RTP4 Antibody

What is RTP4 and why is it important in immunological research?

RTP4 (Receptor Transporter Protein 4), also known as IFRG28, is a 28 kDa interferon-responsive protein that functions as a negative regulator of type I interferon (IFN-I) responses. It plays crucial roles in antimalarial and antiviral immunity, particularly in brain infection and pathology. RTP4 is significant because it binds directly to TBK1 and forms complexes with STING, MAVS, IRF3, and various TRAFs, thereby modulating immune signaling pathways critical to host defense. Studies with RTP4 knockout mice have demonstrated its importance in regulating IFN-I production, as these mice produce higher levels of IFN-I than wild-type mice after malaria parasite infection and show improved resistance to infection. RTP4's importance in brain-specific immune responses makes it a particularly valuable target for research into neuroinflammatory and neuroinfectious diseases .

What types of RTP4 antibodies are available for research purposes?

Researchers have access to several types of RTP4 antibodies optimized for different experimental applications. Commercial suppliers offer polyclonal antibodies raised in rabbits, such as those available from Sigma-Aldrich (anti-RTP4 antibody produced in rabbit, part of their Prestige Antibodies® collection) and Bioss (RTP4 Polyclonal Antibody conjugated with Cy7). These antibodies have been validated for various applications including Western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF). The antibodies target different epitopes of the RTP4 protein; for example, the Bioss antibody targets an immunogen range of 141-246/246 of the human RTP4 protein. These antibodies are typically affinity-isolated and purified using Protein A, ensuring high specificity for RTP4 detection in experimental settings .

What is the subcellular localization of RTP4 and how does this impact antibody selection?

RTP4 primarily localizes to the cell membrane and cytoplasm, where it appears to be associated with membrane-bound vesicles as demonstrated by immunofluorescence assays. This subcellular localization has important implications for antibody selection and experimental design. When selecting an RTP4 antibody, researchers should consider antibodies that have been validated for detecting membrane-associated proteins. For immunofluorescence studies, antibodies conjugated with fluorophores (such as the Cy7-conjugated antibody from Bioss) may provide better visualization of RTP4's membrane and vesicular localization. For applications requiring membrane protein extraction, specialized lysis buffers that effectively solubilize membrane proteins should be used to ensure complete extraction of RTP4. Understanding RTP4's localization also helps in designing colocalization studies with other membrane or vesicular proteins to further elucidate its functional interactions .

What are the validated applications for RTP4 antibodies?

RTP4 antibodies have been validated for multiple experimental applications crucial for investigating RTP4's roles in cellular processes. Based on available commercial antibodies, the validated applications include Western blotting (WB) with recommended dilutions of 1:300-5000, which is useful for detecting RTP4 protein expression levels and molecular weight. Immunofluorescence applications include IHC-P (immunohistochemistry-paraffin), IHC-F (immunohistochemistry-frozen), and ICC (immunocytochemistry), all with recommended dilutions of 1:50-200. These applications allow visualization of RTP4's subcellular localization and expression patterns in tissues and cells. Additionally, RTP4 antibodies have been used successfully in co-immunoprecipitation (co-IP) experiments to study protein-protein interactions, particularly with components of the innate immune signaling pathway such as TBK1, STING, MAVS, and IRF3. The selection of application should be guided by the specific research question and the validated applications listed for each specific antibody .

How should RTP4 antibodies be used in co-immunoprecipitation experiments?

For co-immunoprecipitation (co-IP) experiments investigating RTP4's interactions with other proteins, researchers should follow a structured methodology to ensure reliable results. Begin by lysing cells in a buffer that preserves protein-protein interactions (typically containing 1% NP-40 or similar mild detergent, 150 mM NaCl, 50 mM Tris pH 7.4, and protease inhibitors). When investigating interactions with TBK1, STING, or other signaling molecules, consider stimulating cells with poly(I:C) for approximately 24 hours before lysis to enhance the interactions, as demonstrated in published studies. For the co-IP procedure, use 2-5 μg of anti-RTP4 antibody or antibodies against the interacting partner (e.g., anti-TBK1) per 500-1000 μg of total protein lysate. Incubate with the antibody overnight at 4°C, followed by capturing the immunocomplexes with Protein A/G beads for 1-2 hours. After thorough washing to remove non-specific interactions, elute the complexes with SDS sample buffer and analyze by Western blotting using antibodies against the suspected interacting partners. For detecting weak interactions (such as with TRAF2 or TRAF6), consider using chemical crosslinking before lysis or adjusting salt concentrations in wash buffers .

What immunofluorescence protocols yield optimal results with RTP4 antibodies?

To achieve optimal results in immunofluorescence experiments with RTP4 antibodies, researchers should follow a carefully optimized protocol. For fixed cells (ICC): Culture cells on coverslips and fix with 4% paraformaldehyde for 15 minutes at room temperature. Permeabilize with 0.2% Triton X-100 for 10 minutes, followed by blocking in 3% BSA in PBS for 1 hour. Incubate with primary RTP4 antibody at a dilution of 1:50-1:200 (as recommended) overnight at 4°C in a humidified chamber. For tissue sections (IHC-P or IHC-F): Perform antigen retrieval (for paraffin sections) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) before blocking. Apply the RTP4 antibody at the recommended dilution and incubate overnight at 4°C. For both methods, use appropriate fluorophore-conjugated secondary antibodies if using unconjugated primary antibodies, or proceed directly to nuclear counterstaining if using directly conjugated antibodies like RTP4-Cy7. When studying RTP4's membrane localization, consider performing co-staining with membrane markers for enhanced visualization of its association with membrane-bound vesicles. Counterstain nuclei with DAPI and mount using anti-fade mounting medium to preserve fluorescence. This protocol should be optimized for specific cell types or tissues being examined .

How does RTP4 interact with TBK1 and the type I interferon signaling pathway?

RTP4 acts as a negative regulator of the type I interferon (IFN-I) signaling pathway through its direct interaction with TANK-binding kinase 1 (TBK1). Mechanistically, RTP4 binds directly to TBK1 as demonstrated by both in vivo co-immunoprecipitation and in vitro protein interaction assays using a cell-free protein expression system. While RTP4 also associates with STING, MAVS, and IRF3 in cellular contexts, direct binding has only been conclusively demonstrated with TBK1. This interaction appears to inhibit TBK1's kinase activity, thereby reducing phosphorylation of IRF3 and subsequent IFN-β transcription. Experimental evidence supporting this includes increased IFN-β promoter activity in RTP4-knockdown cells after poly(I:C) or poly(dA:dT) stimulation, and significantly elevated mRNA transcripts for IFN-β and other ISGs (including ISG56, Rantes, and ISG15) in RTP4-knockout cells. Furthermore, RTP4 can reverse TBK1-mediated inhibition of viral replication, as demonstrated in VSV-eGFP replication assays. When designing experiments to study this interaction, researchers should consider using both overexpression systems and genetic knockout approaches, along with stimuli that activate the pathway such as poly(I:C), cGAMP, or viral infection .

What experimental design best examines RTP4's role in neurological infections?

To effectively examine RTP4's role in neurological infections, researchers should implement a comprehensive experimental design that incorporates both in vitro and in vivo approaches. In vitro studies should include: (1) Comparison of viral replication in RTP4-knockout versus wild-type neuronal cells or microglial cells using neurotropic viruses like West Nile virus (WNV); (2) Analysis of IFN-I responses in these cellular models following infection; and (3) Rescue experiments reintroducing RTP4 to confirm phenotype specificity. For in vivo studies, utilize RTP4-knockout mice challenged with neurotropic pathogens (such as WNV or Plasmodium berghei ANKA for cerebral malaria models). Key parameters to measure include: viral or parasite load in the brain versus peripheral tissues, IFN-α/β levels in serum and brain tissue homogenates, expression of ISGs in brain tissue, microglial activation status, neuroinflammation markers, blood-brain barrier integrity, and behavioral/neurological symptoms. Time-course studies are crucial as published research shows differential effects at different infection stages (e.g., higher serum IFN-α/β in RTP4-knockout mice at day 1 post-infection but not at day 4). Additionally, consider cell-specific conditional knockout models to distinguish the role of RTP4 in resident brain cells versus infiltrating immune cells. This approach has successfully demonstrated RTP4's specific impact on neurological infections, as evidenced by reduced WNV titers in the brains, but not hearts or spleens, of RTP4-knockout mice .

How can researchers investigate the differential effects of RTP4 in various tissues?

To investigate the differential effects of RTP4 across various tissues, researchers should employ a multi-faceted approach that integrates tissue-specific analysis techniques. Begin with tissue expression profiling using quantitative PCR and Western blotting to establish baseline RTP4 expression patterns across different tissues under both normal and stimulated (e.g., IFN-treated) conditions. For in vivo studies, utilize global RTP4-knockout mice infected with pathogens that affect multiple organs (such as West Nile virus) and compare viral loads, immune responses, and pathology across different tissues (brain, heart, spleen, liver, etc.) at various time points post-infection. Published data indicates tissue-specific effects of RTP4, with significant differences in viral loads observed in the brain but not in the heart or spleen of RTP4-knockout mice. To further dissect these tissue-specific roles, develop conditional knockout models using tissue-specific promoters (e.g., Nes-Cre for neural cells or Alb-Cre for hepatocytes) to delete RTP4 in specific cell types. Complement in vivo studies with ex vivo organ culture systems and primary cell cultures derived from different tissues of wild-type and RTP4-knockout mice. Additionally, employ immunohistochemistry with RTP4 antibodies on tissue microarrays to visualize RTP4 expression patterns across multiple tissues simultaneously. To understand the molecular basis of tissue-specific effects, analyze the expression of RTP4-interacting partners (TBK1, STING, etc.) in different tissues and investigate whether tissue-specific protein isoforms or post-translational modifications of RTP4 exist .

What are common issues when using RTP4 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with RTP4 antibodies. One common issue is weak or non-specific signal in Western blotting or immunofluorescence. To resolve this, optimize antibody concentration through titration experiments (start with the recommended range of 1:300-5000 for WB and 1:50-200 for IF applications) and consider extended incubation times (overnight at 4°C). For membrane-associated proteins like RTP4, incomplete extraction can lead to poor detection; use specialized lysis buffers containing appropriate detergents (e.g., 1% NP-40, 0.5% sodium deoxycholate) to effectively solubilize membrane proteins. Background noise in immunostaining can be reduced by extending blocking times (2-3 hours), using 5% BSA instead of normal serum for blocking, and including 0.1-0.3% Triton X-100 in antibody dilution buffers to reduce non-specific binding. For tissues with high autofluorescence, consider using Sudan Black B treatment (0.1-0.3% in 70% ethanol) after secondary antibody incubation. Batch-to-batch variability can be addressed by validating each new lot against a previously working lot using positive control samples. If antibody cross-reactivity is suspected, perform validation experiments using RTP4-knockout cells or tissues as negative controls, and conduct peptide competition assays to confirm specificity. For co-immunoprecipitation experiments, weak detection of RTP4-interacting partners may require optimization of lysis conditions, crosslinking prior to lysis, or the use of more sensitive detection methods .

How should researchers validate the specificity of RTP4 antibodies?

Rigorous validation of RTP4 antibody specificity is critical for generating reliable research data. A comprehensive validation approach should include multiple complementary methods. First, perform Western blotting using lysates from cells with endogenous RTP4 expression alongside RTP4-overexpression and RTP4-knockout samples. A specific antibody will show bands of the expected molecular weight (~28 kDa) with intensity corresponding to expression levels and no band in knockout samples. Conduct peptide competition assays by pre-incubating the antibody with its immunogenic peptide before applying to samples; a specific signal should be blocked by this competition. For immunofluorescence applications, compare staining patterns in wild-type versus RTP4-knockout cells or tissues, and perform siRNA knockdown experiments to confirm signal reduction corresponds with reduced RTP4 expression. Validate subcellular localization by co-staining with established markers for cell membrane and vesicular structures, confirming the expected localization pattern. If possible, use different antibodies targeting distinct epitopes of RTP4 to confirm consistent detection patterns. For publication-quality validation, include RTP4-knockout mice tissue samples (generated through CRISPR/Cas9 technology as described in the literature) as definitive negative controls. Additionally, verify that staining intensity increases in interferon-stimulated cells, as RTP4 is an interferon-responsive protein whose expression should be upregulated following interferon treatment. Document all validation steps methodically, including antibody lot numbers, to ensure reproducibility .

What are the optimal storage and handling conditions for RTP4 antibodies?

To maintain the integrity and activity of RTP4 antibodies, proper storage and handling conditions are essential. Store antibody solutions at -20°C as recommended by manufacturers, particularly for conjugated antibodies like RTP4-Cy7. For long-term storage, aliquot the antibody into smaller volumes (10-50 μl depending on usage patterns) to avoid repeated freeze-thaw cycles, which can significantly degrade antibody performance. Each aliquot should be sufficient for a single experiment. When thawing, allow the antibody to warm completely to room temperature before opening the vial to prevent condensation, which can alter antibody concentration and promote bacterial contamination. After thawing, keep antibodies on ice during experiment preparation. For storage buffers, most commercial RTP4 antibodies come in optimal formulations; for example, the Bioss RTP4 antibody is supplied in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol. If diluting the antibody for immediate use, use freshly prepared buffers and include stabilizing proteins (0.1-1% BSA) and preservatives if storing diluted antibody for more than 24 hours. For conjugated antibodies like RTP4-Cy7, minimize exposure to light during handling and storage to prevent photobleaching of the fluorophore. Keep detailed records of antibody handling, including freeze-thaw cycles and dilution dates, to track potential degradation over time. If reduced performance is observed, consider using newer aliquots before troubleshooting experimental conditions .

How should researchers interpret contradictory results between RTP4 antibody experiments and genetic approaches?

When faced with contradictory results between RTP4 antibody-based experiments and genetic approaches (such as knockout or knockdown studies), researchers should implement a systematic analytical framework. First, evaluate antibody specificity thoroughly as described in FAQ 5.2, as non-specific antibody binding can lead to false positives. If using multiple antibodies targeting different epitopes of RTP4, compare their results for consistency. For genetic approaches, verify knockout or knockdown efficiency at both mRNA level (by qPCR) and protein level (by Western blot) using validated antibodies. Consider potential compensatory mechanisms in knockout models; chronic absence of RTP4 may trigger upregulation of functionally redundant proteins, masking the true phenotype. In knockdown experiments, ensure the degree of knockdown is sufficient (typically >70%) to observe functional consequences. Temporal factors can also explain discrepancies—acute knockdown versus chronic knockout may yield different results due to adaptation. Examine the context of experiments; RTP4's effects may be stimulus-dependent or cell type-specific. For instance, published studies show that RTP4's impact on IFN-I responses is more pronounced following specific stimuli like poly(I:C) or cGAMP. To resolve contradictions, perform rescue experiments by reintroducing RTP4 into knockout cells/tissues, which should restore the wild-type phenotype if the observed effects are truly RTP4-dependent. Additionally, consider using orthogonal methods that don't rely on antibodies, such as CRISPR activation/repression systems or tagged RTP4 constructs, to validate findings from antibody-based experiments .

What quantitative methods are recommended for analyzing RTP4 expression levels?

For accurate quantification of RTP4 expression levels, researchers should employ multiple complementary methods with appropriate controls and normalization strategies. Quantitative real-time PCR (qRT-PCR) provides sensitive measurement of RTP4 mRNA levels using validated primers (such as those listed in published datasets). When designing qPCR experiments, select stable reference genes (e.g., GAPDH, β-actin, or 18S rRNA) for normalization, and calculate relative expression using the 2^(-ΔΔCt) method. For protein-level quantification, Western blotting with RTP4 antibodies followed by densitometric analysis provides semi-quantitative data. Use total protein normalization (with stain-free gels or Ponceau staining) rather than single housekeeping proteins for more accurate normalization. For absolute quantification, develop a standard curve using recombinant RTP4 protein at known concentrations. Flow cytometry offers single-cell resolution for analyzing RTP4 expression in heterogeneous cell populations; optimize protocols for intracellular/membrane protein detection since RTP4 localizes to both cytoplasm and membrane. ELISA can provide highly quantitative measurement of RTP4 in solution, though commercial kits may be limited. For tissue samples, quantitative immunohistochemistry with digital image analysis allows spatial mapping of expression levels; use automated image analysis software with consistent thresholding parameters for objective quantification. When comparing RTP4 expression across conditions (e.g., before and after interferon stimulation), establish appropriate time-course studies, as published research shows dynamic changes in expression over time. For all methods, include positive controls (interferon-stimulated cells) and negative controls (RTP4-knockout cells) to validate assay performance .

What are the emerging research areas for RTP4 antibodies in neurological disease studies?

Emerging research areas for RTP4 antibodies in neurological disease studies represent exciting frontiers for investigation. One promising direction is exploring RTP4's role in neurodegenerative diseases with neuroinflammatory components. Given RTP4's negative regulation of type I interferon responses and its significant impact on brain infections, researchers could investigate whether RTP4 modulation affects disease progression in models of Alzheimer's disease, Parkinson's disease, or multiple sclerosis, where neuroinflammation plays a crucial role. Another emerging area is the study of RTP4 in the context of the blood-brain barrier (BBB) integrity during infection or inflammation. Since RTP4-knockout mice show altered responses to neurotropic infections, researchers could use RTP4 antibodies to investigate whether RTP4 influences BBB permeability or immune cell trafficking into the CNS during neuroinflammation. The development of conditional knockout models targeting RTP4 specifically in microglia, astrocytes, or neurons would allow researchers to dissect cell-specific contributions of RTP4 to neurological disease pathogenesis. Additionally, exploring potential RTP4 polymorphisms or expression variations in human neurological disease populations could reveal whether RTP4 represents a risk factor or biomarker for disease susceptibility or progression. Finally, given RTP4's demonstrated potential as a therapeutic target, developing methods to modulate its activity or expression in specific brain regions could lead to novel treatment strategies for neurological diseases with inflammatory components .

How can researchers design experiments to explore novel functions of RTP4 beyond interferon signaling?

To explore novel functions of RTP4 beyond its established role in interferon signaling, researchers should design multifaceted experimental approaches that probe various cellular pathways and contexts. Begin with unbiased interactome studies using techniques such as proximity labeling (BioID or TurboID), immunoprecipitation followed by mass spectrometry, or yeast two-hybrid screening to identify novel RTP4-interacting partners beyond the known TBK1/STING axis. Given RTP4's membrane localization, investigate its potential role in membrane trafficking or receptor transport (suggested by its name, Receptor Transporter Protein 4) using live-cell imaging with fluorescently tagged RTP4 and various membrane compartment markers. Perform comparative transcriptomic and proteomic analyses (RNA-seq and mass spectrometry) in wild-type versus RTP4-knockout cells under both basal and stressed conditions (beyond interferon stimulation) to identify differential gene/protein expression patterns suggesting novel pathways. Since RTP4 shows tissue-specific effects with particular importance in the brain, examine its expression and function in different neural cell types (neurons, astrocytes, microglia, oligodendrocytes) using cell-type-specific isolation methods followed by RTP4 antibody-based detection. Investigate potential roles in processes beyond immune signaling, such as cellular metabolism, by measuring metabolic parameters in RTP4-overexpressing or knockout cells. Design experiments to examine RTP4's potential role in non-viral and non-parasitic conditions, such as sterile inflammation, traumatic injury, or ischemia-reperfusion models. Finally, consider evolutionary approaches by comparing RTP4 function across species to identify conserved versus divergent roles, potentially revealing fundamental biological functions beyond interferon regulation .