MAP3K7CL Antibody

What is MAP3K7CL and what is its relationship to MAP3K7?

MAP3K7CL (MAP3K7 C-terminal like) is a protein encoded by the MAP3K7CL gene, also known by several synonyms including C21orf7, TAKL, TAKL-1, TAKL-2, TAKL-4, HC21ORF7, and TAK1L . The protein has a molecular weight of approximately 27.248 kDa and shares structural similarities with the C-terminal domain of MAP3K7 (also known as TAK1) . MAP3K7 encodes transforming growth factor β (TGF-β)-activated kinase 1 (TAK1), which plays critical roles in cellular signaling pathways including TNF-α and NF-κB signaling . While MAP3K7 functions in viral resistance and immune signaling, MAP3K7CL's specific functions are still being elucidated, though its structural similarity suggests potential related functions in signaling cascades.

What types of MAP3K7CL antibodies are available for research applications?

MAP3K7CL antibodies are available in several formats optimized for different research applications:

Most commercially available MAP3K7CL antibodies are rabbit polyclonal antibodies that recognize epitopes in the C-terminal region of the protein . The choice between different antibodies should be guided by the specific application requirements and the target species in your research model.

What are the optimal storage conditions for maintaining MAP3K7CL antibody activity?

To maintain optimal activity of MAP3K7CL antibodies, follow these evidence-based storage recommendations:

• Short-term storage (up to 6 months): Maintain refrigerated at 2-8°C in the original buffer .

• Long-term storage: Store at -20°C in small aliquots to prevent repeated freeze-thaw cycles that can damage antibody structure and function .

• Storage buffer: MAP3K7CL antibodies are typically supplied in PBS with 0.09% (W/V) sodium azide, pH 7.4 .

• Aliquoting: When receiving a new antibody, divide it into small single-use aliquots before freezing to minimize freeze-thaw cycles.

• Thawing process: Thaw frozen antibodies on ice and centrifuge briefly before use to collect all liquid at the bottom of the tube.

Proper storage is crucial for maintaining antibody specificity and sensitivity in experimental applications.

How should I validate a MAP3K7CL antibody for my specific research application?

Validating a MAP3K7CL antibody is essential for ensuring reliable and reproducible results. Follow this comprehensive validation protocol:

• Positive and negative controls:

  • Use cell lines or tissues with known MAP3K7CL expression levels as positive controls

  • Include samples with knockout or knockdown of MAP3K7CL as negative controls

• Western blot validation:

  • Confirm the antibody detects a band of the expected molecular weight (approximately 27.248 kDa)

  • Evaluate specificity by examining non-specific bands

  • Test antibody performance across a concentration gradient to determine optimal dilution

• Cross-reactivity assessment:

  • Particularly important if studying related proteins like MAP3K7/TAK1

  • Test the antibody against recombinant MAP3K7CL and MAP3K7 to ensure specificity

• Application-specific validation:

  • For immunohistochemistry: Include antigen retrieval optimization steps and test fixation methods

  • For immunoprecipitation: Confirm the antibody can efficiently pull down the target protein

  • For ELISA: Establish standard curves using recombinant protein

• Epitope mapping:

  • Confirm the antibody recognizes the expected epitope region (e.g., C-terminal region amino acids 215 to 241)

Thorough validation ensures reliable experimental outcomes and prevents misinterpretation of data due to antibody artifacts.

What protocols are recommended for Western blot analysis using MAP3K7CL antibodies?

For optimal Western blot results with MAP3K7CL antibodies, follow this detailed protocol:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease and phosphatase inhibitors

  • Determine protein concentration using BCA or Bradford assay

  • Prepare 20-40 μg of total protein per lane

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels (appropriate for the 27.248 kDa MAP3K7CL protein)

  • Include molecular weight markers

• Transfer:

  • Transfer proteins to PVDF or nitrocellulose membrane

  • Use semi-dry or wet transfer systems at 100V for 60-90 minutes

• Blocking:

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

• Primary antibody incubation:

  • Dilute MAP3K7CL antibody (typical starting dilution 1:500-1:1000)

  • Incubate overnight at 4°C with gentle rocking

• Washing and secondary antibody:

  • Wash 3-4 times with TBST, 5-10 minutes each

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000-1:10000)

  • Incubate for 1 hour at room temperature

• Detection:

  • Use ECL reagent for detection

  • Expected band size: ~27.248 kDa

• Controls:

  • Include positive control (cell lines with known MAP3K7CL expression)

  • Consider running parallel blots for loading controls (β-actin, GAPDH)

Optimization of antibody concentration and incubation conditions may be necessary for different sample types and antibody lots.

How can I optimize immunohistochemistry protocols for MAP3K7CL detection?

For successful immunohistochemistry (IHC) detection of MAP3K7CL, follow these methodological guidelines:

• Sample preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Embed in paraffin and section at 4-6 μm thickness

  • Mount sections on positively charged slides

• Deparaffinization and rehydration:

  • Xylene: 3 changes, 5 minutes each

  • Decreasing ethanol gradient (100%, 95%, 80%, 70%)

  • Rinse in distilled water

• Antigen retrieval (critical step):

  • Heat-induced epitope retrieval: Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Pressure cooker method: 125°C for 3 minutes or 95°C for 20 minutes

  • Test both methods to determine optimal conditions for MAP3K7CL epitope exposure

• Blocking:

  • Endogenous peroxidase block: 3% hydrogen peroxide, 10 minutes

  • Protein block: 5% normal goat serum in PBST, 1 hour

  • Avidin/biotin block if using biotin-based detection systems

• Primary antibody incubation:

  • Dilute MAP3K7CL antibody (start with 1:200 dilution)

  • Incubate overnight at 4°C in a humidified chamber

  • Use antibodies specifically validated for IHC applications

• Detection system:

  • Use appropriate HRP-polymer or biotin-based detection system

  • Follow manufacturer's protocol for secondary antibody incubation

  • Develop with DAB substrate and counterstain with hematoxylin

• Controls:

  • Include positive control tissue

  • Include a negative control by omitting primary antibody

  • Consider using tissues with known MAP3K7CL expression patterns

Optimize antibody dilution and antigen retrieval methods for each specific tissue type and fixation protocol.

How can MAP3K7CL antibodies be used to study its potential role in viral resistance pathways?

Based on the relationship between MAP3K7 and viral resistance, investigating MAP3K7CL's potential role requires sophisticated experimental approaches:

• Co-immunoprecipitation studies:

  • Use MAP3K7CL antibodies to pull down protein complexes

  • Analyze interacting partners involved in antiviral signaling

  • Compare MAP3K7CL and MAP3K7 interactomes in virus-resistant vs. sensitive cells

• Proximity ligation assays:

  • Detect in situ protein-protein interactions between MAP3K7CL and components of antiviral pathways

  • Combine MAP3K7CL antibodies with antibodies against interferon-stimulated gene (ISG) products

  • Visualize interactions in response to viral challenge

• ChIP-seq analysis:

  • Similar to studies showing CHD1 binding sites are enriched in TNF-α and NF-κB signaling genes

  • Use MAP3K7CL antibodies for chromatin immunoprecipitation followed by sequencing

  • Identify potential regulatory roles in expression of antiviral genes

• Viral infection models:

  • Examine MAP3K7CL expression and localization before and after viral challenge

  • Compare with MAP3K7's established role in viral resistance

  • Assess impact of MAP3K7CL silencing on viral susceptibility using similar approaches to those used for MAP3K7

• ISG regulation studies:

  • Study the effect of MAP3K7CL modulation on expression of ISGs like MX1, OAS3, EIF2AK2, IFIT1, IFIT5, IFITM3, IRF3, ISG15, and STAT1

  • Use MAP3K7CL antibodies to monitor protein levels in response to interferon stimulation

The experimental design should account for potential functional overlap between MAP3K7CL and MAP3K7, as research has shown MAP3K7 affects viral susceptibility and ISG expression in prostate cancer cells .

What techniques can I use to study MAP3K7CL phosphorylation patterns?

Investigating MAP3K7CL phosphorylation requires specialized approaches:

• Phospho-specific antibody development:

  • If not commercially available, consider developing custom phospho-specific antibodies against predicted phosphorylation sites

  • Base predictions on known sites in the related MAP3K7/TAK1 protein

  • Validate using phosphatase treatment controls

• Mass spectrometry analysis:

  • Immunoprecipitate MAP3K7CL using validated antibodies

  • Perform tryptic digestion followed by phosphopeptide enrichment

  • Use titanium dioxide (TiO2) or immobilized metal affinity chromatography (IMAC)

  • Analyze by LC-MS/MS to identify phosphorylated residues

• Phosphorylation kinetics studies:

  • Stimulate cells with relevant ligands (e.g., TNF-α, IL-1β, TGF-β)

  • Harvest at multiple time points

  • Immunoprecipitate using MAP3K7CL antibodies

  • Analyze phosphorylation changes by Western blot or mass spectrometry

• Kinase prediction and validation:

  • Use bioinformatics tools to predict potential kinases acting on MAP3K7CL

  • Perform in vitro kinase assays with recombinant MAP3K7CL

  • Validate in cells using specific kinase inhibitors

• Functional analysis of phosphorylation sites:

  • Create phosphomimetic and phosphodeficient mutants

  • Assess impact on protein function and signaling

  • Monitor effects on potential downstream targets

These approaches collectively provide comprehensive insights into the regulation of MAP3K7CL through phosphorylation and its potential signaling roles.

How do I design experiments to investigate the relationship between MAP3K7CL and MAP3K7 in immune signaling?

To investigate potential functional relationships between MAP3K7CL and MAP3K7 in immune signaling, implement these methodological approaches:

• Co-expression and co-localization studies:

  • Use validated MAP3K7CL and MAP3K7 antibodies for immunofluorescence

  • Assess subcellular localization under basal and stimulated conditions

  • Quantify co-localization using Pearson's or Mander's coefficients

• Protein interaction analysis:

  • Perform reciprocal co-immunoprecipitation with MAP3K7CL and MAP3K7 antibodies

  • Confirm interactions by proximity ligation assay

  • Use truncation mutants to map interaction domains

• CRISPR-based functional studies:

  • Generate single knockouts of MAP3K7CL and MAP3K7

  • Create double knockout cell lines

  • Compare phenotypes in terms of immune signaling pathway activation

  • Rescue experiments with wild-type or mutant constructs

• Pathway activation analysis:

  • Stimulate cells with immune activators (TNF-α, IL-1β, TLR ligands)

  • Monitor downstream signaling events (NF-κB, JNK, p38 MAPK activation)

  • Compare responses in wild-type, MAP3K7CL-deficient, MAP3K7-deficient, and double-deficient cells

• Protein domain function analysis:

  • Given MAP3K7CL's similarity to MAP3K7's C-terminal domain

  • Create domain-swap constructs

  • Assess functional complementation

• Transcriptional profiling:

  • Perform RNA-seq on cells with modified MAP3K7CL or MAP3K7 expression

  • Focus on immune response genes and interferon-stimulated genes (ISGs)

  • Compare with known MAP3K7-regulated transcriptional programs

Based on research showing MAP3K7's role in viral resistance and ISG expression , these approaches can elucidate whether MAP3K7CL has overlapping, complementary, or distinct functions in immune signaling pathways.

How do I troubleshoot non-specific binding when using MAP3K7CL antibodies?

Non-specific binding is a common challenge when working with antibodies. Follow this systematic troubleshooting approach for MAP3K7CL antibodies:

• Antibody dilution optimization:

  • Test a range of dilutions (e.g., 1:200, 1:500, 1:1000, 1:2000)

  • Excessive antibody concentration often increases background signals

  • Find the optimal balance between specific signal and background

• Blocking protocol improvement:

  • Increase blocking time (from 1 hour to overnight)

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Match blocking agent to the secondary antibody host species

• Sample preparation refinement:

  • Ensure complete cell lysis for Western blots

  • Optimize fixation protocols for immunostaining

  • Consider alternative lysis buffers with different detergent compositions

• Washing optimization:

  • Increase number of washes (minimum 4-5 washes)

  • Extend washing time (10-15 minutes per wash)

  • Use gentle agitation during washing steps

• Cross-reactivity assessment:

  • Perform peptide competition assays using the immunizing peptide

  • Pre-adsorb the antibody with recombinant MAP3K7 to remove cross-reactive antibodies

  • Consider using knockout or knockdown controls

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Test alternative secondary antibodies

  • Consider fluorescent secondaries that may provide better signal-to-noise ratios

• Buffer optimization:

  • Add 0.1-0.3% Triton X-100 or 0.05% Tween-20 to reduce non-specific hydrophobic interactions

  • Adjust salt concentration in wash buffers

Systematic optimization of these parameters should significantly improve specificity when working with MAP3K7CL antibodies.

What controls should I include when designing experiments using MAP3K7CL antibodies?

Proper experimental controls are essential for reliable interpretation of results with MAP3K7CL antibodies:

• Expression controls:

  • Positive control: Samples with confirmed MAP3K7CL expression

  • Negative control: Samples with confirmed absence of MAP3K7CL

  • Gradient control: Sample series with varying levels of MAP3K7CL expression

• Technical controls for Western blot:

  • Loading control: Housekeeping proteins (β-actin, GAPDH, tubulin)

  • Molecular weight marker: To confirm band at expected 27.248 kDa

  • Secondary-only control: Omit primary antibody to assess secondary antibody specificity

  • Peptide competition control: Pre-incubate antibody with immunizing peptide

• Technical controls for immunostaining:

  • Isotype control: Non-specific IgG from same species as primary antibody

  • Blocking peptide control: Antibody pre-incubated with excess target peptide

  • Absorption control: Antibody pre-absorbed against recombinant protein

  • Autofluorescence control: Unstained sample to assess tissue autofluorescence

• Genetic controls:

  • siRNA/shRNA knockdown: Samples with reduced MAP3K7CL expression

  • CRISPR knockout: Samples with complete MAP3K7CL ablation

  • Overexpression: Samples with artificially elevated MAP3K7CL levels

• Specificity controls:

  • Cross-reactivity assessment: Test against related proteins (especially MAP3K7)

  • Multiple antibody validation: Use two different antibodies targeting distinct epitopes

• Application-specific controls:

  • For IP: Input, IgG control IP, flow-through samples

  • For ChIP: Input DNA, IgG ChIP control, positive control locus

Incorporating these controls enables confident interpretation of experimental results and helps distinguish between true signals and artifacts.

How can MAP3K7CL antibodies be used to investigate its potential role in cancer biology?

Based on the relationship between MAP3K7 and cancer (particularly prostate cancer) , investigating MAP3K7CL's potential role in cancer biology can be approached using these methodologies:

• Expression profiling across cancer types:

  • Use validated MAP3K7CL antibodies for tissue microarray analysis

  • Compare expression between normal tissues and corresponding tumors

  • Correlate expression with clinical parameters and patient outcomes

  • Analyze potential co-expression patterns with MAP3K7

• Subcellular localization studies:

  • Perform immunofluorescence with MAP3K7CL antibodies in cancer cell lines

  • Compare localization between normal and cancer cells

  • Assess changes in localization during cancer progression

• Functional studies in cancer models:

  • Given that MAP3K7 and CHD1 silencing affects viral susceptibility and ISG expression in prostate cancer cells

  • Investigate similar roles for MAP3K7CL using knockdown/knockout approaches

  • Assess effects on cancer cell proliferation, migration, and invasion

  • Examine potential roles in resistance to therapy

• Signaling pathway analysis:

  • Based on MAP3K7's role in TNF-α and NF-κB signaling pathways

  • Use phospho-specific antibodies to study activation of relevant pathways

  • Investigate how MAP3K7CL modulation affects these cancer-associated pathways

• Oncolytic virus therapy research:

  • Building on findings that MAP3K7 affects susceptibility to oncolytic viruses

  • Investigate whether MAP3K7CL similarly influences viral sensitivity

  • Potential implications for developing oncolytic virus therapies

• Protein-protein interaction networks:

  • Use MAP3K7CL antibodies for co-immunoprecipitation followed by mass spectrometry

  • Map cancer-specific interaction partners

  • Compare with MAP3K7 interactome in cancer cells

These approaches can help elucidate whether MAP3K7CL plays significant roles in cancer biology, potentially related to or distinct from MAP3K7's established functions.

What are the best practices for multiplexed immunofluorescence including MAP3K7CL detection?

For successful multiplexed immunofluorescence incorporating MAP3K7CL antibodies, follow these methodological guidelines:

• Antibody panel design:

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

  • When using multiple rabbit antibodies (common for MAP3K7CL) , implement sequential staining with stripping steps

  • Choose fluorophores with minimal spectral overlap

  • Include markers for relevant cell types or subcellular compartments

• Sequential staining protocol:

  • First round: MAP3K7CL antibody → secondary antibody → image acquisition

  • Stripping: Glycine buffer (pH 2.5) or commercial antibody stripping buffer

  • Subsequent rounds: Next primary → secondary → image acquisition

  • Validate successful stripping between rounds

• Tyramide signal amplification (TSA) approach:

  • Allows use of multiple antibodies from same species

  • Apply MAP3K7CL antibody at high dilution

  • Use HRP-conjugated secondary and tyramide-fluorophore

  • Inactivate HRP before next antibody application

• Spectral unmixing:

  • Acquire images with spectral detector

  • Create spectral libraries for each fluorophore

  • Apply unmixing algorithms to separate overlapping signals

  • Particularly useful when studying MAP3K7CL alongside related proteins

• Controls for multiplexed detection:

  • Single-color controls for each antibody

  • Fluorescence minus one (FMO) controls

  • Isotype controls for each species

  • Absorption controls for closely related targets (MAP3K7CL vs. MAP3K7)

• Image analysis considerations:

  • Use cell segmentation algorithms

  • Quantify colocalization with appropriate statistical methods

  • Apply consistent thresholding across samples

  • Consider machine learning approaches for complex pattern recognition

Following these practices enables simultaneous detection of MAP3K7CL alongside other proteins of interest, providing valuable insights into its spatial relationships and functional associations.