RNF182 Antibody

What is RNF182 and why is it significant in research?

RNF182 is a RING finger protein that functions as an E3 ubiquitin ligase, playing critical roles in protein degradation via the ubiquitin-proteasome pathway. It is preferentially expressed in brain tissues and has been implicated in several pathological conditions. Research has shown that RNF182 is upregulated in post-mortem Alzheimer's disease brain tissue and in neuronal cells subjected to death-inducing injuries . More recent studies have identified RNF182 as a potential tumor suppressor in lung adenocarcinoma (LUAD), where it induces p65 ubiquitination to suppress PDL1 transcription and immunosuppression . Additionally, RNF182 has been found to function as a negative regulator of TLR signaling in immune responses . Its diverse functions make it an attractive target for research in neuroscience, oncology, and immunology.

What types of RNF182 antibodies are currently available for research applications?

Several types of RNF182 antibodies are available for research use:

• Based on host species: Primarily rabbit polyclonal antibodies, though mouse monoclonal antibodies (e.g., clone 2D8) are also available .

• Based on target region: Antibodies targeting different regions of RNF182 including:

  • N-terminal region antibodies

  • Middle region antibodies (e.g., ABIN2781269 targeting the sequence "LSSTPVVEFYRPASFDSVTTVSHNWTVWNCTSLL FQTSIRVLVWLLGLLY")

  • Full-length or partial protein antibodies (e.g., antibodies against AA 1-160)

• Based on conjugation: Unconjugated, biotin-conjugated, and HRP-conjugated options

The selection depends on the specific application and experimental design requirements.

What is the expression pattern of RNF182 that researchers should consider when using these antibodies?

RNF182 is a weakly expressed gene that is not detectable by Northern blotting, requiring more sensitive detection methods like RT-PCR or specific antibody-based techniques . Its expression displays distinct tissue specificity:

• Brain-enriched expression: Detected in cortex, hippocampus, cerebellum, and spinal cord

• Minimal or undetectable in non-neural tissues: heart, liver, kidney, and skeletal muscle

• Upregulated during retinoic acid (RA)-induced differentiation of human NT2 cells, with increased expression in both neurons and astrocytes

• Elevated expression in Alzheimer's disease brain tissue compared to age-matched controls

• Increased expression during TLR signaling

This limited expression pattern necessitates careful experimental design with appropriate positive controls when using RNF182 antibodies.

What are the validated applications for RNF182 antibodies in current research?

Based on the literature and available products, RNF182 antibodies have been validated for:

• Western Blotting (WB): Used to detect RNF182 protein expression levels and changes under different conditions, typically requiring 100 μg/lane of total cellular protein

• Immunohistochemistry (IHC): Applied to tissue sections at recommended dilutions (e.g., 1:200) for detecting RNF182 expression in tissue samples

• ELISA: Used for quantitative detection of RNF182

• Co-immunoprecipitation: For studying RNF182 interactions with target proteins, such as ATP6V0C and p65

The choice of application should be guided by the specific research question and the validated applications of the particular antibody.

What protocol considerations are important for IHC detection of RNF182?

When performing immunohistochemistry with RNF182 antibodies, researchers should follow these methodological guidelines:

• Tissue preparation:

  • Fix tissues appropriately and embed in paraffin

  • Section tissues at appropriate thickness (typically 4-6 μm)

  • Dewax sections and perform heat-induced epitope retrieval using citrate buffer

  • Block endogenous peroxidase activity with 3% H2O2 for 30 minutes

• Antibody incubation:

  • Incubate with primary RNF182 antibody (1:200 dilution) overnight at 4°C

  • Wash thoroughly and incubate with appropriate secondary antibody (e.g., IgG-HRP at 1:1000) for 45 minutes at room temperature (22-25°C)

• Detection and visualization:

  • Develop signal using 3,3'-diaminobenzidine (DAB)

  • Counterstain with hematoxylin

  • Seal with neutral balsam for microscopy analysis

• Analysis:

  • Score staining intensity using established systems

  • Consider correlating RNF182 expression with clinical parameters as demonstrated in previous studies

How should researchers optimize Western blot protocols for RNF182 detection?

Given that RNF182 is a low abundance protein, Western blot protocols should be optimized as follows:

• Sample preparation:

  • Use sufficient protein (100 μg/lane of total cellular protein is recommended)

  • Include appropriate protease inhibitors to prevent degradation

  • Consider using phosphatase inhibitors if studying phosphorylation events

• Gel electrophoresis and transfer:

  • Use appropriate percentage gels based on RNF182's molecular weight

  • Ensure complete transfer to membrane

• Antibody incubation:

  • Block thoroughly to reduce background

  • Use optimal antibody dilutions (determined empirically for each antibody)

  • Incubate primary antibody at 4°C overnight to maximize sensitivity

• Detection considerations:

  • Use enhanced chemiluminescence (ECL) or other sensitive detection methods

  • Consider longer exposure times due to potentially low expression levels

  • Include positive controls when possible to validate detection

• Storage and handling:

  • Avoid repeated freeze-thaw cycles of antibodies

  • Store antibodies at -20°C for long-term or at 2-8°C for short-term (up to 1 week)

What approaches can resolve weak or absent signal when using RNF182 antibodies?

When encountering weak or no signal with RNF182 antibodies, consider these methodological interventions:

• Antibody-related factors:

  • Verify antibody viability and storage conditions

  • Titrate antibody concentrations to determine optimal working dilution

  • Try alternative antibodies targeting different epitopes of RNF182

• Sample-related factors:

  • Increase protein loading (>100 μg/lane) given RNF182's low expression

  • Use positive control samples where RNF182 is known to be expressed (e.g., brain tissue, NT2 neurons)

  • Consider enrichment steps before detection

• Protocol optimization:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Employ signal amplification methods (e.g., biotin-streptavidin systems)

  • Optimize blocking conditions to improve signal-to-noise ratio

  • Use more sensitive detection systems

• Expression enhancement:

  • Consider using conditions known to upregulate RNF182 (e.g., oxygen-glucose deprivation in neuronal models)

  • For cell culture experiments, verify expression in relevant cell types (RNF182 is preferentially expressed in brain-derived cells)

How can researchers address non-specific binding when using RNF182 antibodies?

Non-specific binding is a common challenge with antibodies targeting low-abundance proteins like RNF182. To address this issue:

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Increase blocking time and/or concentration

  • Include blocking agents in antibody diluent

• Antibody validation:

  • Perform knockdown/knockout controls to confirm specificity

  • Pre-absorb antibody with immunizing peptide when available

  • Test antibody on tissues known to lack RNF182 expression (e.g., liver, kidney)

• Washing optimization:

  • Increase washing duration and/or frequency

  • Test different detergent concentrations in wash buffers

• Secondary antibody considerations:

  • Ensure secondary antibodies are appropriate for the host species and isotype

  • Consider using cross-adsorbed secondary antibodies to reduce cross-reactivity

How can RNF182 antibodies be utilized to study its E3 ubiquitin ligase activity?

Investigating RNF182's E3 ubiquitin ligase activity requires specialized experimental approaches:

• In vitro ubiquitination assays:

  • Immunoprecipitate RNF182 using specific antibodies

  • Combine with E1, E2 enzymes, ubiquitin, ATP, and potential substrates

  • Detect ubiquitinated products via Western blotting

• Substrate identification and validation:

  • Perform co-immunoprecipitation with RNF182 antibodies to identify interacting proteins

  • Conduct follow-up ubiquitination assays with candidate substrates

  • Analyze whether RNF182 overexpression leads to decreased levels of substrate proteins

• Ubiquitin chain specificity:

  • Use antibodies specific for different ubiquitin linkages (K48, K63, etc.)

  • Employ mutant ubiquitin constructs (e.g., K48R) to determine chain specificity

  • Research has shown RNF182 mediates K48-linked polyubiquitination of p65

• Functional consequences:

  • Correlate ubiquitination with protein degradation rates

  • Investigate changes in downstream signaling pathways

  • Examine phenotypic outcomes in cellular or animal models

What experimental designs can effectively investigate RNF182's role in p65 regulation and PDL1 expression?

To study RNF182's involvement in p65 regulation and subsequent PDL1 expression:

• Protein interaction and ubiquitination studies:

  • Co-immunoprecipitation with RNF182 antibodies to confirm p65 interaction

  • Ubiquitination assays to demonstrate RNF182-mediated p65 ubiquitination

  • Analysis of p65 protein levels in response to RNF182 overexpression or knockdown

• Transcriptional regulation analysis:

  • Chromatin immunoprecipitation (ChIP) assays to assess p65 binding to PDL1 promoter

  • Luciferase reporter assays to measure PDL1 transcriptional activity

  • RT-qPCR to quantify PDL1 mRNA levels under various conditions

• Functional studies:

  • Co-culture experiments with CD8+ T cells to assess cytotoxicity and cytokine production

  • Analysis of lactate dehydrogenase release, interferon-γ, and interleukin-2 concentrations

  • In vivo tumor models to evaluate the impact on immunosuppression and tumor growth

• Pathway validation:

  • Rescue experiments where p65 is restored after RNF182 overexpression

  • Analysis of additional NF-κB family members (p50, p52, C-Rel, RelB) to confirm specificity

  • Investigation of upstream components like IKKα/β and IκBα phosphorylation

How can researchers investigate RNF182's role in neurodegenerative diseases using antibody-based approaches?

For studying RNF182 in the context of neurodegenerative diseases:

• Expression analysis in disease models:

  • Compare RNF182 protein levels in affected versus normal brain tissues using Western blotting

  • Perform immunohistochemistry to localize RNF182 in different brain regions

  • Quantify expression changes using image analysis of immunostained sections

• Cellular stress response studies:

  • Subject neuronal cultures to stressors like oxygen-glucose deprivation (OGD)

  • Combine with β-amyloid peptide treatment to model Alzheimer's disease conditions

  • Monitor RNF182 protein levels via Western blotting during stress and recovery phases

• Target protein interaction studies:

  • Investigate RNF182's interaction with ATP6V0C, which is involved in gap junction complexes and neurotransmitter release

  • Examine how this interaction affects neuronal viability and function

  • Analyze the impact on cellular homeostasis through overexpression studies

• Correlation with clinical parameters:

  • Analyze RNF182 expression in relation to disease progression

  • Correlate expression levels with clinical features and pathological markers

  • Consider genetic studies to identify polymorphisms affecting RNF182 function

What methodological approaches can be used to study RNF182's role in TLR signaling and inflammation?

To investigate RNF182's function in TLR signaling:

• Expression analysis during immune activation:

  • Monitor RNF182 expression changes following TLR stimulation (e.g., LPS treatment)

  • Quantify protein levels via Western blotting and mRNA via RT-qPCR

  • Compare expression patterns across different TLR activation scenarios

• Functional studies with gene modulation:

  • Knockdown RNF182 using siRNA approaches

  • Overexpress RNF182 using expression vectors

  • Measure effects on NF-κB activation and proinflammatory cytokine production

• Signaling pathway analysis:

  • Examine phosphorylation status of key components (p65, IKKα/β, IκBα, IRF3)

  • Analyze total protein levels of NF-κB family members (p50, p52, C-Rel, RelB)

  • Perform luciferase reporter assays for NF-κB and IFN-β promoter activity

• Cytokine production assessment:

  • Measure IL-6 and IFN-β levels in cell supernatants via ELISA

  • Compare cytokine profiles between control and RNF182-modulated cells

  • Correlate cytokine production with pathway activation markers

How can RNF182 expression be correlated with clinical parameters in cancer research?

The correlation between RNF182 expression and clinical parameters can be approached methodologically through:

• Patient sample analysis:

  • Collect and categorize patient tissues by clinical stages and parameters

  • Perform immunohistochemistry with RNF182 antibodies

  • Score samples based on staining intensity and distribution

• Statistical evaluation:

  • Group patients by RNF182 expression levels (high vs. low)

  • Analyze correlations with clinical features using appropriate statistical tests

  • As demonstrated in previous studies, Fisher's exact test can be used to evaluate correlations with TNM staging and other clinical parameters

• Correlation table development:

  Clinical Feature	Sample Size	RNF182 Expression	p Value
  		High	Low
  T stage
  N stage
  M stage
  Clinical stage

• Survival analysis:

  • Track patient outcomes based on RNF182 expression levels

  • Generate Kaplan-Meier survival curves

  • Calculate hazard ratios to quantify prognostic significance

This methodological approach has revealed significant correlations between RNF182 expression and T stage (p=0.0092), N stage (p=0.0318), and M stage (p=0.0234) in lung adenocarcinoma patients .