MAP4K4 Antibody, HRP conjugated

What is MAP4K4 and why is it significant in research?

MAP4K4 is a serine/threonine kinase belonging to the Ste20 family that functions as an upstream regulator of the MAPK pathway. It participates in multiple cellular processes including apoptosis, differentiation, and stress response. The significance of MAP4K4 extends to cardiovascular research where it mediates cardioprotection against ischemia/reperfusion injury, and to hematological studies where it regulates neutrophil differentiation in bone marrow . Methodologically, researchers target MAP4K4 to understand how signaling cascades respond to various stimuli, particularly in pathological conditions where MAP4K4 expression is altered.

What applications are MAP4K4 antibodies commonly used for?

MAP4K4 antibodies are utilized in multiple experimental applications including Western blotting (WB), immunohistochemistry (IHC), and flow cytometry . For Western blotting, these antibodies allow researchers to determine relative expression levels and phosphorylation status of MAP4K4 across different experimental conditions. In immunohistochemistry, they enable visualization of MAP4K4 distribution within tissue sections, as demonstrated in human cervical and ovarian cancer tissues . Flow cytometry applications permit analysis of MAP4K4 expression at the single-cell level, facilitating studies of heterogeneous cell populations such as differentiating hematopoietic cells.

What species reactivity should be considered when selecting a MAP4K4 antibody?

When selecting a MAP4K4 antibody, researchers should verify reactivity with their target species. High-quality MAP4K4 antibodies like the Picoband® antibody demonstrate cross-reactivity with human, mouse, and rat MAP4K4 . This cross-reactivity stems from conservation of key epitopes across species. Methodologically, researchers should validate antibody specificity in their particular species of interest using positive and negative controls before conducting full-scale experiments. For novel model organisms, sequence alignment of the antigenic region followed by empirical validation is recommended to ensure accurate detection.

How can I assess the specificity of an HRP-conjugated MAP4K4 antibody in complex signaling pathway studies?

Assessing specificity of HRP-conjugated MAP4K4 antibodies in complex signaling studies requires a multi-step validation approach. First, perform parallel experiments using MAP4K4 knockdown models (siRNA or CRISPR/Cas9) alongside wildtype controls to verify signal reduction corresponds with knockdown efficiency. Second, implement immunoprecipitation coupled with mass spectrometry to confirm antibody captures primarily MAP4K4 and known interacting partners. Third, validate antibody performance across multiple experimental conditions, particularly those that modulate MAP4K4 expression or activation, such as ischemia/reperfusion injury models that show increased MAP4K4 expression . Finally, compare multiple MAP4K4 antibodies targeting different epitopes to ensure consistent detection patterns. This rigorous validation approach minimizes misinterpretation in complex signaling pathway analyses.

What are the optimal conditions for detecting MAP4K4 in cardiac tissue using HRP-conjugated antibodies?

Detecting MAP4K4 in cardiac tissue using HRP-conjugated antibodies requires specific optimization due to cardiac tissue's unique characteristics. The optimal protocol involves: (1) Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) which effectively unmasks MAP4K4 epitopes in formalin-fixed cardiac samples ; (2) Extended blocking with 10% serum (matching the secondary antibody host species) for 1-2 hours to minimize background from endogenous peroxidases abundant in cardiac tissue; (3) Primary antibody incubation at 2 μg/ml concentration overnight at 4°C to ensure sufficient binding while minimizing non-specific signals ; (4) Development using DAB as the chromogen with careful timing optimization to achieve optimal signal-to-noise ratio . For Western blotting applications, cardiac tissue homogenization should include phosphatase inhibitors to preserve MAP4K4 phosphorylation status which may significantly impact antibody recognition.

How can I differentiate between MAP4K4 and other closely related kinases when using antibodies in research?

Differentiating MAP4K4 from closely related kinases (like MAP4K1-3, MAP4K5) requires careful experimental design. Implement these methodological approaches: (1) Select antibodies targeting unique regions of MAP4K4 rather than conserved kinase domains; (2) Perform parallel experiments with recombinant MAP4K4 and related kinases to assess cross-reactivity; (3) Include MAP4K4-specific knockdown controls alongside wildtype samples; (4) Consider competitive binding assays with peptides corresponding to the antibody's epitope region; (5) For advanced validation, employ phosphoproteomic analysis of MAP4K4-specific substrates following immunoprecipitation. When analyzing experimental results, focus on unique protein-protein interactions specific to MAP4K4, such as its binding to RAP2C which has been confirmed through co-immunoprecipitation and immunofluorescence assays . This binding pattern provides an additional verification metric for MAP4K4-specific detection.

What role does MAP4K4 play in the RAP2C-mediated cardioprotection pathways?

MAP4K4 functions as a downstream effector of RAP2C in cardioprotection pathways, particularly in the context of ischemia/reperfusion injury (IRI). Experimental evidence demonstrates that ischemic postconditioning (PostC) reduces MAP4K4 expression that was increased by ischemia/reperfusion . The relationship between RAP2C and MAP4K4 involves direct binding, as confirmed by co-immunoprecipitation assays showing that RAP2C co-elutes with MAP4K4 . This interaction is dynamically regulated, with hypoxia/reoxygenation (H/R) increasing the binding strength while PostC diminishes it . Methodologically, researchers investigating this pathway should monitor both expression levels and binding interactions, as RAP2C knockdown reduces MAP4K4 expression while RAP2C overexpression enhances it . The downstream effects of this pathway involve modulation of MAPK activation (ERK, JNK, P38) and subsequent regulation of apoptotic processes in cardiomyocytes, suggesting potential therapeutic targets for cardioprotection strategies.

What is the optimal protocol for using HRP-conjugated MAP4K4 antibodies in Western blotting?

The optimal Western blotting protocol for HRP-conjugated MAP4K4 antibodies requires careful optimization to ensure specific detection. Sample preparation should include phosphatase inhibitors to preserve phosphorylation states, as MAP4K4 undergoes autophosphorylation and is regulated by phosphorylation events . For protein separation, 8-10% polyacrylamide gels are recommended due to MAP4K4's molecular weight (~140 kDa). After transfer to PVDF membranes (preferred over nitrocellulose for stronger protein binding), blocking should be performed with 5% non-fat milk in TBST for 1 hour at room temperature. Primary antibody incubation with the HRP-conjugated MAP4K4 antibody should be conducted at 1:1000-1:2000 dilution overnight at 4°C. Following thorough washing (5× with TBST), direct development with enhanced chemiluminescence substrate can be performed without secondary antibody incubation. For multiplexing with other proteins of interest, strip and reprobe the membrane after MAP4K4 detection, or consider fluorescent-based Western blotting systems compatible with HRP detection.

How can I optimize immunohistochemistry protocols for MAP4K4 detection in different tissue types?

• For cardiac tissue: Extend antigen retrieval time to 20-25 minutes and block with 10% goat serum for 2 hours to reduce background staining from endogenous peroxidases .

• For cancer tissues (e.g., cervical, ovarian): Use 2 μg/ml antibody concentration with overnight incubation at 4°C followed by 30-minute incubation with HRP-conjugated secondary antibody .

• For bone marrow/hematopoietic tissues: Reduce antibody concentration to 1 μg/ml due to lower background interference and counterstain with hematoxylin for shorter periods (30 seconds) to preserve visualization of nuclear details .

Following antibody incubation, development with DAB chromogen should be carefully timed for each tissue type, with development times typically ranging from 30 seconds (hematopoietic tissues) to 5 minutes (dense tissues like cardiac samples). Consistent optimization and validation across tissue types ensures comparable results when studying MAP4K4 expression in different physiological contexts.

What controls should be included when using MAP4K4 antibodies in research studies?

A comprehensive control strategy for MAP4K4 antibody research should include:

• Positive controls: Tissues or cell lines with verified MAP4K4 expression, such as human cervical or ovarian cancer tissues , or samples from experimental conditions known to upregulate MAP4K4 (e.g., ischemia/reperfusion injury models) .

• Negative controls: MAP4K4 knockdown samples using validated siRNA or CRISPR/Cas9 approaches. The effectiveness of siMAP4K4 has been demonstrated in reducing MAP4K4 expression and downstream MAPK pathway activation .

• Isotype controls: Samples treated with non-specific IgG matching the host species and concentration of the MAP4K4 antibody to identify potential non-specific binding.

• Epitope competition controls: Pre-incubation of the antibody with excess antigenic peptide before application to validate epitope-specific binding.

• Methodological controls: For HRP-conjugated antibodies specifically, include enzyme activity controls to ensure the HRP component remains functional throughout the experiment.

• Biological relevance controls: When studying MAP4K4 in specific pathways like neutrophil differentiation, include complementary markers of differentiation stages to contextualize MAP4K4 findings .

This multi-layered control strategy ensures reliable interpretation of MAP4K4 antibody results across different experimental platforms.

What approaches can resolve inconsistent MAP4K4 detection in Western blotting?

Inconsistent MAP4K4 detection in Western blotting can be resolved through systematic troubleshooting:

• Sample preparation modifications: Include phosphatase inhibitors to preserve phosphorylation states that may affect epitope recognition, particularly as MAP4K4 undergoes autophosphorylation . Adjust lysis buffers to ensure complete solubilization of membrane-associated MAP4K4 complexes.

• Gel percentage optimization: Use 8% gels for better resolution of the ~140 kDa MAP4K4 protein. Extended separation times improve band clarity for high molecular weight proteins.

• Transfer optimization: Implement wet transfer methods for large proteins with extended transfer times (overnight at 30V) and include 0.1% SDS in transfer buffer to facilitate migration of high molecular weight proteins.

• Blocking modifications: Test alternative blocking agents (BSA vs. milk) as MAP4K4 epitopes may be masked differently depending on the blocking reagent. For phosphorylated MAP4K4 detection, 5% BSA is preferable to milk proteins containing phosphatases.

• Signal enhancement: For HRP-conjugated antibodies with weak signals, employ amplification systems like tyramide signal amplification, or consider alternative detection methods like chemifluorescence for higher sensitivity and better quantitation.

• Epitope availability assessment: If inconsistencies persist, evaluate whether different experimental conditions affect post-translational modifications that might mask the epitope recognized by the antibody.

How can MAP4K4 antibodies be employed in studying neutrophil differentiation pathways?

MAP4K4 antibodies can be strategically employed to study neutrophil differentiation through multi-parametric approaches:

• Flow cytometry panels: Combine HRP-conjugated or fluorescently-labeled MAP4K4 antibodies with neutrophil differentiation markers (c-Kit, Ly6G) to track MAP4K4 expression changes during differentiation stages from myeloblasts to segmented neutrophils .

• Sorted population analysis: Use FACS-sorted neutrophil progenitor subpopulations for Western blotting with MAP4K4 antibodies to quantify expression changes across differentiation stages.

• Colony formation assays: Implement MAP4K4 antibody staining in colony formation assays to correlate MAP4K4 expression with colony morphology and differentiation potential of granulocyte-macrophage progenitor cells .

• Reactive oxygen species (ROS) correlation: Simultaneously measure MAP4K4 expression and ROS generation in neutrophil progenitors using flow cytometry to establish functional correlations .

• Single-cell analysis: Apply MAP4K4 antibodies in single-cell protein analysis platforms (e.g., CyTOF) alongside transcriptomic data to correlate protein expression with gene expression patterns during differentiation.

• Inhibition studies: Combine MAP4K4 antibody-based detection with MAP4K4 inhibitor treatments (e.g., PF-06260933) to link expression patterns with functional outcomes in differentiation assays .

These approaches provide comprehensive insights into MAP4K4's role in neutrophil differentiation pathways beyond simple expression analysis.

What are the technical challenges of detecting MAP4K4 in co-immunoprecipitation experiments?

Detecting MAP4K4 in co-immunoprecipitation (co-IP) experiments presents several technical challenges requiring methodological optimization:

• Antibody orientation constraints: When using MAP4K4 antibodies for pull-down, the antibody binding site may interfere with interaction domains, potentially disrupting detection of novel binding partners. Solution: Employ epitope-tagged MAP4K4 constructs or use antibodies targeting different MAP4K4 epitopes for parallel confirmation.

• Transient interaction dynamics: The interaction between MAP4K4 and partners like RAP2C is dynamically regulated by conditions such as hypoxia/reoxygenation . Solution: Implement crosslinking strategies (e.g., DSP, formaldehyde) to capture transient interactions before cell lysis.

• Binding condition sensitivity: MAP4K4 interactions are influenced by phosphorylation states, which can be lost during co-IP procedures. Solution: Preserve phosphorylation states with phosphatase inhibitor cocktails and perform co-IPs at 4°C with minimal washing steps.

• Signal strength variations: Co-IP of MAP4K4 with binding partners like RAP2C shows variable signal strength under different physiological conditions (e.g., normoxia vs. hypoxia/reoxygenation) . Solution: Quantify results using densitometry normalized to input controls and run parallel Pearson's correlation coefficient analyses from immunofluorescence colocalization studies for confirmation .

• Background reduction: When detecting MAP4K4 in co-IPs, non-specific binding can obscure true interactions. Solution: Use pre-clearing steps with protein A/G beads and include stringent controls (IgG isotype, reverse co-IPs) to distinguish specific from non-specific signals.

How can MAP4K4 antibodies be utilized to study cardiomyocyte apoptosis mechanisms?

MAP4K4 antibodies can be strategically employed to elucidate cardiomyocyte apoptosis mechanisms through these methodological approaches:

• Dual immunofluorescence staining: Combine MAP4K4 antibodies with apoptotic markers (cleaved caspase-3, TUNEL) to correlate MAP4K4 expression with apoptotic events in cardiomyocytes under hypoxia/reoxygenation conditions .

• Temporal expression profiling: Monitor MAP4K4 expression kinetics during ischemia/reperfusion using antibody-based Western blotting to establish the temporal relationship between MAP4K4 upregulation and the onset of apoptosis .

• Subcellular localization analysis: Employ confocal microscopy with MAP4K4 antibodies to track subcellular redistribution during stress conditions, particularly focusing on perinuclear localization where MAP4K4 and RAP2C show weak colocalization under normoxic conditions .

• Interaction network analysis: Utilize MAP4K4 antibodies in co-immunoprecipitation experiments to identify and quantify dynamic interactions with partners like RAP2C under normal, stress, and protective conditions (e.g., ischemic postconditioning) .

• Phosphorylation-specific detection: Use phospho-specific MAP4K4 antibodies alongside total MAP4K4 antibodies to determine how MAP4K4 activation state correlates with downstream MAPK pathway activation (p-ERK, p-JNK, p-P38) and subsequent apoptotic signaling .

• Intervention studies: Apply MAP4K4 antibodies to monitor expression changes following genetic manipulations (siRNA knockdown, overexpression) or pharmaceutical interventions to establish causative relationships between MAP4K4 levels and apoptotic outcomes .

How is MAP4K4 research contributing to our understanding of cardiovascular diseases?

MAP4K4 research is substantially advancing cardiovascular disease understanding through several mechanistic pathways:

• Ischemia/reperfusion injury mechanisms: Studies have established that ischemia/reperfusion increases MAP4K4 expression in cardiac tissue, while ischemic postconditioning (PostC) suppresses this effect, identifying MAP4K4 as a key mediator of cardioprotection . Methodologically, this was confirmed through both tissue-level analysis (IHC, Western blotting) and cellular models (cardiomyocytes under hypoxia/reoxygenation conditions) .

• RAP2C-MAP4K4 signaling axis: The identification of RAP2C as a positive regulator of MAP4K4 has established a novel signaling pathway in cardiac pathophysiology . Co-immunoprecipitation assays revealed that hypoxia/reoxygenation enhances RAP2C-MAP4K4 binding, while PostC attenuates this interaction, suggesting a mechanism for PostC-mediated cardioprotection .

• MAPK pathway modulation: MAP4K4 has been shown to regulate downstream MAPK components (ERK, JNK, P38) that control cardiomyocyte apoptosis . Inhibition of MAP4K4 through siRNA knockdown reduces activation of these downstream components, decreasing apoptosis in cardiomyocytes exposed to stress conditions .

• Therapeutic target potential: The identification of MAP4K4 as a regulator of cardiomyocyte apoptosis suggests it could be a valuable therapeutic target. Research shows that MAP4K4 inhibition mimics the cardioprotective effects of ischemic postconditioning, potentially opening new pharmacological approaches to treating ischemic heart disease .

What role does MAP4K4 play in hematopoietic development and related disorders?

MAP4K4 plays crucial roles in hematopoietic development with significant implications for related disorders:

• Neutrophil differentiation regulation: Machine learning-based prediction models have identified MAP4K4 as a novel regulator of neutrophil differentiation . Conditional knockout of Map4k4 in hematopoietic stem and progenitor cells (HSPCs) results in neutropenia, confirming its essential role in neutrophil development .

• Apoptotic control in differentiation: MAP4K4 regulates cell apoptosis during neutrophil differentiation in bone marrow, as demonstrated through single-cell RNA sequencing (scRNA-seq) analysis . Differential expression gene (DEG) analysis revealed that MAP4K4 knockout leads to upregulation of apoptotic signaling pathway genes in neutrophil progenitors .

• Colony formation impact: Experimental evidence shows reduced numbers of granulocyte-macrophage progenitor cell colonies formed by bone marrow cells from Map4k4 conditional knockout mice compared to controls . This indicates MAP4K4's importance in early stages of myeloid differentiation.

• Reactive oxygen species regulation: MAP4K4 affects ROS generation in neutrophil progenitor cells, with altered mean fluorescent intensity observed in Map4k4 knockout mice . This suggests MAP4K4 may link differentiation processes with functional capacity development in neutrophils.

• Clinical implications: The established role of MAP4K4 in neutrophil development suggests potential relevance to neutropenic disorders. Future therapeutic approaches targeting MAP4K4 might address both congenital and acquired neutropenias, though further translational studies are needed to establish clinical applications.

How can researchers integrate MAP4K4 antibody data with transcriptomic and proteomic analyses?

Researchers can implement several methodological approaches to integrate MAP4K4 antibody data with transcriptomic and proteomic analyses:

• Multi-omics correlation analysis: Compare protein expression levels detected by MAP4K4 antibodies with mRNA expression from RNA-seq or microarray data to identify potential post-transcriptional regulation. This approach revealed discrepancies between transcriptional changes and protein levels in neutrophil differentiation studies .

• Single-cell multi-modal analysis: Combine antibody-based protein detection (CyTOF or CITE-seq) with scRNA-seq to simultaneously assess MAP4K4 protein and mRNA levels in individual cells. This technique can reveal heterogeneity in MAP4K4 expression across cell populations and differentiation states as observed in neutrophil lineage cells .

• Pathway integration: Correlate MAP4K4 protein levels with phosphoproteomic data focusing on MAPK pathway components (ERK, JNK, P38) to establish functional relationships between MAP4K4 expression and downstream signaling activation . This integration helps validate antibody-detected changes with functional pathway alterations.

• Temporal dynamics mapping: Align time-course data from antibody-based detection of MAP4K4 with transcriptomic changes to establish cause-effect relationships in signaling cascades. This approach has successfully tracked MAP4K4's role during neutrophil differentiation stages .

• Network analysis: Use computational tools to integrate antibody-derived MAP4K4 protein interaction data with transcriptomic networks to identify novel regulatory relationships. This strategy identified the RAP2C-MAP4K4 signaling axis in cardioprotection .

• Machine learning integration: Apply supervised learning algorithms that incorporate both antibody-detected protein features and transcriptomic signatures to predict functional outcomes, as demonstrated in the Neutrophil Regulatory Gene Identifier (NeuRGI) approach that successfully identified MAP4K4 as a neutrophil differentiation regulator .