MAP4K2 Antibody

What is MAP4K2 and what cellular pathways is it involved in?

MAP4K2, also known as GCK (Germinal Center Kinase) and RAB8IP, belongs to the protein kinase superfamily, specifically the STE Ser/Thr protein kinase family and STE20 subfamily . It functions as a mitogen-activated protein kinase kinase kinase kinase, playing a crucial role in signal transduction pathways.

MAP4K2 enhances MAP3K1 oligomerization, potentially relieving amino-terminal mediated MAP3K1 autoinhibition and leading to activation following autophosphorylation . While expressed in various tissues, its expression in lymphoid follicles is restricted to germinal center cells, suggesting a role in B-cell differentiation .

MAP4K2 can be activated by TNF-alpha and has been shown to specifically activate MAP kinases. Additionally, it interacts with TNF receptor-associated factor 2 (TRAF2), which is involved in activating MAP3K1/MEKK1 . Beyond these canonical roles, MAP4K2 may also function in vesicle targeting or fusion, indicating its multifaceted involvement in cellular processes .

What are the structural and molecular characteristics of MAP4K2?

MAP4K2 is a protein with a calculated molecular weight of 92 kDa, though it is typically observed at 85-91 kDa and sometimes at 58 kDa in Western blot applications . This discrepancy between calculated and observed weights may reflect post-translational modifications or alternative splicing variants.

The protein is primarily localized in the cytoplasm, but can also be found at the basolateral cell membrane and Golgi apparatus membrane . Its subcellular distribution suggests involvement in membrane trafficking and signaling.

The human MAP4K2 gene has the GenBank Accession Number NM_004579, Gene ID 5871, and UNIPROT ID Q12851 . This information is essential for researchers designing experiments targeting specific regions of the protein or gene.

What MAP4K2 antibodies are available and what are their key specifications?

Several MAP4K2-specific antibodies are commercially available for research applications. Key examples include:

Proteintech 55244-1-AP:

• Host/Isotype: Rabbit/IgG

• Class: Polyclonal

• Applications: WB (1:500-1:1000), IHC (1:50-1:500), ELISA

• Reactivity: Human, mouse

• Immunogen: Peptide

• Purification Method: Antigen affinity purification

• Storage: -20°C in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

Elabscience E-AB-10897:

• Host/Isotype: Rabbit/IgG

• Class: Polyclonal

• Applications: WB (1:1000-1:5000)

• Reactivity: Human, mouse

• Immunogen: Recombinant protein of human MAP4K2

• Concentration: 0.3 mg/mL

• Purification Method: Affinity purification

• Storage: -20°C in phosphate buffered solution (pH 7.4) with 0.05% stabilizer and 50% glycerol

When selecting an appropriate antibody, researchers should consider the specific application, species reactivity, and validation data available for each product.

What are the optimal protocols for Western blot applications using MAP4K2 antibodies?

For Western blot applications with MAP4K2 antibodies, researchers should follow these methodological guidelines:

Sample Preparation:

• Positive controls should include mouse brain tissue, human brain tissue, or Raji cells, which have demonstrated consistent MAP4K2 expression .

• Protein extraction should be optimized to maintain protein integrity while ensuring efficient solubilization of membrane-associated MAP4K2.

Dilution and Detection:

• For Proteintech 55244-1-AP: Use a dilution of 1:500-1:1000

• For Elabscience E-AB-10897: Use a dilution of 1:1000-1:5000

• Expect bands at approximately 85-91 kDa and possibly at 58 kDa

Optimization Tips:

• Titrate the antibody in each testing system to obtain optimal results, as performance can be sample-dependent .

• Use appropriate blocking solutions to minimize background signal.

• Consider gradient gels for better resolution of the target protein.

It is recommended that researchers validate the antibody in their specific experimental system before proceeding with larger-scale experiments.

What are the recommended protocols for immunohistochemistry with MAP4K2 antibodies?

For immunohistochemistry applications with MAP4K2 antibodies:

Sample Preparation:

• For validation, human colon cancer tissue and human lung cancer tissue have shown positive results .

• Perform antigen retrieval with TE buffer pH 9.0; alternatively, citrate buffer pH 6.0 may be used .

Protocol Guidelines:

• Use a dilution of 1:50-1:500 for Proteintech 55244-1-AP in IHC applications .

• Follow standard IHC protocols with appropriate blocking, washing, and detection steps.

• Include positive and negative controls to validate staining specificity.

Optimization Considerations:

• Adjust antibody concentration based on signal intensity and background levels.

• Optimize incubation times and temperatures for specific tissue types.

• Consider different detection systems (HRP, AP, fluorescence) based on experimental needs.

The cellular localization of MAP4K2 (cytoplasm, basolateral cell membrane, Golgi apparatus membrane) should be considered when interpreting IHC results .

How can researchers validate the specificity of MAP4K2 antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For MAP4K2 antibodies, consider these approaches:

Positive Controls:

• Use validated samples known to express MAP4K2, such as brain tissue .

• Include cell lines with confirmed MAP4K2 expression, such as Raji cells .

Negative Controls:

• Use isotype controls to identify non-specific binding.

• Consider samples from MAP4K2 knockout models or cells treated with MAP4K2-specific siRNA.

Multiple Antibody Validation:

• When possible, compare results from different antibody clones targeting distinct epitopes of MAP4K2.

• Verify results using alternative detection methods (e.g., mass spectrometry).

Cross-Application Validation:

• If using the antibody for multiple applications (WB, IHC, etc.), verify consistent results across techniques.

• Consider validation through functional assays that correlate with MAP4K2 activity.

Proper validation ensures that experimental observations truly reflect MAP4K2 biology rather than artifacts of non-specific antibody interactions.

How does MAP4K2 function in viral infection pathways, particularly in HCV replication?

Research on hepatitis C virus (HCV) has revealed intriguing roles for MAP4K2 in viral replication:

During HCV infection, the JNK pathway is generally suppressed, with several components including MAP4K2, MAP3K5, MAP2K7, and MAP2K4 showing reduced expression or activity . Despite this suppression, MAP4K2 appears to facilitate viral replication through mechanisms that may be independent of the canonical JNK pathway.

Evidence for this comes from RNAi studies where silencing MAP4K2 significantly reduced virus replication . Interestingly, this effect occurs despite the fact that phosphorylation levels of the effector Jun protein remained unchanged throughout infection, suggesting a JNK pathway-independent mechanism .

The relationship between MAP4K2 and MAP2K7 in this context is noteworthy - while both are downregulated during infection and silencing either reduces viral replication, the suppressive effect of MAP4K2 silencing on virus replication is more pronounced than that of MAP2K7 silencing .

These findings highlight MAP4K2 as a potential therapeutic target for antiviral intervention, particularly against HCV. Researchers studying viral host interactions should consider examining MAP4K2's role in other viral infection models as well.

What is the relationship between MAP4K2 and plant signaling pathways?

Recent research has identified interesting roles for MAP4K2 in plant signaling, particularly in abscisic acid (ABA)-induced pathways:

MAP4K2 has been found to interact directly with MAP4K1, as confirmed through BiFC assays and yeast two-hybrid (Y2H) systems . These kinases function redundantly as positive regulators of ABA-induced stomatal closure in plants .

Phosphoproteomic analysis has identified several phosphorylation sites in MAP4K2, though Ser-488 (which is positionally similar to the functionally important Ser-479 of MAP4K1) was not detected in some studies .

MAP4K1 and MAP4K2 expression is observed in both guard cell protoplasts (GCPs) and mesophyll cell protoplasts (MCPs) . The interaction between SnRK2 and MAP4K1/2 appears to form a signaling module that regulates calcium-mediated stomatal closure .

This research demonstrates conservation of MAP4K-mediated signaling mechanisms across kingdoms and suggests potential evolutionary relationships in stress response pathways between animals and plants.

How do phosphorylation states of MAP4K2 affect its function?

Phosphorylation is a critical post-translational modification that regulates MAP4K2 activity and interactions:

While complete phosphorylation mapping of MAP4K2 is still evolving, studies have identified at least three phosphorylation sites in MAP4K2 . These phosphorylation events likely regulate kinase activity, protein-protein interactions, and subcellular localization.

Of particular interest is Ser-488 of MAP4K2, which is positionally similar to Ser-479 of MAP4K1 . In MAP4K1, Ser-479 phosphorylation is significantly upregulated in response to ABA in wild-type plants but not in certain mutants, suggesting a regulatory role .

Researchers investigating MAP4K2 function should consider:

• The phosphorylation state of the protein in different cellular contexts

• How phosphorylation affects interactions with binding partners like MAP4K1, MAP3K1, and TRAF2

• The relationship between phosphorylation status and subcellular localization

• The impact of phosphorylation on kinase activity toward downstream targets

Advanced methodologies such as phospho-specific antibodies, phosphoproteomics, or phosphomimetic mutations can help elucidate the functional significance of MAP4K2 phosphorylation states.

What are common issues when detecting MAP4K2 and how can they be resolved?

Researchers working with MAP4K2 may encounter several common challenges:

Multiple Bands in Western Blot:

• MAP4K2 is observed at both 85-91 kDa and 58 kDa .

• The presence of multiple bands may reflect post-translational modifications, degradation products, or splice variants.

• Resolution: Use gradient gels, optimize sample preparation to minimize degradation, and consider phosphatase treatment to identify phosphorylation-dependent mobility shifts.

Variable Expression Levels:

• MAP4K2 expression varies across tissues and cell types, with enrichment in germinal center B cells .

• Resolution: Include appropriate positive controls, normalize loading carefully, and consider enrichment steps for low-abundance samples.

Antibody Cross-Reactivity:

• MAP4K2 shares sequence similarities with other MAP4K family members.

• Resolution: Validate antibody specificity using knockout/knockdown approaches, and compare results with multiple antibodies targeting different epitopes.

Fixation Sensitivity in IHC:

• Epitope accessibility may be affected by fixation methods.

• Resolution: Test multiple antigen retrieval methods; TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative .

Careful optimization and validation at each step of the experimental workflow will help ensure reliable detection of MAP4K2.

How should data on MAP4K2 expression and function be interpreted in different disease models?

Interpreting MAP4K2 data requires careful consideration of biological context:

In Viral Infections:

• Despite being downregulated during HCV infection, MAP4K2 supports viral replication through JNK-independent mechanisms .

• When analyzing MAP4K2 function in infectious disease models, consider:

  • The timing of expression changes relative to infection progression

  • Interactions with viral proteins

  • Pathway cross-talk that may compensate for altered MAP4K2 activity

In Cancer Research:

• MAP4K2 antibodies have been validated in colon and lung cancer tissues .

• When studying MAP4K2 in cancer models, consider:

  • Expression differences between tumor and normal tissues

  • Correlation with cancer progression markers

  • Potential as a therapeutic target or biomarker

In Signaling Studies:

• MAP4K2 interacts with multiple partners including MAP4K1, MAP3K1, and TRAF2 .

• When analyzing signaling data:

  • Examine multiple components of the pathway

  • Consider both phosphorylation-dependent and independent functions

  • Validate with pharmacological inhibitors and genetic approaches

What are the best practices for storing and handling MAP4K2 antibodies to maintain reactivity?

Proper storage and handling of MAP4K2 antibodies is essential for maintaining their performance over time:

Storage Conditions:

• Store antibodies at -20°C as recommended by manufacturers .

• Most MAP4K2 antibodies are stable for one year after shipment when properly stored .

• For some formulations, aliquoting is unnecessary for -20°C storage, but this depends on the specific product .

Buffer Composition:

• MAP4K2 antibodies are typically supplied in PBS with preservatives such as sodium azide and stabilizers like glycerol .

• The buffer pH is typically maintained around pH 7.3-7.4 for optimal stability .

Handling Recommendations:

• Avoid repeated freeze-thaw cycles to prevent antibody degradation .

• When removing from storage, thaw antibodies on ice or at 4°C rather than at room temperature.

• For diluted working solutions, prepare fresh or store for minimal periods at 4°C.

• Follow manufacturer's recommendations for specific products, as formulations may vary.

Proper storage and handling practices will help ensure consistent antibody performance across experiments and maximize the useful lifetime of these valuable reagents.

What are promising areas for future research on MAP4K2 function?

Based on current knowledge, several promising research directions for MAP4K2 include:

Novel Signaling Pathways:

• Further investigation of JNK-independent functions of MAP4K2 in viral infection models .

• Exploration of MAP4K2's role in vesicle trafficking and membrane dynamics .

• Study of MAP4K2-MAP4K1 interactions in diverse biological systems beyond plant ABA responses .

Therapeutic Targeting:

• Development of selective MAP4K2 inhibitors for antiviral applications, particularly against HCV .

• Evaluation of MAP4K2 as a potential target in cancer therapy, given its detection in colon and lung cancer tissues .

• Investigation of MAP4K2 modulation in immune cell function, particularly in B-cell germinal centers .

Technological Advances:

• Development of phospho-specific antibodies targeting key regulatory sites like Ser-488 .

• Application of CRISPR/Cas9 to generate MAP4K2 knockout or knock-in models.

• High-throughput screening for MAP4K2 modulators with therapeutic potential.

These research directions could significantly advance our understanding of MAP4K2 biology and potentially lead to new therapeutic strategies.

How can advanced imaging techniques enhance MAP4K2 research?

Advanced imaging approaches offer powerful tools for investigating MAP4K2 localization and dynamics:

Super-Resolution Microscopy:

• Techniques like STED, PALM, or STORM can resolve MAP4K2 localization to specific subcellular compartments (cytoplasm, basolateral cell membrane, Golgi apparatus membrane) .

• These approaches could reveal previously undetected protein complexes involving MAP4K2.

Live-Cell Imaging:

• Fluorescently tagged MAP4K2 combined with time-lapse microscopy can track dynamic relocalization during signaling events.

• FRET/FLIM approaches could monitor real-time interactions with binding partners like MAP4K1 or TRAF2 .

Correlative Microscopy:

• Combining fluorescence microscopy with electron microscopy can provide both molecular specificity and ultrastructural context for MAP4K2 localization.

• This approach is particularly valuable for understanding membrane associations.

Considerations for Implementation:

• Validate that tags or labeling strategies do not interfere with MAP4K2 function.

• Combine imaging with functional assays to correlate localization with activity.

• Use appropriate controls to distinguish specific from non-specific signals.

Advanced imaging approaches, when properly implemented and validated, can provide unique insights into MAP4K2 biology that complement biochemical and genetic approaches.