RNF125 Antibody, Biotin conjugated

What is RNF125 and what cellular functions does it regulate?

RNF125 (also known as RING finger protein 125, T-cell RING activation protein 1, or TRAC-1) is an E3 ubiquitin-protein ligase that mediates ubiquitination and subsequent proteasomal degradation of target proteins . It functions in several key cellular pathways:

• It acts as a negative regulator of type I interferon production by mediating ubiquitination of RIG-I at 'Lys-181', which leads to RIG-I degradation

• It mediates K48-linked ubiquitination and destabilization of RIG-I, thereby negatively regulating RIG-I-mediated antiviral signaling

• It can target multiple immune signaling proteins for ubiquitination, including MDA5 and IPS1/MAVS/VISA/Cardif

• It mediates ubiquitination and subsequent degradation of p53/TP53 and JAK1

• Despite its negative regulation of antiviral pathways, RNF125 acts as a positive regulator of T-cell activation

The diverse functions of RNF125 make it an important target for studying immune regulation, particularly in the context of viral infections and T-cell-mediated immunity.

What are the technical specifications of the RNF125 Antibody, Biotin conjugated?

The RNF125 Antibody, Biotin conjugated is a polyclonal antibody with the following specifications:

• Antibody Type: Polyclonal

• Raised In: Rabbit

• Species Reactivity: Human

• Immunogen: Recombinant human E3 ubiquitin-protein ligase RNF125 protein (143-231AA)

• Tested Applications: ELISA (not yet tested in other applications)

• Isotype: IgG

• Conjugate: Biotin

• Purity: >95%, Protein G purified

• Storage Buffer: Preservative: 0.03% Proclin 300, Constituents: 50% Glycerol, 0.01M PBS, pH 7.4

• Uniprot ID for target protein: Q96EQ8

This biotin-conjugated antibody provides researchers with a tool for detecting and studying RNF125 in human samples, particularly through ELISA-based detection methods.

How should the RNF125 Antibody, Biotin conjugated be stored to maintain optimal activity?

Proper storage of the RNF125 Antibody, Biotin conjugated is crucial for maintaining its activity and specificity:

• Shipping Condition: The antibody is shipped at 4°C

• Long-term Storage: Upon delivery, aliquot the antibody and store at -20°C or -80°C

• Critical Consideration: Avoid repeated freeze-thaw cycles as they can denature the antibody and reduce its activity

• Working Solution: When preparing working dilutions, it's advisable to prepare them fresh on the day of use

• Storage Buffer Composition: The antibody is supplied in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

The glycerol in the storage buffer helps prevent freeze-damage during storage, while the preservative inhibits microbial growth. For optimal performance in sensitive applications, researchers should adhere strictly to these storage recommendations to ensure the stability and functionality of the antibody.

What controls should be included when using this antibody for RNF125 expression analysis?

When designing experiments using the RNF125 Antibody, Biotin conjugated, several controls should be implemented to ensure reliable and interpretable results:

• Positive Control: Include lysates from cells known to express RNF125 (lymphoid tissues express higher levels of RNF125/TRAC-1)

• Negative Control: Use samples from cells where RNF125 is absent or has been knocked down using siRNA (such as siRNF125-3 mentioned in the literature)

• Isotype Control: Include a biotin-conjugated rabbit IgG that is not specific to any target to assess non-specific binding

• Blocking Peptide Control: Where possible, pre-incubate a portion of the antibody with the immunizing peptide (143-231AA of RNF125) to demonstrate specificity

• Antibody Titration: Perform a titration series to determine the optimal antibody concentration that provides the best signal-to-noise ratio

• Cross-reactivity Assessment: If working with multiple species, include samples from non-target species to confirm species specificity

These controls help validate experimental findings and provide confidence in the specificity and sensitivity of the detected signals, which is particularly important when studying proteins involved in complex signaling pathways like RNF125.

What are the optimal conditions for using RNF125 Antibody, Biotin conjugated in ELISA?

For optimal ELISA performance using the RNF125 Antibody, Biotin conjugated, consider the following methodological approaches:

• Antibody Dilution: Begin with a 1:500 to 1:2000 dilution range and optimize based on signal strength and background levels

• Blocking Solution: Use a 3-5% BSA or milk protein solution in TBS/PBS with 0.05% Tween-20 to minimize non-specific binding

• Incubation Time and Temperature:

  • Primary incubation: 1-2 hours at room temperature or overnight at 4°C

  • Detection system (streptavidin-HRP/AP): 30-60 minutes at room temperature

• Washing Protocol: Implement 4-5 washing steps with TBS/PBS containing 0.05% Tween-20 after each antibody incubation

• Detection System: Utilize streptavidin-coupled enzymes (HRP or AP) to recognize the biotin conjugate

• Substrate Selection: Choose an appropriate substrate based on desired sensitivity (TMB for HRP, pNPP for AP)

• Signal Enhancement: Consider amplification systems if detecting low-abundance RNF125

The biotin conjugation provides advantages for detection through strong biotin-streptavidin interactions, potentially enhancing sensitivity compared to conventional detection methods. Researchers should perform preliminary optimization experiments to establish ideal conditions for their specific experimental system.

How can this antibody be applied to study RNF125's role in the RIG-I signaling pathway?

The RNF125 Antibody, Biotin conjugated can be instrumental in investigating RNF125's regulatory role in RIG-I signaling through several experimental approaches:

• Co-immunoprecipitation Studies:

  • Use the antibody to immunoprecipitate RNF125 and analyze its interaction with RIG-I, MDA5, and IPS1 under various stimulation conditions

  • Investigate how viral infection alters the association between RNF125 and its substrate proteins

• Ubiquitination Assays:

  • Employ the antibody to detect RNF125 in ubiquitination assays examining K48-linked polyubiquitination of RIG-I

  • Compare ubiquitination patterns in cells overexpressing or depleted of RNF125

• Protein Degradation Analysis:

  • Monitor RIG-I protein levels in relation to RNF125 expression during viral infection

  • Study how proteasome inhibitors affect this relationship

• Signaling Pathway Activation:

  • Correlate RNF125 levels with downstream signaling events such as IRF3 activation and IFN-β production

  • Examine how RNF125 affects the competition between K63-linked (activating) and K48-linked (degradative) ubiquitination of RIG-I

• Viral Infection Models:

  • Assess RNF125 expression dynamics during infection with RNA viruses that activate RIG-I signaling

  • Compare with TRIM4 and TRIM25 expression patterns to understand their redundant roles

This antibody provides researchers with a tool to investigate the molecular mechanisms underlying RNF125's negative regulation of antiviral signaling, potentially revealing new insights into immune response modulation.

How does RNF125 functionally differ from other E3 ligases that target RIG-I, and how can this antibody help distinguish these differences?

RNF125 functions distinctly from other E3 ligases in the regulation of RIG-I, and the RNF125 Antibody, Biotin conjugated can help elucidate these differences:

• Opposing Regulatory Functions:

  • RNF125 mediates K48-linked ubiquitination of RIG-I, leading to its proteasomal degradation and negative regulation of signaling

  • In contrast, TRIM25 and Riplet/REUL catalyze K63-linked ubiquitination of RIG-I at K154, K164, and K172, which positively regulates RIG-I signaling

  • TRIM4 also mediates K63-linked ubiquitination similar to TRIM25, promoting RIG-I activation

• Experimental Approaches to Distinguish Functions:

  • Co-expression Studies: Use the antibody to detect RNF125 in cells co-expressing different E3 ligases to analyze competitive binding to RIG-I

  • Ubiquitin Linkage Analysis: Combine with K48-specific and K63-specific ubiquitin antibodies to distinguish the type of ubiquitin chains added by different E3 ligases

  • Domain-specific Interactions: Map the specific regions of RIG-I targeted by RNF125 versus TRIM25/TRIM4/Riplet

• Redundancy Assessment:

  • Investigate potential redundancy between multiple E3 ligases as suggested by research showing TRIM4, TRIM25, and Riplet are co-expressed in several cell types

  • Explore competitive interactions, as studies have shown TRIM4 and TRIM25 competitively interact with RIG-I-CARD

• Methodological Approach:

  • Combine RNF125 antibody detection with siRNA knockdown of individual or combinations of E3 ligases

  • Use the antibody in chromatin immunoprecipitation studies to determine if different E3 ligases regulate RNF125 expression

This antibody provides a valuable tool for dissecting the complex interplay between different E3 ligases in fine-tuning RIG-I signaling during viral infections.

What methodological approaches can resolve contradictory findings regarding RNF125's role in T-cell activation versus its inhibitory effect on antiviral signaling?

The literature presents an apparent paradox where RNF125 acts as both a positive regulator of T-cell activation and a negative regulator of antiviral signaling . Researchers can employ the following methodological approaches using the RNF125 Antibody, Biotin conjugated to resolve these seemingly contradictory functions:

• Cell-type Specific Analysis:

  • Compare RNF125 expression, localization, and binding partners in T cells versus innate immune cells (e.g., dendritic cells, macrophages)

  • Investigate whether RNF125 targets different substrates in different cell types

• Temporal Dynamics Investigation:

  • Examine RNF125 expression kinetics during T-cell activation versus viral infection

  • Analyze changes in RNF125 localization and binding partners at different time points after stimulation

• Substrate Identification Studies:

  • Use the antibody for immunoprecipitation followed by mass spectrometry to identify cell-type specific RNF125 substrates

  • Compare the ubiquitination profiles of various potential targets (RIG-I, MDA5, T-cell signaling components) in different cellular contexts

• Pathway Cross-talk Analysis:

  • Investigate potential cross-regulation between T-cell receptor signaling and antiviral pathways

  • Examine whether RNF125 serves as a switch between these pathways depending on cellular context

• Post-translational Modification Profiling:

  • Determine if RNF125 itself undergoes different modifications in T cells versus innate immune cells

  • Analyze how these modifications might alter its substrate specificity or E3 ligase activity

• Experimental Design:

  • Use dual reporter systems to simultaneously monitor T-cell activation and antiviral signaling in the same cells

  • Apply CRISPR-Cas9 to create domain-specific mutants of RNF125 to identify regions responsible for different functions

Resolving this apparent contradiction would provide significant insights into how a single E3 ligase can perform context-dependent functions in immune regulation, potentially uncovering new therapeutic targets for immune modulation.

What are common technical challenges when using biotin-conjugated antibodies like RNF125 Antibody, and how can they be addressed?

Researchers using the RNF125 Antibody, Biotin conjugated may encounter several technical challenges that require specific troubleshooting approaches:

• High Background in Detection Systems:

  • Cause: Endogenous biotin in biological samples competing with the biotinylated antibody

  • Solution: Implement a biotin blocking step using streptavidin/avidin followed by free biotin before adding the biotinylated antibody

  • Methodology: Incubate samples with unconjugated streptavidin (10-20 μg/ml) for 15-20 minutes, followed by excess free biotin (50-100 μg/ml) for 15-20 minutes before antibody application

• Weak Signal Despite Expected Expression:

  • Cause: The biotin conjugation might mask crucial epitopes or alter antibody binding capacity

  • Solution: Compare with unconjugated RNF125 antibody; optimize antibody concentration; use amplification systems

  • Alternative Approach: Employ a two-step detection where primary unconjugated anti-RNF125 is followed by biotinylated secondary antibody

• Non-specific Binding:

  • Cause: Cross-reactivity with off-target proteins

  • Solution: Increase blocking time/concentration; pre-adsorb antibody with irrelevant proteins; optimize washing steps

  • Validation: Confirm specificity using RNF125 knockdown controls

• Inconsistent Results Between Experiments:

  • Cause: Antibody degradation due to improper storage or handling

  • Solution: Strictly adhere to storage recommendations; prepare fresh working dilutions; minimize freeze-thaw cycles

  • Quality Control: Include a standard positive control in each experiment to normalize between runs

• Interference with Protein-Protein Interactions:

  • Cause: Biotin conjugation near binding interfaces can disrupt protein interactions

  • Solution: For co-immunoprecipitation studies, consider using the antibody for detection rather than precipitation

  • Alternative Approach: Compare results with unconjugated antibody to assess potential interference

These methodological solutions provide researchers with strategies to optimize experimental conditions and ensure reliable, reproducible results when working with the biotin-conjugated RNF125 antibody.

How can researchers distinguish between specific and non-specific signals when analyzing RNF125 in complex biological samples?

Distinguishing specific from non-specific signals is critical when analyzing RNF125 in complex biological samples. Here are methodological approaches to ensure signal specificity:

• Comprehensive Control Panel Implementation:

  • Genetic Controls: Compare wild-type samples with RNF125 knockout/knockdown samples (e.g., using siRNF125-3 as mentioned in the literature)

  • Absorption Controls: Pre-incubate antibody with immunizing peptide (143-231AA of RNF125) to block specific binding sites

  • Isotype Controls: Use biotin-conjugated non-specific rabbit IgG at the same concentration

  • Secondary-only Controls: Include samples treated only with streptavidin-conjugated detection reagent

• Signal Validation Through Multiple Detection Methods:

  • Orthogonal Techniques: Confirm findings using alternative methods (e.g., mass spectrometry)

  • Alternative Antibodies: Validate with antibodies recognizing different epitopes of RNF125

  • Molecular Weight Verification: Ensure detected bands match the expected molecular weight of RNF125 (~34 kDa)

• Biological Validation Strategies:

  • Expression Modulation: Analyze samples where RNF125 expression is induced (e.g., after T-cell activation)

  • Stimulus Response: Verify that signal changes correlate with known regulators of RNF125 expression

  • Co-localization Studies: For imaging applications, confirm co-localization with known RNF125 interacting partners

• Technical Optimization:

  • Titration Series: Perform antibody dilution series to identify concentration that maximizes specific signal while minimizing background

  • Blocking Optimization: Test different blocking agents (BSA, milk, serum) to reduce non-specific binding

  • Stringent Washing: Implement additional washing steps with increasing detergent concentrations

• Data Analysis Approaches:

  • Signal-to-Noise Ratio Calculation: Quantify the ratio between specific signal and background

  • Statistical Validation: Apply appropriate statistical tests to distinguish true signals from random variations

  • Expression Correlation: Verify that RNF125 signals correlate with expected biological contexts (e.g., inverse correlation with RIG-I levels during viral infection)

These methodological approaches provide a systematic framework for researchers to confidently distinguish genuine RNF125 signals from artifacts, ensuring experimental rigor and reproducibility.

How can researchers design experiments to study the interplay between different ubiquitination types (K48 vs. K63) on RIG-I regulated by RNF125 versus TRIM4/TRIM25?

The current literature indicates a complex interplay between different E3 ligases mediating distinct ubiquitination patterns on RIG-I, with RNF125 promoting K48-linked (degradative) ubiquitination and TRIM4/TRIM25 promoting K63-linked (activating) ubiquitination . Here's a methodological approach to investigate this interplay:

• Ubiquitin Linkage-Specific Analysis:

  • Experimental Design: Utilize the RNF125 Antibody, Biotin conjugated alongside linkage-specific antibodies (anti-K48-Ub and anti-K63-Ub)

  • Methodology: Immunoprecipitate RIG-I and probe with these antibodies under different conditions:

    • Overexpression of individual E3 ligases (RNF125, TRIM4, TRIM25)

    • Combinatorial expression of multiple E3 ligases

    • Viral infection time course

  • Controls: Include ubiquitin mutants (K48R and K63R) to validate linkage specificity

• Competition Assay Development:

  • Experimental Approach: Design a time-course experiment to track the dynamic competition between these E3 ligases for RIG-I binding

  • Technique: Use fluorescently-tagged E3 ligases and RIG-I in live-cell imaging with FRET analysis

  • Quantification: Measure association/dissociation rates and binding affinities between RIG-I and different E3 ligases

• Sequential Ubiquitination Analysis:

  • Hypothesis Testing: Determine if K63-linked ubiquitination by TRIM4/TRIM25 precedes K48-linked ubiquitination by RNF125

  • Methodology: Perform pulse-chase ubiquitination assays with temporal control of E3 ligase activation

  • Analysis: Track the transition from activation to degradation signals on individual RIG-I molecules

• Site-Specific Ubiquitination Mapping:

  • Technical Approach: Employ mass spectrometry to identify which lysine residues on RIG-I are preferentially modified by each E3 ligase

  • Mutational Analysis: Create lysine-to-arginine mutants at positions K154, K164, K172, and K181 to determine if these residues are differentially targeted

  • Functional Assessment: Correlate site-specific modifications with RIG-I signaling output

• Physiological Context Examination:

  • Viral Infection Models: Compare the dynamics of K48 versus K63 ubiquitination during infection with different RNA viruses

  • Cell Type Comparison: Analyze this interplay in various immune cell types where these E3 ligases are co-expressed

  • Cytokine Environment: Determine how type I interferons feed back to regulate the expression and activity of these competing E3 ligases

This methodological framework enables researchers to dissect the complex regulatory mechanism where the same lysine residues on RIG-I may be subject to competing ubiquitination events that determine the balance between activation and degradation of this critical antiviral sensor.

What experimental approaches can be used to investigate the potential therapeutic targeting of RNF125 in viral infections or autoimmune disorders?

Given RNF125's role as a negative regulator of antiviral signaling and positive regulator of T-cell activation , it presents an intriguing therapeutic target. Here are methodological approaches to investigate its therapeutic potential:

• High-Throughput Screening for RNF125 Modulators:

  • Assay Development: Create cell-based systems where RNF125 activity is linked to reporter gene expression

  • Screening Platform: Test compound libraries for molecules that inhibit or enhance RNF125 E3 ligase activity

  • Validation: Use the RNF125 Antibody, Biotin conjugated to confirm target engagement through cellular thermal shift assays

• Structure-Function Analysis for Rational Drug Design:

  • Structural Biology Approach: Determine crystal structures of RNF125 RING domain (residues 1-76) alone and in complex with E2 enzymes

  • Virtual Screening: Identify small molecules that could disrupt the RNF125-E2 interface

  • Mutagenesis Studies: Focus on cysteine residues 72 and 75, which are critical for ubiquitin ligase activity

• Disease Model Testing:

  • Viral Infection Models: Evaluate how RNF125 modulation affects viral clearance and inflammatory responses in:

    • RNA virus infection models (influenza, coronavirus)

    • DNA virus models to assess pathway specificity

  • Autoimmune Disease Models: Test RNF125 modulation in models of systemic lupus erythematosus or type I interferonopathies

  • Measurement Parameters: Monitor viral titers, inflammatory markers, and tissue damage

• Cell-Specific Targeting Strategies:

  • Nanotechnology Approach: Develop antibody-conjugated nanoparticles carrying RNF125 modulators

  • Cell-Type Selectivity: Target delivery to specific immune cell populations

  • Validation: Use the RNF125 Antibody, Biotin conjugated to assess cellular uptake and target modulation

• Combinatorial Therapeutic Approaches:

  • Synergy Testing: Combine RNF125 modulators with existing antivirals or immunomodulators

  • Sequential Treatment Protocols: Time RNF125 modulation to either enhance initial antiviral response or resolve inflammation

  • Biomarker Development: Identify predictive biomarkers for treatment response using the antibody

• Translational Research Plan:

  • Ex vivo Human Sample Testing: Use patient-derived cells to validate findings in clinical specimens

  • Humanized Mouse Models: Test therapeutic approaches in mice with humanized immune systems

  • Safety Assessment: Evaluate potential consequences of RNF125 modulation on T-cell function and autoimmunity risk

These methodological approaches provide a comprehensive framework for investigating RNF125 as a therapeutic target, potentially leading to novel treatments for infectious diseases or autoimmune conditions through precise modulation of innate immune signaling pathways.

How can the RNF125 Antibody, Biotin conjugated be utilized to investigate the cross-talk between RNF125 and JAK-STAT signaling pathways?

Recent research has identified JAK1 as a target for RNF125-mediated ubiquitination and degradation , suggesting an important role for RNF125 in regulating JAK-STAT signaling. Here's a methodological framework to investigate this cross-talk:

• Dynamic Interaction Analysis:

  • Co-immunoprecipitation Studies: Use the RNF125 Antibody, Biotin conjugated to pull down RNF125 complexes and probe for JAK1 and STAT proteins

  • Temporal Analysis: Monitor the dynamics of these interactions during cytokine stimulation (e.g., IFN-α/β, IL-6)

  • Subcellular Localization: Track the co-localization of RNF125 and JAK1 using confocal microscopy during receptor activation

• Ubiquitination Profiling:

  • Site-specific Analysis: Identify the lysine residues on JAK1 targeted by RNF125 using mass spectrometry

  • Ubiquitin Chain Analysis: Determine if RNF125 mediates K48-linked ubiquitination of JAK1 similar to its action on RIG-I

  • E2 Enzyme Identification: Identify which E2 ubiquitin-conjugating enzymes cooperate with RNF125 in JAK1 ubiquitination

• Signaling Pathway Modulation:

  • Gain/Loss of Function Studies: Examine how RNF125 overexpression or knockdown affects:

    • JAK1 protein levels and phosphorylation status

    • STAT1/STAT3 activation and nuclear translocation

    • Transcription of interferon-stimulated genes (ISGs)

  • Cytokine Response Profiling: Assess how RNF125 modulation affects cellular responses to type I and II interferons

• Feedback Regulation Investigation:

  • Expression Analysis: Determine if JAK-STAT pathway activation regulates RNF125 expression

  • Post-translational Modification: Investigate whether RNF125 activity is modulated by JAK-mediated phosphorylation

  • Protein Stability Assessment: Examine if active JAK-STAT signaling affects RNF125 protein stability

• Disease-Relevant Context Testing:

  • Viral Infection Models: Analyze how the RNF125-JAK1 interaction changes during viral infection

  • Cytokine Storm Conditions: Examine this regulatory axis under conditions mimicking hyperinflammatory states

  • Cancer Cell Signaling: Investigate whether this interaction is altered in malignant cells with constitutive JAK-STAT activation

These methodological approaches would provide mechanistic insights into how RNF125 may function as a negative regulator of both RIG-I-mediated antiviral signaling and JAK-STAT-mediated cytokine signaling, potentially representing a coordinated mechanism to fine-tune immune responses.

What methodological considerations are important when designing experiments to understand the role of RNF125 in the balance between K48 and K63 ubiquitination on its targets?

The research indicates that RNF125 mediates K48-linked ubiquitination leading to protein degradation, while other E3 ligases like TRIM4/TRIM25 mediate K63-linked ubiquitination promoting signaling activation . Investigating this balance requires careful methodological considerations:

• Ubiquitin Chain-Specific Detection Systems:

  • Antibody Selection: Use chain-specific antibodies that exclusively recognize K48 or K63 linkages

  • Ubiquitin Mutants: Employ ubiquitin constructs where only K48 or K63 is available for chain formation

  • Mass Spectrometry Approach: Develop targeted methods to quantify specific ubiquitin linkages on immunoprecipitated substrates

• Competition Analysis Between Ubiquitin Chain Types:

  • Sequential Ubiquitination Assays: Determine if pre-existing K63 chains protect against or promote K48 chain formation

  • E3 Ligase Competition Experiments: Analyze how varying concentrations of RNF125 versus TRIM4/TRIM25 affect the balance of ubiquitin chain types

  • Substrate Modification Sites: Investigate whether the same or different lysine residues are targeted for different ubiquitin chain types

• Temporal Dynamics Investigation:

  • Pulse-Chase Ubiquitination Assays: Track the formation and turnover of different ubiquitin chain types over time

  • Single-Molecule Approaches: Develop methods to visualize the transition between different ubiquitination states on individual substrate molecules

  • Computational Modeling: Create predictive models of how the K48/K63 balance shifts under different conditions

• Deubiquitinating Enzyme (DUB) Contribution Analysis:

  • DUB Specificity Testing: Identify DUBs that preferentially remove K48 versus K63 chains from RNF125 targets

  • DUB Manipulation: Determine how DUB inhibition or overexpression affects the K48/K63 balance

  • Complex Formation Analysis: Investigate if RNF125 associates with specific DUBs to coordinate ubiquitin chain editing

• Physiological Trigger Response:

  • Viral Infection Time Course: Monitor how the K48/K63 balance on RIG-I changes throughout viral infection

  • Pathway Activation States: Correlate ubiquitin chain profiles with downstream signaling metrics

  • Cellular Compartmentalization: Examine whether ubiquitination patterns differ in distinct subcellular locations

• Technical Controls and Validations:

  • Proteasome Inhibition: Use proteasome inhibitors to stabilize K48-ubiquitinated proteins

  • Linkage-Specific Controls: Include known substrates with well-characterized ubiquitination patterns

  • Antibody Validation: Regularly validate the specificity of linkage-specific antibodies

These methodological considerations provide a framework for investigating the complex interplay between different ubiquitination types mediated by RNF125 and other E3 ligases, which may reveal important regulatory mechanisms in immune signaling pathways.