TOLLIP Antibody

What is TOLLIP and what is its biological function?

TOLLIP (Toll-interacting protein) is a 30 kDa inhibitory adaptor protein that plays multiple important roles in cellular pathways. It functions as a negative regulator of the IL-1R and Toll-like receptors (TLRs) signaling pathway, directly impacting inflammatory immune responses. TOLLIP inhibits IRAK1 phosphorylation and kinase activity, effectively dampening inflammatory signaling . Beyond immune regulation, TOLLIP connects the ubiquitin pathway to autophagy by functioning as a ubiquitin-ATG8 family adapter, mediating autophagic clearance of ubiquitin conjugates. This TOLLIP-dependent selective autophagy pathway plays a significant role in clearing cytotoxic polyQ protein aggregates, which has implications for neurodegenerative diseases . TOLLIP also participates in early endosomal trafficking of ubiquitinated proteins in complex with TOM1 and binds to phosphatidylinositol 3-phosphate (PtdIns(3)P) .

What applications can TOLLIP antibodies be used for?

TOLLIP antibodies are versatile tools applicable to multiple experimental techniques as shown in the following table:

These applications enable researchers to study TOLLIP expression, localization, and functional interactions in various experimental contexts .

What samples and cell lines show positive reactivity with TOLLIP antibodies?

TOLLIP antibodies have demonstrated positive reactivity in diverse biological samples, making them suitable for cross-species research applications:

For immunohistochemistry, 11315-1-AP antibody shows positive detection in human gliomas tissue, with recommended antigen retrieval using TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 .

How should TOLLIP antibodies be stored for optimal stability?

Proper storage of TOLLIP antibodies is crucial for maintaining their reactivity and specificity. Based on manufacturer recommendations, TOLLIP antibodies should be stored at -20°C, where they remain stable for one year after shipment . The antibodies are typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. Aliquoting is generally unnecessary for -20°C storage, simplifying laboratory handling procedures. Some preparations (20 μl sizes) contain 0.1% BSA as a stabilizing agent . Following these storage guidelines ensures antibody integrity for reproducible experimental results.

What are the optimal conditions for Western Blot detection of TOLLIP?

For optimal Western Blot detection of TOLLIP, researchers should follow these methodological guidelines:

• Antibody selection and dilution: Use antibodies like 11315-1-AP at 1:5000-1:50000 dilution or #4748 at 1:1000 dilution .

• Expected molecular weight: Look for a band at approximately 30-33 kDa, which is the observed molecular weight of TOLLIP protein .

• Sample preparation: TOLLIP has been successfully detected in various samples including cell lysates (HepG2, HEK-293, PC-3) and tissue extracts (human liver, mouse/rat brain) .

• Blocking and antibody incubation: Follow standard Western Blot protocols as provided by manufacturers. For example, Proteintech offers specific WB protocols for their TOLLIP antibodies that can be downloaded from their website .

• Optimization recommendations: As stated in the product information, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" as outcomes may be sample-dependent .

For consistent results, always include appropriate positive controls such as lysates from HepG2 or HEK-293 cells, which have been verified to express detectable levels of TOLLIP .

How can researchers validate the specificity of a TOLLIP antibody?

Validating antibody specificity is essential for reliable research outcomes. For TOLLIP antibodies, consider these validation approaches:

• Knockout/knockdown controls: Several publications have utilized TOLLIP knockdown/knockout approaches to validate antibody specificity. Both antibody 11315-1-AP and others have been cited in KD/KO validation studies .

• Multiple antibody comparison: Compare results from different antibodies targeting distinct epitopes of TOLLIP. The search results mention multiple antibodies (11315-1-AP, 84711-5-RR, ab37155, #4748) that could be used for cross-validation .

• Recombinant protein controls: Use TOLLIP recombinant proteins as positive controls. The TOLLIP fusion protein Ag1864 is mentioned as an immunogen and could serve as a control .

• Epitope analysis: Confirm that the observed molecular weight matches the calculated weight (30 kDa for TOLLIP) .

• Cross-species reactivity: Test the antibody in samples from different species to confirm consistent detection patterns. The antibodies show reactivity with human, mouse, and rat samples, which provides an opportunity for cross-species validation .

Documentation of these validation steps strengthens the reliability of experimental findings and should be included in publications involving TOLLIP antibody usage.

What is the best approach for immunohistochemical detection of TOLLIP?

For optimal immunohistochemical (IHC) detection of TOLLIP, researchers should follow these methodological recommendations:

• Antibody selection: The 11315-1-AP antibody has been validated for IHC applications with a recommended dilution of 1:50-1:500 .

• Antigen retrieval method: For human gliomas tissue, suggested antigen retrieval should be performed with TE buffer pH 9.0. Alternatively, citrate buffer pH 6.0 can be used, though possibly with different efficacy .

• Tissue preparation: Proper fixation and processing of tissues is crucial. Based on successful detection in human gliomas tissue, standard formalin fixation and paraffin embedding protocols are suitable .

• Detection system: While not explicitly stated in the search results, standard detection systems compatible with rabbit polyclonal antibodies would be appropriate, such as biotin-streptavidin HRP or polymer-based detection systems.

• Protocol resources: Proteintech offers specific IHC protocols for their TOLLIP antibody (11315-1-AP) that can be downloaded from their website for detailed methodology .

For robust experimental design, include appropriate positive control tissues that are known to express TOLLIP, such as human liver tissue, which has been validated for Western Blot applications and likely expresses detectable levels for IHC as well .

How can TOLLIP antibodies be utilized to study autophagy pathways?

TOLLIP antibodies are valuable tools for investigating autophagy mechanisms due to TOLLIP's crucial role as a mediator between ubiquitin and autophagy pathways. Research applications include:

• Selective autophagy studies: TOLLIP functions as a ubiquitin-ATG8 family adapter that mediates autophagic clearance of ubiquitin conjugates. Antibodies can help visualize and quantify this process through co-localization studies with autophagy markers .

• Polyglutamine (polyQ) protein aggregate clearance: The TOLLIP-dependent selective autophagy pathway is particularly important in clearing cytotoxic polyQ protein aggregates. Researchers can use TOLLIP antibodies to study neurodegenerative disease mechanisms involving these aggregates .

• Co-immunoprecipitation experiments: TOLLIP antibodies can be used for IP to identify interaction partners in autophagy pathways. Publications have cited the use of TOLLIP antibodies for IP applications .

• Quantification of autophagy flux: By monitoring TOLLIP levels and localization during autophagy induction or inhibition, researchers can gain insights into regulatory mechanisms of selective autophagy.

• Disease model applications: Recent publications highlight TOLLIP's role in conditions like triple-negative breast cancer progression through autophagy mechanisms, suggesting applications in cancer research .

This approach has been demonstrated in research exploring SARS-CoV-2 infection, where TOLLIP mediates selective autophagy in disease pathogenesis .

What role does TOLLIP play in inflammatory signaling pathways that can be studied with these antibodies?

TOLLIP antibodies enable detailed investigation of inflammatory pathway regulation through several mechanistic approaches:

• IL-1R and TLR signaling inhibition: TOLLIP is a negative regulator of IL-1R and Toll-like receptors signaling. Antibodies can be used to study how TOLLIP modulates these pathways through immunoprecipitation and co-localization studies .

• IRAK1 interaction and inhibition: TOLLIP inhibits IRAK1 phosphorylation and kinase activity, a critical step in inflammatory signaling. Researchers can use antibodies to study this interaction through Western blotting, IP, and proximity ligation assays .

• NF-κB pathway regulation: Overexpression of TOLLIP results in impaired NF-κB signaling. Antibodies can help quantify how varying TOLLIP levels affect downstream NF-κB activation and inflammatory gene expression .

• Adaptor complex formation: TOLLIP associates with the IRAK complex following IL-1 stimulation. Antibodies can track the dynamics of this complex formation through time-course experiments and co-immunoprecipitation .

• Differential response in knockout models: Studies of Tollip-deficient mice suggest it plays a role in regulating inflammatory cytokines in response to IL-1 and LPS. Antibodies can confirm knockout efficacy and study compensatory mechanisms .

These approaches have been utilized in studying viral infections, such as African swine fever virus, where TOLLIP has been shown to mediate the autophagic degradation of IKKα and IKKβ .

How can TOLLIP antibodies help investigate viral infection mechanisms?

TOLLIP antibodies provide valuable insights into viral infection mechanisms as evidenced by recent publications:

• Viral immune evasion studies: Several viruses target TOLLIP to evade host immune responses. For example, African swine fever virus proteins MGF300-2R and L83L have been shown to manipulate TOLLIP to promote autophagic degradation of immune signaling components. TOLLIP antibodies can be used to track these interactions and visualize subcellular localization changes during infection .

• SARS-CoV-2 infection research: Studies have revealed that suppression of ACE2 SUMOylation protects against SARS-CoV-2 infection through TOLLIP-mediated selective autophagy. Antibodies enable researchers to monitor this protective mechanism through Western blotting and immunofluorescence approaches .

• Innate immune signaling modulation: Viruses frequently target innate immune pathways. TOLLIP's role in TLR and IL-1R signaling makes it a critical regulatory point that viruses may manipulate. For instance, the L83L protein of African swine fever virus negatively regulates the cGAS-STING-mediated IFN-I pathway by recruiting TOLLIP to promote STING autophagic degradation .

• Autophagy manipulation by pathogens: Many viruses subvert autophagy for their benefit. TOLLIP antibodies can be used to study how viral proteins interact with TOLLIP to redirect autophagy machinery toward degrading antiviral factors rather than viral components .

These applications demonstrate the utility of TOLLIP antibodies in uncovering novel virus-host interactions and potential therapeutic targets for viral infections.

How should researchers interpret multiple bands in Western Blot when using TOLLIP antibodies?

When encountering multiple bands in Western Blot experiments with TOLLIP antibodies, consider the following interpretative framework:

• Expected band size: The primary TOLLIP band should appear at approximately 30-33 kDa, which is both the calculated and observed molecular weight reported across multiple antibodies .

• Possible causes of multiple bands:

  • Post-translational modifications: TOLLIP may undergo phosphorylation, ubiquitination, or other modifications that alter its migration pattern.

  • Splice variants: Alternative splicing might generate TOLLIP isoforms of different sizes.

  • Degradation products: Improper sample handling or storage may result in protein degradation and appearance of lower molecular weight bands.

  • Non-specific binding: Some antibodies may cross-react with structurally similar proteins.

• Validation approaches:

  • Positive controls: Compare band patterns with validated positive controls such as HepG2 cells, HEK-293 cells, or human liver tissue .

  • Knockout/knockdown validation: Use TOLLIP knockout or knockdown samples to identify which bands are specific to TOLLIP. Publications citing TOLLIP antibodies in KD/KO experiments can provide guidance .

  • Alternative antibodies: Test multiple antibodies targeting different epitopes of TOLLIP to confirm band specificity .

  • Blocking peptide competition: For peptide-raised antibodies like ab37155, using the immunizing peptide to block antibody binding can help identify specific bands .

• Documentation best practices: When reporting results, clearly indicate which band(s) were considered for quantification and provide rationale based on molecular weight and validation experiments.

What approaches can resolve contradictory results when using different TOLLIP antibodies?

When faced with contradictory results from different TOLLIP antibodies, implement these systematic resolution strategies:

• Epitope mapping comparison: Different antibodies target distinct epitopes of TOLLIP, which may be differentially accessible based on protein conformation, interactions, or modifications. Comparing the immunogens used (e.g., TOLLIP fusion protein Ag1864 vs. synthetic peptides) may explain discrepancies.

• Antibody class and specificity assessment: Compare results between polyclonal (11315-1-AP, ab37155) and recombinant (84711-5-RR) antibodies. Polyclonal antibodies recognize multiple epitopes and may detect TOLLIP in various conformational states, while recombinant antibodies offer higher specificity but might miss certain protein states .

• Validation using genetic approaches:

  • Employ TOLLIP knockout/knockdown controls to definitively identify specific signals

  • Implement TOLLIP overexpression systems to confirm antibody sensitivity

  • Reference the multiple publications citing KD/KO validation of TOLLIP antibodies

• Application-specific optimization: Different antibodies may perform optimally in distinct applications. For example, some may excel in Western blot but underperform in IHC or IF. Select antibodies specifically validated for your application of interest .

• Sample preparation effects: Variations in sample preparation (lysis buffers, fixation methods, antigen retrieval) can affect epitope accessibility. Standardize these protocols when comparing antibodies .

• Biological context consideration: TOLLIP's localization, interactions, and modifications may vary across cell types and physiological conditions, affecting antibody recognition. Consider using multiple cell types including validated positive samples like HepG2, HEK-293, or brain tissues .

By systematically implementing these approaches, researchers can resolve contradictions and establish reliable TOLLIP detection protocols for their specific experimental systems.

What are the best practices for quantifying TOLLIP expression levels?

For accurate quantification of TOLLIP expression, researchers should implement these methodological best practices:

• Optimal antibody selection and dilution:

  • For Western Blot: Use validated antibodies at appropriate dilutions (e.g., 11315-1-AP at 1:5000-1:50000 or #4748 at 1:1000)

  • For Flow Cytometry: Consider 84711-5-RR at 0.25 μg per 10^6 cells for accurate quantification of intracellular TOLLIP levels

• Loading control normalization: Always normalize TOLLIP signals to appropriate loading controls:

  • For total protein: Housekeeping proteins like β-actin, GAPDH, or tubulin

  • For subcellular fractions: Compartment-specific markers (e.g., HDAC1 for nuclear, VDAC for mitochondrial)

• Standard curve calibration: For absolute quantification, include a standard curve using recombinant TOLLIP protein at known concentrations.

• Multiple detection methods: Combine complementary techniques:

  • Western Blot: For total protein levels and molecular weight confirmation

  • Flow Cytometry: For single-cell quantification and population heterogeneity assessment

  • IF/ICC: For subcellular localization and spatial distribution analysis

• Biological replicate considerations: TOLLIP expression may vary across:

  • Cell types: Include validated positive samples like HepG2, HEK-293, or tissue samples

  • Physiological conditions: Consider standardizing culture conditions and stimulation protocols

  • Species differences: Be aware that antibody affinity may vary across human, mouse, and rat samples

• Dynamic range assessment: Ensure measurements fall within the linear range of detection by testing serial dilutions of samples.

• Image analysis recommendations: For Western Blot or IF quantification:

  • Use appropriate software (ImageJ, CellProfiler, etc.)

  • Subtract background signal

  • Avoid saturated signals

  • Define consistent region-of-interest selection criteria

By adhering to these quantification guidelines, researchers can generate reproducible and biologically meaningful data on TOLLIP expression levels across experimental conditions.