene expression profiling and correlate these with MAP2K3 expression levels .

• Immune checkpoint correlation analysis: Compare expression of various immunological checkpoints between high and low MAP2K3-expressing groups using appropriate statistical tests like the Wilcoxon rank sum test .

• Therapy response prediction: Calculate Tumor Immune Dysfunction and Exclusion (TIDE) scores for high and low MAP2K3 expression groups to predict sensitivity to immune checkpoint inhibitor therapy .

What troubleshooting strategies are recommended when working with MAP2K3 antibodies in Western blot applications?

When encountering challenges with MAP2K3 detection in Western blot applications, consider:

• Antibody dilution optimization: Titrate the antibody (e.g., 1:1000-1:4000 range) to determine optimal concentration for your specific sample type .

• Sample preparation variables: Different cell/tissue types may require adjusted lysis conditions to effectively extract MAP2K3. The protein has been successfully detected in various samples including A431 cells, mouse skeletal muscle tissue, A549 cells, THP-1 cells, and rat skeletal muscle tissue .

• Molecular weight verification: Confirm bands appear at the expected molecular weight range of 36-40 kDa (as observed) compared to the calculated 39 kDa .

• Phosphorylation state considerations: When studying activated MAP2K3, ensure samples are properly preserved to maintain phosphorylation at sites Ser189 and Thr193 .

• Positive controls inclusion: Include validated positive controls such as A431 or A549 cells that are known to express detectable levels of MAP2K3 .

• Loading control selection: Choose appropriate loading controls that don't overlap with MAP2K3's molecular weight range (36-40 kDa) to avoid signal interference.

How can researchers effectively analyze MAP2K3 promoter regulation in experimental settings?

To investigate MAP2K3 promoter regulation, researchers can employ these methodological approaches:

• Promoter cloning and reporter assays: The MAP2K3 gene 5′-regulatory region can be PCR-amplified from genomic DNA (e.g., a 1.0 kb fragment from positions −989 to −2 relative to the translational start site) and cloned into a luciferase reporter vector like pGL3-Luc .

• PCR amplification protocols: For human MAP2K3 promoter amplification, researchers can use primers such as:

  • Forward primer including SacI restriction site: 5′-tatagactat-GAGCTCACCACCGACCC-3′

  • Reverse primer including BglII restriction site: 5′-tataagatct-TGCAAGTGGGTCCTGGAC-3′

• ChIP assays: Chromatin immunoprecipitation can identify transcription factors binding to the MAP2K3 promoter. Quantitative PCR can be performed using primers specific to the hMAP2K3 promoter:

  • Forward: 5′-TTAACCCCCGCCCACTTC-3′

  • Reverse: 5′-TGCGTCGTCTGGAAAAAACC-3′

• Expression verification: After promoter studies, cellular MAP2K3 expression can be verified by Western blot using appropriate antibodies to confirm the functional consequences of promoter regulation .

How can MAP2K3 antibodies be used to characterize mutant forms identified in cancer?

MAP2K3 antibodies can be instrumental in characterizing cancer-associated MAP2K3 mutants through:

What methodological considerations are important when studying post-translational modifications of MAP2K3?

When investigating post-translational modifications of MAP2K3, researchers should consider:

• Phospho-specific antibody selection: For MAP2K3 activation studies, use antibodies that specifically recognize phosphorylated Ser189 and Thr193 sites, which are critical for its activation by upstream MKKK proteins .

• Sample preparation optimization: Phosphorylation states are labile, so samples must be collected and processed with phosphatase inhibitors to preserve modification status.

• Positive control stimulation: Include samples treated with known activators of the p38 MAPK pathway, such as cytokines or environmental stress inducers, as positive controls for MAP2K3 phosphorylation .

• Kinase assay integration: Complement antibody-based detection with in vitro kinase assays to directly assess MAP2K3 enzymatic activity toward p38 MAPK substrates.

• Mass spectrometry validation: For comprehensive phosphorylation site mapping, combine immunological methods with mass spectrometry analysis.

How can researchers design experiments to investigate the role of MAP2K3 in immune regulation?

To study MAP2K3's role in immune regulation, researchers can design experiments that:

• Utilize gene expression manipulation:

  • Employ siRNA, shRNA, or CRISPR-Cas9 to knockdown/knockout MAP2K3

  • Use expression vectors for overexpression studies

  • Validate knockdown/overexpression efficiency via Western blot with MAP2K3 antibodies

• Assess immune infiltration consequences:

  • Apply computational deconvolution methods like CIBERSORT to analyze immune cell populations

  • Calculate immune scores using the ESTIMATE algorithm

  • Compare immune checkpoint molecule expression between experimental conditions

• Determine clinical implications:

  • Correlate experimental findings with patient outcomes data

  • Calculate TIDE scores to predict immunotherapy response

  • Evaluate interferon gamma scores, T cell receptor abundance, and other immune parameters

• Implement pathway analysis:

  • Use available tools to perform functional and pathway enrichment analysis

  • Focus on immunomodulatory pathways associated with MAP2K3 expression

What methodological approaches can be used to validate MAP2K3 antibody specificity?

To ensure antibody specificity in MAP2K3 research, consider:

• Knockout/knockdown controls:

  • Use MAP2K3 knockout or knockdown samples as negative controls

  • Validate antibody specificity by demonstrating loss of signal in these samples

• Multiple antibody validation:

  • Compare results using antibodies targeting different epitopes of MAP2K3

  • Verify consistent results across different antibody clones or sources

• Peptide competition assays:

  • Pre-incubate the antibody with excess MAP2K3 immunizing peptide

  • Demonstrate signal reduction/elimination in the presence of competing peptide

• Cross-reactivity assessment:

  • Test against samples from multiple species to confirm the antibody's specificity across human, mouse, and rat samples as indicated in product information

  • Evaluate potential cross-reactivity with closely related proteins like MAP2K6

• Molecular weight verification:

  • Confirm detection at the expected molecular weight (36-40 kDa for MAP2K3)

  • Use purified recombinant MAP2K3 protein as a positive control

How can MAP2K3 antibodies be applied in translational research for cancer biomarker development?

MAP2K3 antibodies offer valuable tools for translational cancer research through:

• Tissue microarray analysis:

  • Immunohistochemical staining of patient cohort samples

  • Correlation of MAP2K3 expression with clinicopathological features and survival outcomes

  • Standardized scoring systems to quantify expression levels

• Prognostic biomarker validation:

  • Assessment of MAP2K3 expression levels in tumor versus normal tissues

  • Correlation with patient survival data

  • Multivariate analysis to determine independent prognostic value

• Predictive biomarker development:

  • Correlation of MAP2K3 expression with treatment response

  • Integration with immune checkpoint expression data to predict immunotherapy efficacy

  • Calculation of TIDE scores to assess potential benefit from immune checkpoint inhibitors

• Therapeutic target evaluation:

  • Use MAP2K3 antibodies to monitor protein expression changes after drug treatment

  • Assessment of pathway inhibition through both MAP2K3 and downstream p38 phosphorylation status

  • Correlation of target inhibition with phenotypic outcomes

What protocols are recommended for analyzing MAP2K3 expression in patient-derived samples?

For analyzing MAP2K3 in clinical samples, researchers should consider:

• Immunohistochemistry protocol optimization:

  • Standardize antigen retrieval methods (heat-induced epitope retrieval is often effective)

  • Determine optimal antibody dilution through titration experiments

  • Include appropriate positive and negative control tissues in each run

  • Use The Human Protein Atlas database for reference images of normal brain tissue, LGG, and HGG samples

• Sample preservation considerations:

  • Formalin-fixed paraffin-embedded (FFPE) versus fresh-frozen tissue processing

  • Immediate fixation to preserve protein phosphorylation status

  • Standardized fixation times to ensure consistent results

• Quantification methods:

  • Implement digital pathology tools for objective quantification

  • Establish scoring criteria for staining intensity and percentage of positive cells

  • Validate scoring through multiple independent observers

• Multi-omic integration:

  • Combine protein expression data with genomic and transcriptomic analyses

  • Correlate MAP2K3 protein levels with mRNA expression

  • Integrate with mutation data to identify potential structure-function relationships