RNF122 Antibody, HRP conjugated

What is RNF122 and why is it significant in immunological research?

RNF122 (Ring Finger Protein 122) is an E3 ubiquitin ligase containing a transmembrane (TM) domain in its N-terminus and a RING finger domain in its C-terminus. Its significance in immunological research stems from its role as a selective negative regulator of RIG-I-triggered antiviral innate immune responses. RNF122 directly binds to the caspase activation and recruitment domains (CARDs) of RIG-I through its transmembrane domain and catalyzes the K48-linked ubiquitination of RIG-I at residues Lys115 and Lys146 . This interaction promotes RIG-I degradation via the proteasome, resulting in significant inhibition of downstream signaling. RNF122 expression is upregulated following RNA virus infection, where it serves as a feedback mechanism to prevent excessive inflammatory reactions while maintaining appropriate immune homeostasis . Understanding RNF122's regulatory function provides critical insights into how innate immunity against viral infections is controlled at the molecular level.

What does HRP conjugation mean for an RNF122 antibody and what advantages does it provide?

HRP (horseradish peroxidase) conjugation refers to the covalent attachment of the enzyme horseradish peroxidase to an antibody molecule, in this case, an anti-RNF122 antibody. This chemical conjugation creates a direct detection system that eliminates the need for secondary antibody incubation steps in various immunoassays. The primary advantages of using HRP-conjugated RNF122 antibodies include:

• Enhanced sensitivity - HRP catalyzes reactions that produce colorimetric, chemiluminescent, or fluorescent signals, greatly amplifying detection capabilities compared to unconjugated antibodies.

• Reduced background - By eliminating secondary antibody steps, non-specific binding is minimized.

• Simplified workflows - Protocols become more streamlined with fewer incubation and washing steps.

• Quantitative analysis - HRP conjugates provide superior signal linearity for quantitative applications like ELISA.

• Time efficiency - Immunodetection procedures can be completed more rapidly.

Several commercial RNF122 antibodies are available with HRP conjugation, including those targeting amino acid regions 65-145, which have demonstrated utility in ELISA applications .

What are the common applications for RNF122 antibody, HRP conjugated?

HRP-conjugated RNF122 antibodies are versatile tools in molecular and cellular biology research, with applications including:

• Enzyme-Linked Immunosorbent Assay (ELISA): HRP-conjugated RNF122 antibodies are particularly well-suited for ELISA applications where they can detect RNF122 protein in cell or tissue lysates, providing quantitative measurements of protein expression . The direct HRP conjugation eliminates secondary antibody requirements, simplifying the workflow.

• Western Blotting: While unconjugated RNF122 antibodies are typically used at 1:1000 dilution for Western blotting , HRP-conjugated versions offer the advantage of direct detection, enabling visualization of RNF122 expression patterns and post-translational modifications, particularly after viral infection when expression levels change dynamically .

• Immunohistochemistry: HRP-conjugated antibodies are valuable for tissue section analysis, allowing researchers to visualize RNF122 expression patterns across different cell types and in response to viral challenges. This is particularly relevant given RNF122's differential expression across immune tissues and preferential expression in macrophages .

• Protein-Protein Interaction Studies: These antibodies can be employed in co-immunoprecipitation experiments followed by direct detection to study RNF122's interactions with RIG-I and other potential binding partners in the antiviral signaling pathway .

• Viral Infection Studies: For monitoring changes in RNF122 expression during viral infections, HRP-conjugated antibodies provide sensitive detection of expression kinetics .

What are the optimal conditions for using HRP-conjugated RNF122 antibody in Western blotting?

To achieve optimal results when using HRP-conjugated RNF122 antibody in Western blotting, researchers should consider the following protocol recommendations:

Sample Preparation:

• Include proteasome inhibitors (e.g., MG132) in lysis buffers since RNF122 undergoes self-ubiquitination and proteasomal degradation . This is especially critical when examining RNF122 levels after viral infection, as both expression and degradation rates change dynamically.

• For detecting endogenous RNF122, macrophages are recommended as they express relatively high levels of the protein compared to other immune cells .

Blocking and Antibody Incubation:

• Use 5% non-fat dry milk or BSA in TBST for blocking membranes

• Optimal dilution for HRP-conjugated RNF122 antibodies typically ranges from 1:1000 to 1:2000, though this may vary between different antibody products

• Incubate at 4°C overnight or room temperature for 2 hours with gentle agitation

Signal Detection:

• Enhanced chemiluminescence (ECL) substrates are preferred for optimal sensitivity

• For low expression levels, consider using super-sensitive ECL substrates

• When studying RNF122 after viral infection, be aware that levels fluctuate with a general decrease within 4-8 hours post-infection due to self-ubiquitination

Controls:

• Include lysates from cells expressing recombinant RNF122 as positive controls

• Peritoneal macrophages make excellent positive controls due to their high endogenous RNF122 expression

• Consider including VSV-infected samples (4h post-infection) to demonstrate upregulation of RNF122

How should I validate the specificity of an RNF122 antibody in my experiments?

Validating antibody specificity is crucial for generating reliable experimental data. For RNF122 antibodies, including HRP-conjugated versions, consider the following validation approaches:

• Positive and Negative Control Tissues/Cells:

  • Use peritoneal macrophages as positive controls due to their preferential expression of RNF122

  • Include samples from various immune tissues (spleen, lymph nodes) where RNF122 is abundantly expressed

  • Include RNF122-deficient cells or tissues as negative controls when possible

• Overexpression and Knockdown Validation:

  • Perform parallel experiments with RNF122 overexpression in HEK293T cells (with proteasome inhibitor MG132 to prevent self-degradation)

  • Use siRNA knockdown or CRISPR/Cas9 knockout models to confirm antibody specificity

  • Compare detection patterns before and after manipulation of RNF122 expression

• Immunoprecipitation Followed by Mass Spectrometry:

  • Perform immunoprecipitation using the RNF122 antibody

  • Verify the pulled-down protein by mass spectrometry to confirm identity as RNF122

• Multiple Antibody Comparison:

  • Test multiple antibodies targeting different epitopes of RNF122 (e.g., those targeting amino acids 65-145 and those targeting 84-110)

  • Compare banding patterns in Western blots to ensure consistency

• Domain-Specific Detection:

  • Confirm antibody reactivity against full-length RNF122 versus truncated versions (TM domain alone or RING finger domain alone)

  • Verify that the antibody recognizes the expected molecular weight protein (~25-30 kDa)

What controls should I include when studying RNF122 in viral infection models?

When investigating RNF122 in the context of viral infections, particularly its role in regulating RIG-I-mediated antiviral responses, the following controls are essential:

• Temporal Controls:

  • Include multiple time points post-infection (0h, 4h, 8h, 12h, 24h) to capture the dynamic changes in RNF122 expression and activity

  • RNF122 mRNA and protein levels are typically upregulated within 4 hours after RNA virus infection

  • K48-linked ubiquitination of RNF122 increases within 4-8 hours post-infection, followed by decreased protein levels

• Virus-Type Controls:

  • Include both RNA viruses (VSV, Sendai virus) and DNA viruses (HSV-1) to demonstrate selectivity of RNF122's effect on RIG-I-mediated responses

  • Different RNA viruses should be tested, including those recognized by RIG-I (VSV, SeV) versus those recognized by MDA5 (encephalomyocarditis virus)

• Molecular Pathway Controls:

  • Monitor IRF3 and p65 phosphorylation as downstream indicators of RIG-I pathway activation

  • Include controls for other innate immune pathways (TLR activation with LPS, poly(I:C), CpG ODN) to confirm specificity

• Cell Type Controls:

  • Compare responses in macrophages (high RNF122 expression) versus conventional BMDCs and plasmacytoid BMDCs (low RNF122 expression)

  • This comparison helps confirm the cell type-specific effects of RNF122 on antiviral responses

• Proteasome Inhibition Control:

  • Include MG132-treated samples to distinguish between changes in protein levels due to transcriptional regulation versus proteasomal degradation

How can RNF122 antibodies be used to study its E3 ubiquitin ligase activity?

RNF122 antibodies, including HRP-conjugated versions, can be instrumental in studying the E3 ubiquitin ligase activity of RNF122 through several sophisticated experimental approaches:

• In Vitro Ubiquitination Assays:

  • Immunoprecipitate RNF122 using specific antibodies from cell lysates

  • Perform in vitro ubiquitination reactions with purified E1, E2 enzymes, ubiquitin, and potential substrate proteins

  • Use Western blotting with the HRP-conjugated RNF122 antibody to detect both RNF122 and its self-ubiquitination activity

• Self-Ubiquitination Analysis:

  • RNF122 exhibits self-ubiquitination activity that leads to its own degradation

  • To capture this, transfect cells with RNF122 expression vectors with or without MG132

  • Immunoprecipitate RNF122 and perform Western blotting with anti-ubiquitin antibodies

  • The self-ubiquitination activity increases dramatically after MG132 treatment

• Substrate Ubiquitination Assays:

  • Co-transfect cells with RNF122 and potential substrate (e.g., RIG-I)

  • Use RNF122 and substrate-specific antibodies to immunoprecipitate protein complexes

  • Perform Western blotting with anti-ubiquitin antibodies to detect ubiquitination patterns

  • RNF122 specifically catalyzes K48-linked ubiquitination of RIG-I CARDs at Lys115 and Lys146

• Domain Function Analysis:

  • Generate RNF122 mutants lacking the RING finger domain or carrying point mutations at catalytic residues

  • Compare ubiquitination activities between wild-type and mutant RNF122

  • HRP-conjugated antibodies that recognize the TM domain of RNF122 would be particularly useful for detecting both wild-type and RING finger domain mutants

• Virus-Induced Ubiquitination Dynamics:

  • Monitor changes in RNF122's self-ubiquitination and substrate ubiquitination following viral infection

  • K48-linked ubiquitination of RNF122 significantly increases within 4-8 hours after VSV infection

  • Correlate these changes with downstream signaling events like IRF3 and p65 phosphorylation

What experimental approaches can reveal the interaction between RNF122 and RIG-I?

The interaction between RNF122 and RIG-I represents a critical mechanism for regulating antiviral responses. The following experimental approaches can effectively characterize this interaction:

• Co-Immunoprecipitation (Co-IP):

  • Perform reciprocal Co-IPs using anti-RNF122 and anti-RIG-I antibodies

  • This approach has confirmed RNF122-RIG-I interaction in both uninfected and VSV-infected cells

  • HRP-conjugated RNF122 antibodies can simplify detection in Western blot analysis of immunoprecipitates

• GST Pull-Down Assays:

  • Use purified GST-tagged RIG-I or its domains (particularly CARDs) and test binding with purified RNF122

  • This approach has confirmed direct interaction between RNF122 and RIG-I

  • The transmembrane (TM) domain of RNF122 directly interacts with CARDs of RIG-I

• Domain Mapping:

  • Generate truncation mutants of both RNF122 and RIG-I to identify minimal interaction domains

  • Both full-length and CARDs of RIG-I bind to RNF122

  • Both full-length and TM domain of RNF122 bind to RIG-I

• Proximity Ligation Assay (PLA):

  • Detect protein-protein interactions in situ with spatial resolution

  • Use specific antibodies against RNF122 and RIG-I to visualize their colocalization in intact cells

  • This technique can reveal physiological interactions at endogenous protein levels

• Immunofluorescence Microscopy:

  • Visualize colocalization of RNF122 and RIG-I in cells with or without viral infection

  • This approach has demonstrated their colocalization in the cytoplasm of mouse peritoneal macrophages

How can RNF122 antibodies help investigate immune regulation in RNF122-deficient models?

RNF122 antibodies are invaluable tools for investigating immune regulation in RNF122-deficient models, providing insights into the consequences of removing this negative regulator from antiviral signaling pathways:

• Verification of Knockout Efficiency:

  • HRP-conjugated RNF122 antibodies are essential for confirming complete absence of the protein in RNF122-deficient mouse models or cells

  • Antibodies targeting different epitopes should be used to ensure no truncated forms of the protein remain

• Pathway Activation Analysis:

  • Monitor enhanced activation of RIG-I signaling in RNF122-deficient cells through:

    • Increased phosphorylation of IRF3 and p65 transcription factors

    • Elevated type I IFN and proinflammatory cytokine production

    • Reduced viral replication in RNF122-deficient cells and tissues

• Substrate Stabilization Studies:

  • RNF122 antibodies can be used alongside RIG-I antibodies to demonstrate increased stability of RIG-I in the absence of RNF122-mediated degradation

  • Pulse-chase experiments with cycloheximide can reveal extended half-life of RIG-I in RNF122-deficient cells

• Reconstitution Experiments:

  • Re-introduce wild-type or mutant RNF122 into knockout models to restore negative regulation

  • Use RNF122 antibodies to confirm expression of the reconstituted protein

  • Correlate expression levels with the ability to suppress RIG-I signaling and type I IFN production

• Tissue-Specific and Cell-Type-Specific Analysis:

  • Compare RNF122 expression and function across different immune cell populations

  • RNF122 is preferentially expressed in macrophages compared to other immune cells

  • This explains why RNF122 deficiency affects antiviral responses in macrophages but not in conventional or plasmacytoid BMDCs

Why might I observe variable signal intensity when using RNF122 antibody in virus-infected vs. uninfected cells?

When using RNF122 antibodies in experimental systems comparing virus-infected and uninfected cells, researchers often encounter variable signal intensity that requires careful interpretation. Several factors contribute to this variability:

What strategies can help improve signal-to-noise ratio when using HRP-conjugated RNF122 antibodies?

Achieving optimal signal-to-noise ratio with HRP-conjugated RNF122 antibodies can be challenging, particularly when detecting endogenous protein levels or studying dynamic changes during viral infection. The following strategies can help improve signal quality:

• Sample Preparation Optimization:

  • Include proteasome inhibitors (MG132) in lysis buffers to prevent RNF122 degradation

  • Use phosphatase inhibitors to preserve phosphorylation states of RIG-I pathway components

  • Consider cell fractionation to enrich for membrane-associated RNF122, as it contains a transmembrane domain

• Blocking Optimization:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers) to identify optimal conditions

  • For phospho-specific detection of downstream signaling molecules, use BSA instead of milk

  • Consider including 0.05% Tween-20 in blocking solutions to reduce non-specific binding

• Antibody Dilution and Incubation:

  • Perform careful titration experiments to determine optimal antibody concentration

  • Extended incubation at 4°C (overnight) often improves signal quality compared to shorter room temperature incubations

  • For weak signals, consider using signal enhancers compatible with HRP detection systems

• Enhanced Chemiluminescence Options:

  • For low abundance targets, use high-sensitivity ECL substrates

  • Consider femto-level detection reagents for visualizing endogenous RNF122 in cell types with lower expression

• Controls and Normalization:

  • Include positive controls from peritoneal macrophages, which have high RNF122 expression

  • When comparing infected vs. uninfected samples, normalize to appropriate housekeeping proteins

  • Consider loading increasing amounts of protein to establish detection thresholds

How should I interpret contradictory results between RNF122 mRNA and protein levels?

Researchers studying RNF122 often encounter apparent contradictions between mRNA and protein levels, particularly in the context of viral infection. The following guidelines help interpret these seemingly contradictory results:

• Understanding the Biphasic Response:

  • RNF122 mRNA levels increase within 4 hours after RNA virus infection

  • Protein levels initially increase but then decrease due to enhanced self-ubiquitination and proteasomal degradation within 4-8 hours post-infection

  • This creates a temporal disconnect between peak mRNA and protein levels

• Proteasome-Dependent Regulation:

  • To determine whether discrepancies are due to post-translational degradation, perform parallel experiments with and without proteasome inhibitors (MG132)

  • If protein levels increase dramatically with MG132 treatment while mRNA remains unchanged, this confirms post-translational regulation

• Analysis of Ubiquitination Status:

  • Assess K48-linked ubiquitination of RNF122 when mRNA and protein levels appear contradictory

  • Increased K48-linked ubiquitination with decreased protein levels (despite increased mRNA) indicates active proteasomal degradation

• Time-Course Experiments:

  • Perform detailed time-course analyses measuring both mRNA and protein levels at multiple timepoints (0h, 2h, 4h, 6h, 8h, 12h, 24h)

  • This approach can reveal the dynamic relationship between transcriptional induction and subsequent protein degradation

• Cell-Type Specific Considerations:

  • The relationship between RNF122 mRNA and protein levels may vary across cell types

  • In cells with lower baseline expression (e.g., conventional BMDCs), the dynamic range of both mRNA and protein changes may differ from macrophages

Table 2: Temporal Dynamics of RNF122 Expression After VSV Infection

How might RNF122 antibodies contribute to developing antiviral therapeutics?

RNF122 antibodies can play pivotal roles in the development of antiviral therapeutics targeting the RIG-I pathway, offering several promising research avenues:

• Therapeutic Target Validation:

  • RNF122 antibodies can help validate this E3 ligase as a potential drug target

  • Temporary inhibition of RNF122 could enhance antiviral immunity, as demonstrated by the increased resistance to RNA virus infection in RNF122-deficient mice

  • Antibodies help map critical functional domains (particularly the TM domain that interacts with RIG-I) for targeted drug development

• High-Throughput Screening Assays:

  • Develop cell-based screening assays using HRP-conjugated RNF122 antibodies to identify compounds that:

    • Disrupt RNF122-RIG-I interaction

    • Inhibit RNF122's E3 ligase activity

    • Prevent RNF122-mediated degradation of RIG-I

  • These assays could lead to small molecule inhibitors as potential antiviral therapeutics

• Biomarker Development:

  • RNF122 antibodies can help establish whether RNF122 expression or activity correlates with susceptibility to RNA virus infections

  • Changes in RNF122 levels might serve as biomarkers for antiviral response efficacy

  • Monitoring these changes could help predict therapeutic outcomes

• Engineering Therapeutic Antibodies:

  • Develop cell-penetrating antibodies or antibody fragments that inhibit RNF122 function

  • These could enhance antiviral responses during acute viral infections

  • Target delivery to specific cell types like macrophages where RNF122 plays significant regulatory roles

• Combination Therapy Approaches:

  • Use RNF122 antibodies to investigate synergistic effects between RNF122 inhibition and existing antiviral therapies

  • Evaluate whether temporary enhancement of innate immunity through RNF122 suppression improves vaccine efficacy

What aspects of RNF122 biology remain unexplored and how can antibodies help address these questions?

Despite significant advances in understanding RNF122 function, several aspects of its biology remain unexplored. RNF122 antibodies will be critical tools for addressing these knowledge gaps:

• Tissue-Specific Functions:

  • RNF122 is widely expressed across tissues, with particularly high expression in immune organs

  • HRP-conjugated antibodies could enable high-resolution immunohistochemistry to map expression patterns across tissues and cell types

  • This could reveal additional functions beyond innate immune regulation

• Regulation of RNF122 Expression:

  • While viral infection upregulates RNF122 , the transcriptional mechanisms remain unclear

  • Chromatin immunoprecipitation experiments using transcription factor antibodies combined with RNF122 antibodies could identify regulators

• Additional Substrates Beyond RIG-I:

  • RNF122 may target proteins beyond RIG-I for ubiquitination

  • Immunoprecipitation with RNF122 antibodies followed by mass spectrometry could identify novel interaction partners and substrates

• RNF122 in Human Disease:

  • The role of RNF122 in human viral infections and inflammatory diseases requires investigation

  • Antibodies specific to human RNF122 could enable studies with patient samples to correlate expression levels with disease severity or outcomes

• Post-Translational Modifications:

  • Beyond self-ubiquitination, RNF122 may undergo other modifications that regulate its function

  • Phospho-specific or other modification-specific antibodies could reveal regulatory mechanisms

• Subcellular Localization Dynamics:

  • RNF122 contains a transmembrane domain and partially localizes to the ER

  • High-resolution imaging with RNF122 antibodies could track its subcellular redistribution during infection or other cellular stresses