TMOD1 Antibody, HRP conjugated

What is TMOD1 and what are its key cellular functions?

Tropomodulin-1 (TMOD1) is an actin-capping protein that regulates actin filament dynamics by capping the pointed (slow-growing) end of actin filaments. TMOD1 functions by:

• Binding to tropomyosin at its N-terminus

• Inhibiting depolymerization and elongation of actin filaments

• Regulating actin cytoskeleton organization

• Influencing cell membrane skeleton structure, particularly in erythrocytes

TMOD1 plays critical roles in multiple cellular processes including:

• Regulating inflammatory responses in macrophages by modulating TLR4 endocytosis

• Controlling dendritic cell maturation and immune functions

• Influencing cell motility and proliferation in cancer cells

• Maintaining cardiac muscle structure and function

Expression of TMOD1 varies across tissues, with notable presence in erythrocytes, cardiomyocytes, lens fiber cells, neurons, and immune cells like monocytes and dendritic cells .

How should researchers select the appropriate TMOD1 antibody for their specific experimental needs?

Selection should be based on:

1. Application compatibility: Verify the antibody has been validated for your intended application. For instance:

2. Species reactivity: Confirm reactivity with your experimental model. Available antibodies show reactivity with:

• Human TMOD1

• Mouse TMOD1

• Rat TMOD1

3. Immunogen information: Review the specific region of TMOD1 used as immunogen:

• Recombinant Human Tropomodulin-1 protein (105-359 AA)

• Synthesized peptide derived from human TMOD1, corresponding to amino acid residues E31-R81

• TMOD1 fusion protein Ag0192

This information is crucial when studying specific domains of TMOD1, particularly when investigating mutations like p.R189W that affect actin filament regulation .

What are the optimal protocols for using TMOD1 Antibody, HRP conjugated in cellular and molecular studies?

ELISA Applications:

• Coating: Immobilize the target antigen (recombinant TMOD1 or cell/tissue lysate) on the plate

• Blocking: Use appropriate blocking buffer (typically BSA-based) to reduce non-specific binding

• Primary antibody: Apply TMOD1 Antibody, HRP conjugated at manufacturer-recommended dilutions

• Detection: Add substrate (TMB for HRP) and measure colorimetric signal

• Controls: Include negative controls (no primary antibody) and positive controls (known TMOD1-positive samples)

Optimal sample preparation for detecting endogenous TMOD1:

• For tissues with high TMOD1 expression (brain, heart, skeletal muscle), standard lysis buffers (RIPA) are effective

• For immune cells (macrophages, dendritic cells), consider isolating BMDMs or Raw264.7 cells and treating with LPS (100 ng/mL or 1 μg/mL) to induce TMOD1 expression changes

• For cell types with lower expression, consider enrichment techniques before analysis

Validation approaches:

• Perform knockdown experiments (as demonstrated with TMOD1 shRNAs in cervical cancer cell lines)

• Include rescue experiments by re-expressing TMOD1 in knockdown models

• Use adenovirus-mediated overexpression (Ad-TMOD1) as a positive control

How can researchers effectively measure changes in TMOD1 expression during immune cell activation?

Based on research involving dendritic cells and macrophages :

Experimental approach:

• Cell preparation:

  • Isolate bone marrow cells from mice and differentiate them into immature DCs using IL-4 and GM-CSF

  • Alternatively, use established cell lines like Raw264.7 for macrophage studies

• Activation protocol:

  • Treat immature DCs or macrophages with LPS (100 ng/mL) for specific time points:

    • For protein expression: 2, 4, 6, 24, and 48 hours

    • For signaling pathways: 5, 15, 30 minutes

  • Compare with unstimulated controls

• Expression analysis:

  • qPCR for mRNA expression (shows ~4-fold upregulation in mature DCs)

  • Western blotting for protein expression

  • Flow cytometry for surface marker correlation

• Experimental controls:

  • Use different TLR agonists (Pam3csk4, poly(I:C), CpG ODN) to compare TMOD1 response specificity

  • Include genetic models (TMOD1+/+ vs. TOT/TMOD1-/-) to confirm specificity

Expected results:

• TMOD1 expression increases significantly upon DC maturation

• Expression correlates with activation of MyD88-dependent pathway and inhibition of TRIF-dependent pathway

• Changes in TMOD1 affect inflammatory cytokine production (TNF-α, IL-6, IFN-β)

How does TMOD1 regulate TLR4 endocytosis and inflammatory responses in macrophages?

TMOD1 serves as a critical regulator of inflammatory responses in macrophages through several mechanisms:

1. Regulation of TLR4 endocytosis:

• TMOD1 inhibits LPS-induced TLR4 endocytosis and intracellular trafficking

• This leads to increased surface TLR4 and prolonged signaling

• TMOD1 deficiency promotes TLR4 endocytosis, reducing surface TLR4 levels

2. Differential regulation of TLR4 signaling pathways:

• TMOD1 enhances MyD88-dependent pathway activation:

  • Increased NF-κB and MAPK activation

  • Enhanced production of inflammatory cytokines (TNF-α, IL-6)

• TMOD1 suppresses TRIF-dependent pathway:

  • Reduced IRF3 phosphorylation

  • Decreased type I interferon (IFN-β) production

3. Molecular mechanism:

• TMOD1 modulates the CD14/Syk/PLCγ2/IP3/Ca2+ signaling pathway

• Affects actin cytoskeleton reorganization necessary for receptor-mediated endocytosis

• Regulates membrane tension, which influences endocytic processes

4. Physiological significance:

• TMOD1-deficient macrophages showed reduced inflammatory response in LPS-induced acute lung injury model

• This suggests TMOD1 as a potential therapeutic target for excessive inflammation and sepsis

What is the relationship between TMOD1 expression and cancer progression?

Research indicates complex and context-dependent roles for TMOD1 in cancer:

TMOD1 as a potential tumor suppressor:

• In cervical cancer:

  • Downregulation of TMOD1 promoted cell motility and proliferation

  • TMOD1 knockdown enhanced G1/S phase transition in HeLa and CaSki cells

  • High TMOD1 expression associated with good pathological status in patients with cervical cancer

  • These findings suggest TMOD1 may act as a tumor suppressor in this context

Examples of TMOD1 expression in different cervical cancer stages:

TMOD1 with potential oncogenic functions:

• Has been reported to enhance regional lymph node metastasis in human oral cancer

• May promote breast cancer development via the NF-κB-TMOD1-β-catenin-MMP13 axis

• TMOD1 loci identified as a potential risk loci in esophageal adenocarcinoma

Reconciling contradictory findings:

• TMOD1's role appears to be cancer type-specific and stage-dependent

• Its function in actin dynamics may have different consequences depending on the cellular context

• Researchers should carefully consider cancer type, stage, and molecular subtype when investigating TMOD1

How can researchers design experiments to investigate the mechanistic relationship between TMOD1 and actin dynamics?

Recommended experimental approaches:

• TMOD1 manipulation strategies:

  • Genetic approaches:

    • Knockdown using shRNAs as demonstrated in cervical cancer studies

    • Knockout models (TOT/TMOD1-/- mice)

    • Rescue experiments with wild-type or mutant TMOD1

  • Pharmacological approaches:

    • Actin-modulating compounds like PIC (75 μM) or JASP (200 nM) for 30 min prior to further treatments

• Actin dynamics assessment:

  • F-actin quantification in cells with modified TMOD1 expression

  • Live-cell imaging of actin filament formation using fluorescent actin probes

  • Biochemical assays measuring:

    • Actin polymerization/depolymerization rates

    • Pointed-end capping activity

    • Actin filament length distribution

• Functional readouts:

  • Cell migration assays (random and chemotactic migration)

  • Membrane dynamics (endocytosis rates, receptor trafficking)

  • Cell mechanical properties (stiffness measurements)

  • Inflammatory response metrics (cytokine production)

• Advanced techniques for mechanistic insights:

  • Structure-function analysis using TMOD1 mutations (e.g., p.R189W mutation affects actin filament length regulation)

  • Protein-protein interaction studies with actin and tropomyosin

  • Subcellular localization of TMOD1 during dynamic cellular processes

What are the critical considerations when interpreting contradictory findings about TMOD1's role in different experimental systems?

When encountering contradictory data regarding TMOD1 function, researchers should consider:

1. Cellular context differences:

• TMOD1 functions differently in various cell types:

  • In immune cells, it regulates inflammatory responses

  • In cancer cells, it affects proliferation and motility differently across cancer types

  • In cardiomyocytes, mutations can cause cardiomyopathy

2. Experimental approach variations:

• Expression level considerations:

  • Complete knockout vs. partial knockdown

  • Overexpression levels (physiological vs. supraphysiological)

• Temporal aspects:

  • Acute vs. chronic modifications

  • Developmental timing of manipulation

3. Readout specificity:

• Direct vs. indirect effects:

  • Primary effects on actin dynamics

  • Secondary consequences on signaling pathways

  • Compensatory mechanisms in chronic models

4. Technical considerations:

• Antibody specificity and validation

• Detection method sensitivity

• Sample preparation differences

• Statistical power and biological replicates

5. Integration strategies:

• Combine multiple methodological approaches

• Validate key findings across different model systems

• Consider systems biology approaches to model complex interactions

• Directly test competing hypotheses in the same experimental system

For example, when reconciling contradictory findings regarding TMOD1's role in signaling, researchers should examine both MyD88-dependent and TRIF-dependent pathways simultaneously, as TMOD1 has been shown to differentially regulate these interconnected pathways .

How might TMOD1 be exploited as a therapeutic target in inflammatory and immune-related disorders?

The emerging role of TMOD1 in immune regulation suggests several therapeutic possibilities:

1. Anti-inflammatory applications:

• TMOD1 inhibition strategies:

  • Macrophages deficient in TMOD1 showed reduced inflammatory response in LPS-induced acute lung injury model

  • This suggests TMOD1 inhibitors might reduce excessive inflammation in sepsis and acute inflammatory conditions

  • Targeting TMOD1 could modulate TLR4-mediated inflammatory responses without completely blocking pathogen recognition

2. Immune tolerance induction:

• TMOD1 inhibition in dendritic cells:

  • TMOD1-deficient mDCs secreted high levels of IFN-β and IL-10

  • These cells induced immune tolerance in an experimental autoimmune encephalomyelitis (EAE) mouse model

  • Potential applications in autoimmune disease treatment

3. Cancer immunotherapy approaches:

• Context-dependent strategies:

  • In cancers where TMOD1 acts as a tumor suppressor (e.g., cervical cancer), enhancing its expression might be beneficial

  • In contexts where TMOD1 promotes metastasis, inhibition might be preferred

  • Modulating TMOD1 in tumor-associated macrophages could reshape the tumor microenvironment

4. Technical challenges to address:

• Achieving cell-type specificity in TMOD1 targeting

• Balancing actin dynamics without disrupting essential cellular functions

• Developing high-specificity modulators of TMOD1 function

What experimental approaches would best determine the role of TMOD1 mutations in cardiomyopathy pathogenesis?

Recent identification of the TMOD1 p.R189W mutation in childhood cardiomyopathy suggests several research directions:

1. Structural and functional protein analysis:

• Biochemical characterization of mutant TMOD1:

  • Actin filament pointed-end capping activity

  • Tropomyosin binding affinity

  • Protein stability and folding properties

• Structural modeling:

  • Analysis of potential defects in local protein folding

  • Evaluation of actin binding interfaces

2. Cellular models:

• Cardiomyocyte-specific effects:

  • Expression of GFP-TMOD1 (wild-type vs. R189W) in cardiomyocytes

  • Assessment of thin filament length regulation

  • Sarcomere organization analysis

  • Contractile function measurement

• iPSC-derived cardiomyocytes from affected patients

3. Animal models:

• Generation of knock-in mouse models with the R189W mutation

• Comparative analysis with existing TMOD1 models:

  • Complete knockout (embryonic lethal)

  • Heterozygous models

  • Cardiac-specific conditional models

4. Clinical correlation studies:

• Detailed phenotype-genotype analysis of patients with TMOD1 mutations

• Long-term follow-up to determine disease progression

• Screening for TMOD1 variants in larger cohorts of childhood cardiomyopathy

5. Therapeutic testing:

• Evaluation of actin-stabilizing compounds

• Gene therapy approaches to restore normal TMOD1 function

• Small molecule screens to identify compounds that might correct mutant TMOD1 function

By integrating these approaches, researchers can develop a comprehensive understanding of how TMOD1 mutations lead to cardiomyopathy and potentially identify therapeutic interventions.

What are the optimal storage and handling conditions for maintaining TMOD1 antibody, HRP conjugated activity?

For maximum stability and performance:

Storage recommendations:

• Store at -20°C upon receipt

• Avoid repeated freeze-thaw cycles by preparing small aliquots

• Some formulations may be stored at -80°C for longer-term storage

• Product-specific storage buffers typically include:

  • 50% Glycerol

  • 0.01M PBS, pH 7.4

  • 0.03% Proclin 300 as preservative

Working solution preparation:

• Thaw aliquots completely before use

• Mix gently to ensure homogeneity without foaming

• Return unused portion to -20°C immediately after use

• Do not store diluted antibody solutions for extended periods

Stability considerations:

• HRP conjugation may decrease stability compared to unconjugated antibodies

• Protect from light during storage and handling

• Monitor pH changes that might affect HRP activity

• Consider adding stabilizing proteins for diluted working solutions

How should researchers validate and troubleshoot TMOD1 antibody specificity in their experimental systems?

Validation approaches:

• Positive and negative control samples:

  • Positive controls:

    • Mouse brain tissue, heart, skeletal muscle (high TMOD1 expression)

    • Adenovirus-mediated TMOD1 overexpression samples

  • Negative controls:

    • TMOD1 knockout or knockdown samples

    • Tissues known to express minimal TMOD1

• Antigen pre-absorption test:

  • Pre-incubate antibody with purified recombinant TMOD1 protein

  • Compare staining/signal with and without pre-absorption

  • Specific signal should be significantly reduced after pre-absorption

• Multiple antibody verification:

  • Use antibodies targeting different epitopes of TMOD1

  • Compare staining patterns to confirm consistency

  • Consider antibodies raised in different host species

Troubleshooting guidance: