TICAM2 Antibody

What is TICAM2 and what are its primary functions in immune signaling?

TICAM2 (Toll-Like Receptor Adaptor Molecule 2), also known as TRAM, TIRP, or TIRAP3, functions as a critical sorting adapter in various signaling pathways, particularly in Toll-like receptor (TLR) signaling. Its primary functions include:

• Physically bridging TLR4 and TICAM1 (TRIF) in endosomes following TLR4 endocytosis

• Facilitating the transmission of lipopolysaccharide (LPS) signaling from TLR4 to TICAM1

• Regulating the MyD88-independent pathway during innate immune responses to LPS

• Activating interferon regulatory factor 3 (IRF-3), leading to type I interferon production

• Participating in IL-1 and IL-18 signaling pathways by functioning upstream of IRAK1, IRAK2, TRAF6, and IKBKB

• Forming complexes with RAB11FIP2 to promote phagocytosis of Gram-negative bacteria through activation of actin-regulatory GTPases RAC1 and CDC42

TICAM2's proper functioning ensures effective immune responses against pathogens by linking innate and adaptive immunity mechanisms.

What criteria should researchers consider when selecting a TICAM2 antibody for their specific application?

When selecting a TICAM2 antibody, researchers should evaluate:

• Target species reactivity: Confirm the antibody reacts with your species of interest. Many TICAM2 antibodies are validated for human, mouse, and rat samples , but cross-reactivity varies between products.

• Application validation: Verify the antibody is validated for your specific application (WB, IHC, IF, ELISA, Flow Cytometry). For example:

  • For Western blotting: Antibodies like AF4348 detect TICAM2 at approximately 31 kDa

  • For immunohistochemistry: Products like 19524-1-AP are validated for specific tissues with recommended dilutions (1:20-1:200)

• Epitope location: Some antibodies target specific regions:

  • C-terminal targeted antibodies (ABIN6990461)

  • N-terminal targeted antibodies

  • Full-length protein recognition (antibodies raised against recombinant proteins covering amino acids 1-235)

• Clonality:

  • Polyclonal antibodies offer broader epitope recognition

  • Monoclonal antibodies provide higher specificity and batch consistency

  • Recombinant monoclonal antibodies ensure unrivaled batch-to-batch consistency and future supply security

• Host species: Consider secondary antibody compatibility based on the host (rabbit, mouse, goat) to avoid cross-reactivity in multi-labeling experiments .

How should researchers optimize Western blot protocols for TICAM2 detection?

For optimal Western blot detection of TICAM2:

• Sample preparation:

  • Use appropriate lysis buffers (e.g., Immunoblot Buffer Group 2 has been validated for TICAM2 detection)

  • Include protease inhibitors to prevent degradation

  • For cellular samples, validated cell lines include Raji human Burkitt's lymphoma, C2C12 mouse myoblast, and NRK rat normal kidney cell lines

• Protein loading and separation:

  • Load 25μg protein per lane for cellular lysates

  • Use PVDF membrane for optimal protein binding

  • The observed molecular weight of TICAM2 is approximately 31-32 kDa, which is slightly higher than the calculated 27 kDa

• Antibody dilutions and incubation:

  • Primary antibody: Optimal dilutions range from 1:500-1:2000 depending on the specific antibody

  • Example protocol: Incubate with 1 μg/mL of TICAM2 antibody followed by HRP-conjugated secondary antibody

  • For Proteintech's 19524-1-AP: Use dilution 1:500-1:1000

  • For Novus Biologicals' NBP224638: Use dilution 0.5-2 μg/ml

• Detection systems:

  • Both chemiluminescence and fluorescence-based detection systems are suitable

  • Confirm appropriate secondary antibody selection based on host species (anti-goat, anti-rabbit, etc.)

• Controls:

  • Include positive controls from validated cell lines

  • Consider using recombinant TICAM2 protein as a positive control

What are the optimal conditions for immunohistochemical detection of TICAM2 in tissue samples?

For successful immunohistochemical detection of TICAM2:

• Tissue preparation and fixation:

  • Paraffin-embedded sections are suitable for most TICAM2 antibodies

  • Fixed cell preparations can be used for immunocytochemistry applications

• Antigen retrieval:

  • For Proteintech's antibody 19524-1-AP: Use TE buffer pH 9.0 for optimal results

  • Alternative method: Citrate buffer pH 6.0 can also be effective

• Antibody dilutions and incubation:

  • For Proteintech's 19524-1-AP: Use dilution 1:20-1:200

  • For Novus Biologicals' NBP224638: Use 5 μg/ml

  • Incubation time: 3 hours at room temperature has been validated for some antibodies

• Detection and visualization:

  • For fluorescent detection: Secondary antibodies like NorthernLights™ 557-conjugated Anti-Goat IgG have been validated

  • Counter-staining with DAPI helps visualize nuclei in relation to TICAM2 expression

• Validated tissue types:

  • Human testis tissue and human prostate hyperplasia tissue have been validated for TICAM2 detection

  • Specific staining is typically localized to cytoplasm

• Controls:

  • Include isotype controls to assess non-specific binding

  • Use tissues known to express TICAM2 as positive controls

How can researchers effectively employ flow cytometry for TICAM2 detection in various cell populations?

For optimal flow cytometric detection of TICAM2:

• Cell preparation:

  • For intracellular staining, cells must be fixed with paraformaldehyde and permeabilized with saponin

  • Single-cell suspensions are required for accurate analysis

• Antibody selection and dilution:

  • Validated antibodies include R&D Systems' AF4348 and Santa Cruz Biotechnology's sc-376076

  • Mouse monoclonal antibodies (like clone mAbcam77169) are specifically validated for flow cytometry

• Staining protocol:

  • Primary antibody staining followed by fluorochrome-conjugated secondary antibody

  • Validated secondary antibodies include Phycoerythrin-conjugated Anti-Goat IgG (F0107) and Allophycocyanin-conjugated Anti-Goat IgG (F0108)

  • Some antibodies are available directly conjugated to fluorochromes like FITC and PE

• Controls and gating strategy:

  • Use isotype control antibodies (e.g., AB-108-C) to establish background staining levels

  • Include unstained and single-stained controls for compensation

  • Validated positive control cell lines: Raji, C2C12, and NRK cell lines

• Data analysis considerations:

  • TICAM2 shows primarily intracellular expression pattern

  • Daudi cells express low levels of TICAM2 mRNA and can serve as a negative control

  • Comparison of median fluorescence intensity rather than percent positive cells may better reflect expression level differences

What strategies should be employed when addressing non-specific binding or high background issues with TICAM2 antibodies?

When encountering non-specific binding or high background:

• Antibody validation and specificity:

  • Verify antibody specificity through knockout/knockdown controls

  • Consider using alternative antibodies targeting different epitopes of TICAM2

  • For polyclonal antibodies, antigen affinity purification (as used in Proteintech's 19524-1-AP) can reduce non-specific binding

• Blocking optimization:

  • For Western blot: Test different blocking agents (3% nonfat dry milk in TBST has been validated)

  • For IHC/ICC: Optimize blocking time and buffer composition

  • Consider adding 0.1-0.3% Triton X-100 to reduce non-specific hydrophobic interactions

• Antibody dilution and incubation conditions:

  • Further dilute primary antibody if background persists

  • Reduce incubation temperature (4°C overnight instead of room temperature)

  • Include 0.05-0.1% Tween-20 in antibody diluent to reduce non-specific binding

• Washing protocols:

  • Increase washing duration and number of washes

  • Use buffers with appropriate ionic strength (PBS or TBS with 0.05-0.1% Tween-20)

• Detection system considerations:

  • For HRP-based detection, dilute substrate solution or reduce exposure time

  • For fluorescence, minimize autofluorescence through quenching agents or spectral unmixing

How can researchers effectively distinguish between TICAM2 isoforms in experimental systems?

To distinguish between TICAM2 isoforms:

• Isoform-specific detection strategies:

  • Isoform 2 has been identified as a potential inhibitor of LPS-TLR4 signaling that interacts with isoform 1 and disrupts its association with TICAM1

  • Select antibodies targeting regions with sequence differences between isoforms

  • Antibodies targeting the C-terminus, like ABIN6990461, may detect different patterns among isoforms

• Western blot optimization:

  • Use higher resolution gel systems (gradient gels or longer running times) to separate closely sized isoforms

  • Extended running times on SDS-PAGE can help resolve small molecular weight differences

  • Different isoforms may show subtle variations from the observed 31-32 kDa typical for TICAM2

• PCR-based approaches:

  • Design isoform-specific primers for RT-PCR to quantify relative expression levels

  • Consider qPCR with isoform-specific probes for accurate quantification

• Co-immunoprecipitation strategies:

  • Use antibodies that preferentially recognize specific isoforms for pull-down experiments

  • Follow with Western blot analysis using a different TICAM2 antibody to confirm identity

• Functional validation:

  • Create isoform-specific expression constructs to study their differential roles

  • The E87/D88/D89 acidic motif in TICAM-2 provides interaction surfaces between TICAM-2 and TICAM-1, which may differ between isoforms

  • The D91/E92 motif guides membrane localization and self-activation for signaling

What considerations are important when designing experiments to study TICAM2 phosphorylation or other post-translational modifications?

For studying TICAM2 post-translational modifications:

• Sample preparation to preserve modifications:

  • Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate) in lysis buffers

  • Use deubiquitinase inhibitors (N-ethylmaleimide) if studying ubiquitination

  • Minimize sample processing time and maintain cold temperatures

• Enrichment strategies:

  • For phosphorylation: Use phospho-protein enrichment columns or immunoprecipitation with phospho-specific antibodies

  • For ubiquitination: Use tandem ubiquitin binding entities (TUBEs) or immunoprecipitation under denaturing conditions

• Detection methods:

  • For Western blot: Use phospho-specific antibodies if available

  • Run parallel samples treated with/without phosphatase

  • Consider Phos-tag™ acrylamide gels to detect mobility shifts due to phosphorylation

  • For mass spectrometry: Use TiO₂ enrichment for phosphopeptides

• Experimental controls:

  • Include treatment conditions known to modify TICAM2 (e.g., LPS stimulation)

  • Use site-directed mutagenesis of potential modification sites as negative controls

  • Compare wild-type to D91A/E92A mutant, which fails to activate TICAM-1 despite retaining ability to pass LPS-mediated IRF3 activation

• Functional validation:

  • Correlate modifications with changes in TICAM2 localization (membrane vs. cytosolic)

  • Assess impact on protein-protein interactions, particularly with TICAM1 and TLR4

  • Evaluate effects on downstream signaling events like IRF3 activation

How should researchers interpret TICAM2 localization patterns observed in immunofluorescence studies?

When interpreting TICAM2 localization patterns:

• Normal subcellular distribution:

  • TICAM2 typically shows cytoplasmic localization as demonstrated in Raji, C2C12, and NRK cell lines

  • Distribution patterns should be analyzed in relation to membrane compartments, as TICAM2 functions at early endosomes after TLR4 endocytosis

• Colocalization analysis:

  • Consider co-staining with markers for:

    • Early endosomes (EEA1, Rab5)

    • Plasma membrane (PM marker)

    • TLR4 to assess interaction during signaling

  • Quantify colocalization using coefficient metrics (Pearson's, Mander's)

  • D91A/E92A TICAM2 mutant induces TLR4-mediated IRF3 activation with high coefficient colocalization upon LPS stimulation

• Dynamic trafficking considerations:

  • TICAM2 distribution changes upon LPS stimulation

  • Wild-type TICAM2 forms self-aggregates, while the D91A/E92A mutant distributes largely to the cytosol despite myristoylation

  • Time-course experiments after stimulation can reveal trafficking patterns

• Technical considerations for accurate interpretation:

  • Fixation method impacts observed localization patterns

  • Confocal microscopy provides better resolution of subcellular compartments than widefield

  • Validate antibody specificity for immunofluorescence applications specifically

• Functional correlation:

  • Correlate localization patterns with functional readouts (e.g., IRF3 activation)

  • Mutants like D91A/E92A show altered localization that correlates with functional changes

What experimental designs best address the role of TICAM2 in various TLR signaling pathways?

To effectively study TICAM2's role in TLR signaling:

• Genetic manipulation approaches:

  • CRISPR/Cas9 knockout of TICAM2 in relevant cell lines

  • siRNA or shRNA knockdown for transient depletion

  • Reconstitution experiments in TICAM2 knockout cells with wild-type or mutant constructs

  • Site-directed mutagenesis of key functional domains:

    • The E87/D88/D89 acidic motif that mediates TICAM1 interaction

    • The D91/E92 motif that guides membrane localization

• Stimulation protocols:

  • LPS stimulation to activate TLR4-dependent pathways

  • Time-course experiments to differentiate early (MyD88-dependent) vs. late (TICAM2-dependent) responses

  • Dose-response studies to assess sensitivity thresholds

• Readout selection:

  • IRF3 phosphorylation and nuclear translocation

  • Type I interferon production (IFNβ reporter assays or ELISA)

  • NF-κB activation (reporter assays or IκBα degradation)

  • Inflammasome activation where relevant

• Protein-protein interaction studies:

  • Co-immunoprecipitation of TICAM2 with TLR4, TICAM1, or other signaling components

  • Proximity ligation assays to visualize interactions in situ

  • FRET or BRET approaches for real-time interaction dynamics

• Comparative pathway analysis:

  • Study both MyD88-dependent and MyD88-independent pathways

  • Compare TLR4 signaling with other TLRs where TICAM2 may play different roles

  • Examine IL-18 signaling pathways where TICAM2 functions as a sorting adapter for MyD88

How can researchers effectively validate TICAM2 antibody specificity in their experimental systems?

To validate TICAM2 antibody specificity:

• Genetic validation approaches:

  • CRISPR/Cas9 knockout cell lines as negative controls

  • siRNA knockdown with multiple siRNAs targeting different regions

  • Overexpression systems with tagged TICAM2 constructs as positive controls

• Immunoblotting validation:

  • Verify single band at expected molecular weight (~31-32 kDa)

  • Compare multiple antibodies targeting different epitopes

  • Include positive control lysates from validated cell lines:

    • Raji human Burkitt's lymphoma cells

    • C2C12 mouse myoblast cells

    • NRK rat normal kidney cells

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide before application

  • Signal should be significantly reduced or eliminated

  • Particularly useful for polyclonal antibodies raised against peptide immunogens

• Cross-species validation:

  • Test in multiple species where sequence homology is known

  • The amino acid sequence used as immunogen for some antibodies shows 100% homology in human, chimpanzee, and rhesus monkey, and 93% homology in rat and mouse

• Application-specific validation:

  • For flow cytometry: Compare with isotype controls (e.g., AB-108-C)

  • For IHC: Include no-primary-antibody controls

  • For IF: Evaluate subcellular localization pattern (should be predominantly cytoplasmic)

  • Daudi cells express low levels of TICAM2 mRNA and can serve as a negative control in flow cytometry

How can TICAM2 antibodies be effectively employed in studying the relationships between TLR4 endocytosis and TICAM2-dependent signaling?

To investigate TLR4 endocytosis and TICAM2-dependent signaling:

• Co-localization studies:

  • Use multi-color immunofluorescence with markers for:

    • TLR4 (cell surface and endocytosed)

    • TICAM2

    • Early endosome markers (EEA1, Rab5)

    • Late endosome/lysosome markers (LAMP1)

  • Time-course imaging after LPS stimulation to track receptor trafficking

  • Super-resolution microscopy for precise localization analysis

• Endocytosis inhibition approaches:

  • Use pharmacological inhibitors (dynasore, chlorpromazine)

  • Express dominant-negative dynamin to block endocytosis

  • Evaluate impact on TICAM2-dependent signaling outputs (IRF3 activation, type I IFN production)

• Live-cell imaging techniques:

  • Fluorescently tagged TICAM2 constructs for dynamic trafficking studies

  • FRET-based biosensors to detect TICAM2-TLR4-TICAM1 interactions in real-time

  • Photoactivatable or photoconvertible fusion proteins to track specific protein populations

• Membrane fractionation approaches:

  • Separate plasma membrane, early endosomal, and late endosomal fractions

  • Analyze TICAM2 distribution across fractions before and after LPS stimulation

  • Compare wild-type TICAM2 with the D91A/E92A mutant, which shows altered membrane localization

• Functional validation:

  • Compare wild-type cells with those expressing mutant TICAM2 (e.g., D91A/E92A)

  • The D91A/E92A mutant maintains ability to respond to LPS but fails to self-activate, revealing two distinct steps in endosomal LPS signaling

  • Analyze downstream signaling (IRF3 activation) in relation to endocytosis kinetics

What considerations are important when using TICAM2 antibodies in multiplex immunoassays or high-throughput screening applications?

For multiplex and high-throughput applications:

• Antibody compatibility assessment:

  • Test for cross-reactivity between antibodies in multiplex panels

  • Evaluate species compatibility of primary and secondary antibodies

  • Proteintech offers matched antibody pairs specifically validated for multiplex assays:

    • MP00979-1: 84064-2-PBS capture and 84064-4-PBS detection

    • MP00979-3: 84064-2-PBS capture and 84064-1-PBS detection

• Signal optimization for high-throughput platforms:

  • Determine optimal antibody concentration to maximize signal-to-noise ratio

  • Evaluate detection limits for quantitative applications

  • Test linearity range for quantitative assessment

  • Consider conjugation-ready formats (e.g., 84064-2-PBS) for direct labeling

• Multiplexing strategies:

  • For flow cytometry: Careful fluorophore selection to avoid spectral overlap

  • For imaging: Select compatible fluorophores or chromogens

  • For bead-based assays: Validate capture and detection antibody pairs

  • Cytometric bead array (CBA) applications have been validated for some TICAM2 antibody pairs

• Validation in complex samples:

  • Test in relevant biological matrices (cell lysates, tissue homogenates)

  • Evaluate matrix effects on antibody performance

  • Include appropriate controls for each sample type

• Data analysis considerations:

  • Establish standard curves with recombinant TICAM2 protein

  • Normalize data to account for technical variations

  • Implement appropriate statistical methods for high-dimensional data analysis

How can researchers utilize TICAM2 antibodies to investigate the cross-talk between different pattern recognition receptor pathways?

To study PRR pathway cross-talk using TICAM2 antibodies:

• Co-stimulation experimental designs:

  • Combine TLR4 ligands (LPS) with ligands for other PRRs:

    • TLR2 ligands (TICAM2 bridges TLR2 and MyD88)

    • IL-1R/IL-18R activators (TICAM2 is involved in these pathways)

    • NOD-like receptors

  • Time-course stimulation to identify sequential activation patterns

  • Dose-response matrices to identify synergistic or antagonistic effects

• Protein complex analysis:

  • Immunoprecipitate TICAM2 after various stimulation conditions

  • Analyze co-precipitating proteins by mass spectrometry or Western blotting

  • Compare complex composition between different activation states

  • Proximity-based labeling techniques (BioID, APEX) to identify proximal proteins

• Signaling pathway dissection:

  • Monitor multiple downstream readouts simultaneously:

    • IRF3 activation

    • NF-κB pathway components

    • MAPK pathway activation

    • Type I IFN induction

  • Use pathway-specific inhibitors to isolate contribution of each pathway

• Genetic manipulation approaches:

  • Generate cell lines with mutations in key TICAM2 domains:

    • E87/D88/D89 acidic motif (TICAM1 interaction)

    • D91/E92 motif (membrane localization)

  • Create knockout/knockdown cells for TICAM2 and other pathway components

  • Reconstitute with wild-type or mutant constructs to assess functional rescue

• Advanced imaging techniques:

  • Multi-color confocal microscopy to visualize co-localization of TICAM2 with various PRRs

  • Live-cell imaging of signaling complex formation

  • Single-molecule localization microscopy for high-resolution spatial organization analysis