MAP4K4 Antibody, FITC conjugated

What is the optimal storage condition for MAP4K4 antibody, FITC conjugated?

MAP4K4 antibody, FITC conjugated should be stored at -20°C or -80°C upon receipt to maintain stability and activity. Avoid repeated freeze-thaw cycles as this can degrade the antibody and diminish fluorescence intensity. The antibody is typically provided in a buffer containing 50% glycerol, 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative . For long-term storage, aliquoting the antibody before freezing is recommended to minimize freeze-thaw cycles.

What are the confirmed applications for MAP4K4 antibodies?

MAP4K4 antibodies have been validated for various applications with specific dilution recommendations:

For fluorescently conjugated versions, specifically FITC-conjugated antibodies, ELISA applications have been confirmed . Additional applications such as Immunofluorescence (IF) and Co-Immunoprecipitation (CoIP) have been reported in published literature .

What is the molecular weight of MAP4K4 and how does this affect antibody detection?

MAP4K4 has a calculated molecular weight of 151 kDa, but the observed molecular weight in experimental conditions typically ranges between 130-160 kDa . This variation can be attributed to post-translational modifications or alternative splicing. When performing Western blot analysis, researchers should expect bands within this range rather than precisely at the calculated molecular weight. Sample preparation and experimental conditions can influence the apparent molecular weight, so including positive controls such as Jurkat or PC-3 cell lysates is recommended for accurate band identification.

How can I optimize MAP4K4 antibody for detecting protein-protein interactions within the STRIPAK complex?

The MAP4K4-STRIPAK complex represents a significant research area as it functions as a central hub orchestrating tissue invasion and growth in various cancers. For optimizing detection of these interactions:

• Cross-linking approach: Use membrane-permeable crosslinkers like DSP (dithiobis(succinimidyl propionate)) prior to cell lysis to stabilize transient interactions.

• Co-immunoprecipitation optimization: Based on research findings, the citron homology domain (CNH, residues 954-1273) of MAP4K4 is required for MAP4K4-STRIPAK interaction . Design your co-IP strategy to preserve this interaction by:

  • Using mild lysis buffers (e.g., containing 0.5% NP-40)

  • Including phosphatase inhibitors to maintain phosphorylation states

  • Testing both N- and C-terminal targeted antibodies, as MAP4K4's interaction with STRIPAK components may mask certain epitopes

• Proximity labeling: Consider BioID approaches as used in published research, where biotinylation of proteins in close proximity to MAP4K4 identified 156 MAP4K4-specific proteins including STRIPAK components .

When using FITC-conjugated antibodies for visualizing these interactions, implement appropriate controls for autofluorescence and optimize fixation methods to preserve both protein complexes and fluorophore activity.

What strategies can address discrepancies between MAP4K4 antibody detection in different tissue types?

Researchers may encounter variability in MAP4K4 detection across different tissues. To address these discrepancies:

• Antigen retrieval optimization: For IHC applications, research indicates that TE buffer at pH 9.0 is recommended for MAP4K4 detection, but citrate buffer at pH 6.0 may be used as an alternative . Systematic comparison of both methods on your specific tissue samples may be necessary.

• Validation through multiple approaches: Combine techniques (e.g., IHC, IF, WB) to confirm expression patterns. Research has demonstrated MAP4K4 expression in multiple human tissues including skeletal muscle, placenta, and liver .

• Expression level quantification: Establish a standardized scoring system based on fluorescence intensity when using FITC-conjugated antibodies, accounting for tissue-specific autofluorescence.

• Positive and negative controls: Include tissues with known high expression (e.g., placenta) and tissues where MAP4K4 is minimally expressed as controls in each experiment.

• Alternative epitope targeting: If detection issues persist, consider antibodies targeting different epitopes of MAP4K4, as protein conformation or post-translational modifications may mask certain regions in specific tissues.

How can I effectively use MAP4K4 antibodies to investigate its role in cancer progression pathways?

MAP4K4 has been implicated in various cancer types, including gastric cancer and medulloblastoma. To investigate its role:

• Pathway analysis strategy: Research indicates MAP4K4 involvement in multiple signaling pathways:

  • It activates the JNK pathway

  • It regulates the PI3K-Akt signaling pathway in gastric cancer

  • It interacts with the Hippo pathway by increasing expression of YAP/TAZ target genes (CTGF, CYR61, ANKRD1)

• Dual labeling approach: Combine FITC-conjugated MAP4K4 antibodies with antibodies against pathway components (e.g., phospho-JNK, YAP/TAZ) to assess co-localization and activation status.

• Functional validation: Complement antibody-based detection with functional assays:

  • siRNA knockdown followed by rescue experiments

  • Kinase activity assays to measure MAP4K4's effect on downstream targets

  • Migration/invasion assays to assess phenotypic consequences, as MAP4K4 promotes invasion in medulloblastoma cells

• Immune microenvironment assessment: Recent findings show positive correlation between immune scores and MAP4K4 expression in gastric cancer , suggesting evaluation of immune infiltrates in tumors with varying MAP4K4 expression levels.

What is the current understanding of MAP4K4's role in cellular signaling networks?

MAP4K4, also known as HGK, NIK, or RAB8IPL1, is a serine/threonine kinase belonging to the STE20 family. Current research provides several insights into its signaling functions:

• MAPK pathway regulation: MAP4K4 acts upstream of the JNK pathway, potentially linking environmental stress and cytokine signals to cellular responses .

• Cytoskeletal regulation: It directly phosphorylates ezrin, radixin, moesin (ERM) proteins to mediate lamellipodium formation in response to growth factors. MAP4K4 also couples growth factor signaling to actin polymerization through phosphorylation of the Arp2 subunit of the Arp2/3 complex .

• Receptor trafficking: MAP4K4 promotes turnover and activation of receptor tyrosine kinases (e.g., c-MET) and adhesion receptors (e.g., β-1 integrin) .

• SMAD signaling: It phosphorylates SMAD1 on Thr-322, potentially affecting TGF-β family signaling .

• Hippo pathway regulation: MAP4K4 depletion increases expression of YAP/TAZ target genes, suggesting it activates the Hippo pathway in certain contexts .

• STRIPAK complex interaction: MAP4K4 interacts with the STRIPAK complex, a supramolecular scaffold held together by striatin family proteins (STRN, STRN3, STRN4) , which may regulate its diverse cellular functions.

Understanding these multiple signaling roles is essential when designing experiments to investigate MAP4K4 function in specific cellular contexts.

How does MAP4K4 contribute to vascular development and lymphatic function?

Research indicates that endothelial MAP4K4 (EC Map4k4) plays a crucial role in lymphatic vascular development through:

• Regulation of endothelial cell quiescence: MAP4K4 appears to control the balance between endothelial cell proliferation and quiescence .

• Lymphatic endothelial cell fate determination: EC Map4k4 is critical for lymphatic endothelial cell specification and development .

For researchers investigating these aspects:

• Consider using combination staining with lymphatic markers (e.g., LYVE-1, VEGFR-3) alongside FITC-conjugated MAP4K4 antibodies

• Examine both developmental contexts and adult tissues to distinguish between developmental and homeostatic functions

• Implement loss-of-function studies in relevant model systems

The specific mechanisms through which MAP4K4 regulates lymphatic development may involve integration of growth factor signaling, cytoskeletal reorganization, and control of cell-cell junctions, though further research is needed to fully elucidate these pathways.

What controls should be included when using MAP4K4 antibody, FITC conjugated in immunofluorescence experiments?

For robust immunofluorescence experiments with FITC-conjugated MAP4K4 antibodies, include these essential controls:

• Positive tissue controls: Use tissues with confirmed MAP4K4 expression, such as:

  • Human skeletal muscle tissue

  • Human placenta tissue

  • Human liver tissue

  • Jurkat or PC-3 cell lines

• Negative controls:

  • Isotype control (rabbit IgG-FITC) to assess non-specific binding

  • Secondary-only control (when using a primary-secondary approach)

  • Tissues or cells with MAP4K4 knockdown/knockout (if available)

• Autofluorescence controls:

  • Unstained tissue sections to identify natural autofluorescence

  • Consider adding a quenching step (e.g., Sudan Black B treatment) particularly for tissues with high autofluorescence

• Absorption controls: Pre-incubate the antibody with immunizing peptide to demonstrate specificity

• Channel bleed-through controls: When performing multi-color IF, include single-stained samples to establish proper exposure settings and confirm absence of spectral overlap

Remember that FITC fluorescence is sensitive to pH changes and photobleaching, so optimize your fixation protocol and minimize exposure to light during sample processing.

How can I troubleshoot weak or inconsistent MAP4K4 signal in Western blot applications?

When experiencing weak or inconsistent MAP4K4 detection in Western blots:

• Sample preparation optimization:

  • Ensure complete protein extraction with appropriate lysis buffers

  • Include protease inhibitors to prevent degradation

  • Avoid excessive sample heating which may cause protein aggregation

• Loading amount adjustment:

  • MAP4K4 may require higher protein loading (20-50 μg) for clear detection

  • Maintain protein concentration within linear detection range

• Transfer parameters:

  • For high molecular weight proteins like MAP4K4 (130-160 kDa), extend transfer time or reduce voltage

  • Consider using low-percentage gels (6-8%) for better resolution

• Antibody incubation conditions:

  • Start with the recommended 1:200-1:1000 dilution range

  • Extend primary antibody incubation to overnight at 4°C

  • Optimize blocking conditions to reduce background while maintaining specific signal

• Detection system sensitivity:

  • Use enhanced chemiluminescence (ECL) substrates designed for low-abundance proteins

  • Consider using signal amplification systems for extremely low expression

• Positive control inclusion:

  • Always include Jurkat or PC-3 cell lysates as positive controls

  • Consider using recombinant MAP4K4 protein as a standard

• Membrane stripping considerations:

  • Avoid harsh stripping conditions that may remove target proteins

  • If reprobing is necessary, document initial blot before stripping

What are the best practices for multiplex analysis using MAP4K4 antibody, FITC conjugated alongside other antibodies?

For effective multiplex analysis:

• Spectral compatibility planning:

  • FITC has excitation/emission peaks at approximately 495/519 nm

  • Choose companion fluorophores with minimal spectral overlap (e.g., Cy5, Texas Red)

  • Design panels based on expression levels (use brightest fluorophores for lowest-expressed targets)

• Sequential staining approach:

  • For challenging combinations, implement sequential rather than simultaneous staining

  • Test order of antibody application to optimize signal for all targets

• Cross-reactivity prevention:

  • Use antibodies raised in different host species to avoid cross-reactivity

  • Employ isotype-specific secondary antibodies when using multiple primary antibodies from the same species

  • Block between sequential staining steps with relevant host serum

• Compensation and unmixing:

  • Acquire single-stained controls for each fluorophore

  • Apply proper compensation in flow cytometry or spectral unmixing in imaging applications

• Biological controls for co-expression:

  • Include samples with known co-expression patterns

  • Validate antibody combinations on control tissues before experimental samples

• Target selection considerations:

  • For studying MAP4K4 signaling, consider multiplex staining with:

    • Phosphorylated forms of downstream targets (p-JNK)

    • STRIPAK complex components (STRN3, STRN4, STRIP1)

    • Markers relevant to specific research context (e.g., lymphatic markers if studying vascular development )

By following these guidelines, researchers can generate reliable and informative data when using MAP4K4 antibody, FITC conjugated in complex experimental designs.

How can MAP4K4 antibodies be used to investigate its role in cancer progression?

MAP4K4 has been implicated in multiple cancer types, with specific research showing:

• Gastric cancer implications:

  • MAP4K4 expression is significantly higher in tumor samples compared to adjacent normal tissue

  • High expression correlates with enrichment in tumor-related pathways, particularly PI3K-Akt signaling

  • Immune infiltration analysis reveals positive correlation between immune scores and MAP4K4 expression

• Medulloblastoma progression:

  • MAP4K4 promotes receptor tyrosine kinase-induced cell dissemination

  • It regulates membrane protrusion at the leading edge of invading cells

  • The MAP4K4-STRIPAK complex functions as a central hub orchestrating tissue invasion and growth

For investigating these roles:

• Implement tissue microarray analysis with FITC-conjugated MAP4K4 antibodies to assess expression in patient cohorts

• Correlate expression with clinical parameters (tumor stage, grade, patient survival)

• Combine with markers of proliferation, invasion, and angiogenesis for comprehensive phenotyping

• Consider dual staining with immune cell markers based on the correlation with immune infiltration

What methodological approaches can detect alterations in MAP4K4 phosphorylation status?

MAP4K4 phosphorylation status can significantly affect its function. To investigate this:

• Phospho-specific antibody selection:

  • While the search results don't mention specific phospho-MAP4K4 antibodies, research indicates that site-specific phosphorylation of MAP4K4 in ID1 and ID2 domains correlates with STRIPAK complex activity

  • Use phospho-specific antibodies for key regulatory sites when available

• Phosphatase treatment controls:

  • Include lambda phosphatase-treated samples to confirm phosphorylation-specific signals

  • Compare migration patterns before and after phosphatase treatment

• Phos-tag™ gel electrophoresis:

  • Implement Phos-tag™ acrylamide gels to separate phosphorylated from non-phosphorylated forms

  • This technique allows visualization of multiple phosphorylation states without phospho-specific antibodies

• Mass spectrometry approach:

  • For comprehensive phosphorylation mapping, immunoprecipitate MAP4K4 using validated antibodies

  • Perform phospho-peptide enrichment followed by mass spectrometry analysis

• Kinase inhibitor studies:

  • Treat cells with appropriate kinase inhibitors to identify upstream regulators of MAP4K4 phosphorylation

  • Monitor changes in MAP4K4 activity and downstream signaling

This multilayered approach can provide comprehensive information about MAP4K4 phosphorylation dynamics in different cellular contexts.

How should researchers interpret conflicting MAP4K4 expression data across different experimental systems?

When confronted with conflicting MAP4K4 expression data:

• Contextual evaluation:

  • Cell/tissue type specificity: MAP4K4 functions may vary significantly between cell types

  • Developmental stage considerations: Expression patterns may differ between embryonic, postnatal, and adult tissues

  • Disease state influence: Pathological conditions may alter expression levels and localization

• Methodological reconciliation:

  • Compare antibody epitopes: Different antibodies may recognize distinct isoforms or conformations

  • Assess detection methods: Transcript (qPCR) vs. protein (WB, IHC) measurements may not correlate due to post-transcriptional regulation

  • Evaluate quantification approaches: Absolute vs. relative quantification may yield apparently conflicting results

• Experimental design analysis:

  • In vitro vs. in vivo discrepancies: Cell culture conditions may not recapitulate in vivo regulation

  • Acute vs. chronic manipulations: Transient knockdown may produce different effects than stable knockout

  • 2D vs. 3D culture systems: Spatial organization can influence MAP4K4 expression and function

• Resolution strategies:

  • Implement multiple detection methods in parallel

  • Use genetically modified systems with tagged endogenous MAP4K4 for consistent detection

  • Perform time-course studies to capture dynamic expression changes

  • Consider single-cell analysis to identify subpopulation differences that might explain apparent contradictions

By systematically addressing these factors, researchers can develop more nuanced interpretations of seemingly conflicting data on MAP4K4 expression and function.