rnf170 Antibody

What is RNF170 and why is it important in research?

RNF170 (Ring Finger Protein 170) is an E3 ubiquitin ligase primarily located in the endoplasmic reticulum (ER) membrane. It plays a crucial role in several cellular processes, notably in the ubiquitination and degradation of inositol 1,4,5-trisphosphate receptors . More recently, RNF170 has been identified as a regulator of innate immune responses through its interaction with Toll-like receptor 3 (TLR3). Research has demonstrated that RNF170 selectively inhibits TLR3-triggered immune responses by promoting TLR3 degradation through the K48-linked polyubiquitination of K766 in TLR3 . This function makes RNF170 an important target for immunological research, particularly in studies investigating innate immunity, viral response pathways, and inflammatory conditions.

How do I select the appropriate RNF170 antibody for my research?

Selection of an appropriate RNF170 antibody depends on several experimental factors:

Species reactivity: Determine which species your samples originate from and choose an antibody with corresponding reactivity. Available RNF170 antibodies show reactivity to various species including human, mouse, rat, dog, cow, horse, rabbit, guinea pig, bat, chicken, monkey, and Xenopus laevis .

Target epitope: Different antibodies target different regions of the RNF170 protein. For instance, some antibodies target amino acids 121-193, others target the C-terminal region, and some target amino acids 1-200 . The choice of epitope matters particularly when:

• Studying specific domains of RNF170

• Investigating splice variants

• Examining post-translational modifications

Antibody format: Consider whether you need polyclonal or monoclonal antibodies. Polyclonal antibodies offer broader epitope recognition, while monoclonal antibodies provide higher specificity for a single epitope .

Applications: Ensure the antibody is validated for your intended application. Available RNF170 antibodies are validated for Western Blotting (WB), ELISA, Immunohistochemistry (IHC), and Immunofluorescence (IF) .

What are the key differences between polyclonal and monoclonal RNF170 antibodies?

When selecting between polyclonal and monoclonal RNF170 antibodies, consider these functional differences:

Polyclonal RNF170 antibodies:

• Recognize multiple epitopes on the RNF170 protein

• Typically generated in rabbits or mice against recombinant RNF170 protein fragments

• Offer higher sensitivity due to recognition of multiple epitopes

• Beneficial for detecting low expression levels of RNF170

• Can continue to detect the protein even if some epitopes are altered by experimental conditions

• Example: Rabbit polyclonal antibodies targeting amino acids 1-200 or the C-terminal region of RNF170

Monoclonal RNF170 antibodies:

• Recognize a single epitope on the RNF170 protein

• Generated from a single B-cell clone, ensuring consistency between batches

• Provide higher specificity for a particular region of RNF170

• Particularly useful when distinguishing between closely related proteins

• Essential for quantitative applications requiring consistent recognition

• Example: Mouse monoclonal antibody 2D6 that targets amino acids 121-193 of RNF170

The choice between these types should be guided by your experimental needs - use polyclonal antibodies when sensitivity is paramount, and monoclonal antibodies when specificity and consistency are critical.

What are the optimal conditions for Western Blotting with RNF170 antibodies?

Achieving optimal Western Blotting results with RNF170 antibodies requires careful protocol optimization:

Sample preparation:

• Extract proteins from cells using RIPA buffer supplemented with protease inhibitors

• For membrane proteins like RNF170, consider specialized extraction buffers containing mild detergents

• Sonicate briefly to shear genomic DNA and reduce sample viscosity

Dilution optimization:

• Most RNF170 antibodies perform optimally at dilutions between 1:500-1:2000 for Western Blotting

• Begin with the manufacturer's recommended dilution and adjust as needed

• Perform a dilution series (e.g., 1:500, 1:1000, 1:2000) to determine optimal signal-to-noise ratio

Detection considerations:

• RNF170 typically appears at approximately 25-30 kDa on Western blots, though different forms may appear at different sizes due to post-translational modifications or splicing variants

• When using tissues or cell lines not previously tested, include positive controls

• For low abundance samples, consider enrichment through immunoprecipitation prior to Western Blotting

Band interpretation:

• Multiple bands may represent different post-translational modifications or splicing forms in different cells

• Verify specificity using RNF170 knockout samples (such as those generated through CRISPR/Cas9 system) as negative controls

How can RNF170 antibodies be used to investigate protein-protein interactions?

RNF170 antibodies are valuable tools for studying protein-protein interactions, particularly for understanding RNF170's role in immune signaling:

Co-immunoprecipitation (Co-IP):

• Lyse cells in a non-denaturing buffer to preserve protein complexes

• Pre-clear the lysate with appropriate control IgG and protein A/G beads

• Incubate cleared lysate with RNF170 antibody overnight at 4°C

• Capture antibody-protein complexes using protein A/G beads

• Wash stringently to remove non-specific interactions

• Elute and analyze by SDS-PAGE and Western Blotting with antibodies against suspected interaction partners

This approach has successfully demonstrated direct interactions between RNF170 and TLR3, as confirmed by GST pull-down assays .

Proximity Ligation Assay (PLA):
For in situ detection of RNF170 interactions:

• Fix cells on coverslips

• Permeabilize and block non-specific binding

• Incubate with RNF170 antibody and antibody against suspected interaction partner

• Apply species-specific PLA probes and perform ligation and amplification

• Visualize interaction points by fluorescence microscopy

Immunofluorescence colocalization:
Immunofluorescence studies have demonstrated that RNF170 colocalizes with TLR3 in both resting cells and after poly(I:C) stimulation . Both proteins colocalize with ER marker KDEL in resting cells and with early endosome marker EEA1 after poly(I:C) stimulation .

What controls should be included when using RNF170 antibodies for immunohistochemistry?

When conducting immunohistochemistry (IHC) with RNF170 antibodies, proper controls are essential to ensure result validity:

Positive controls:

• Tissue samples known to express RNF170 (e.g., immune cells like macrophages or dendritic cells)

• Cell lines with confirmed RNF170 expression

• Recombinant RNF170-expressing cells

Negative controls:

• Isotype controls matching the RNF170 antibody's host species and isotype (e.g., rabbit IgG for rabbit polyclonal antibodies)

• RNF170 knockout tissues generated using CRISPR/Cas9 system

• Primary antibody omission control

• Blocking peptide competition assay using the immunizing peptide

Dilution optimization:

• For IHC applications, RNF170 antibodies typically work at dilutions between 1:20-1:200

• Perform a titration series to determine optimal dilution for your specific tissue

• Consider differences in fixation methods (formalin, paraformaldehyde, etc.) when optimizing

Signal validation approach:

• Perform parallel staining with two different RNF170 antibodies targeting distinct epitopes

• Compare staining patterns between knockout and wild-type tissues

• Correlate IHC results with other methods (e.g., Western Blotting, qPCR)

• Assess subcellular localization (primarily ER and endosomes for RNF170)

How can RNF170 antibodies be used to investigate the role of RNF170 in innate immune responses?

RNF170 antibodies are instrumental in elucidating the function of RNF170 in innate immunity:

Monitoring RNF170-TLR3 interactions:
Studies have demonstrated that RNF170 selectively inhibits TLR3-triggered innate immune responses . To investigate this:

• Stimulate cells with poly(I:C), a TLR3 agonist

• Immunoprecipitate TLR3 using specific antibodies

• Probe for co-precipitated RNF170 using RNF170 antibodies

• Compare interaction dynamics before and after stimulation

• Correlate with downstream signaling events (phosphorylation of IRF3 and P65)

Ubiquitination assays:
To examine RNF170-mediated ubiquitination of TLR3:

• Co-express TLR3 and ubiquitin constructs

• Immunoprecipitate TLR3

• Probe for ubiquitin modifications

• Compare ubiquitination patterns between wild-type and RNF170-deficient cells

• Use RNF170 antibodies to confirm RNF170 expression levels and correlation with ubiquitination intensity

Signaling pathway analysis:
Compare signaling in wild-type and RNF170-deficient cells:

• Stimulate cells with various TLR agonists (poly(I:C), LPS, CpG) or infect with viruses (HSV, VSV, SeV)

• Assess cytokine production (IFN-β, IL-6, TNF-α, IFN-α)

• Examine phosphorylation of signaling components (IRF3, P65, JNK)

• Correlate findings with RNF170 expression levels as determined by RNF170 antibodies

This approach has revealed that RNF170 specifically regulates TLR3-TRIF signaling but not RIG-I-MAVS signaling .

What approaches can be used to verify RNF170 antibody specificity in CRISPR/Cas9 knockout models?

Verifying antibody specificity using CRISPR/Cas9 knockout models is critical for research integrity:

Generation of RNF170 knockout models:

• Design guide RNAs targeting exon 4 of RNF170 (as successfully used in previous studies)

• Transfect cells with CRISPR/Cas9 components

• Select and isolate clones

• Verify knockout by PCR and sequencing

Antibody validation protocol:

• Prepare protein lysates from wild-type and RNF170 knockout cells/tissues

• Run parallel Western blots with identical loading

• Probe with different RNF170 antibodies targeting distinct epitopes

• Compare staining patterns - true RNF170 bands should be absent in knockout samples

• Document any non-specific bands that persist in knockout samples

Immunofluorescence validation:

• Grow wild-type and knockout cells on coverslips

• Perform immunofluorescence using RNF170 antibodies

• Include co-staining for ER markers (KDEL) to identify expected localization

• Compare staining patterns - specific staining should be absent in knockout cells

• Document any background or non-specific staining

Quantitative assessment:
Create a validation table documenting each antibody's performance in knockout validation:

How can RNF170 antibodies be used to investigate the spatiotemporal dynamics of RNF170 during immune activation?

Understanding the subcellular localization and trafficking of RNF170 during immune activation provides critical insights into its regulatory function:

Time-course immunofluorescence:

• Stimulate cells with poly(I:C) for various timepoints (0, 15, 30, 60, 120 minutes)

• Fix cells and perform co-immunofluorescence using:

  • RNF170 antibodies

  • TLR3 antibodies

  • Organelle markers (KDEL for ER, EEA1 for early endosomes)

• Analyze colocalization coefficients at each timepoint

• Quantify changes in subcellular distribution

Previous research has demonstrated that both RNF170 and TLR3 relocate from the ER to early endosomes after poly(I:C) stimulation .

Live-cell imaging:
For dynamic visualization:

• Generate cell lines expressing fluorescently-tagged RNF170

• Validate tagged protein functionality by rescue experiments in RNF170 knockout cells

• Perform live-cell imaging during immune stimulation

• Quantify protein movement and interaction dynamics

• Validate key findings using RNF170 antibodies in fixed-cell immunofluorescence

Biochemical fractionation:
To quantitatively assess protein redistribution:

• Stimulate cells for various timepoints

• Perform subcellular fractionation to isolate distinct organelles

• Analyze RNF170 distribution by Western Blotting with RNF170 antibodies

• Quantify changes in RNF170 levels in different fractions over time

• Correlate with functional outcomes (e.g., cytokine production, TLR3 degradation)

Why might Western Blotting with RNF170 antibodies show multiple bands, and how should these be interpreted?

Multiple bands in Western Blotting with RNF170 antibodies can have several causes requiring careful interpretation:

Expected patterns:

• RNF170 may appear as multiple bands due to different post-translational modifications or splicing forms in different cell types

• The primary band for RNF170 should appear at approximately 25-30 kDa

• Additional bands may represent:

  • Post-translationally modified forms (ubiquitinated, phosphorylated)

  • Alternative splice variants

  • Degradation products

Verification approaches:

• Compare with knockout controls: Genuine RNF170 bands should disappear in RNF170 knockout samples

• Epitope blocking: Pre-incubate antibody with immunizing peptide - specific bands should disappear

• Cross-verification: Use multiple antibodies targeting different RNF170 epitopes

• Deglycosylation/dephosphorylation: Treat samples with appropriate enzymes to resolve modification-based bands

Optimization strategies:

• Adjust protein loading (10-30 μg typically optimal)

• Optimize antibody dilution (1:500-1:2000 recommended range)

• Reduce background by increasing blocking stringency and wash duration

• Consider gradient gels for better separation of closely migrating bands

Remember that different antibodies targeting different epitopes may reveal distinct banding patterns depending on the accessibility of the epitope in different RNF170 forms.

What are the key considerations when using RNF170 antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) with RNF170 antibodies requires optimization to maintain interaction integrity:

Lysis buffer considerations:

• Use non-denaturing buffers to preserve protein-protein interactions

• For membrane proteins like RNF170, include appropriate detergents:

  • NP-40 (0.5-1%)

  • Digitonin (1%) for milder extraction

  • Avoid harsh detergents like SDS

• Include protease inhibitors to prevent degradation

• Consider phosphatase inhibitors to preserve phosphorylation-dependent interactions

Antibody selection criteria:

• Confirm the antibody's suitability for immunoprecipitation

• Polyclonal antibodies often perform better for Co-IP due to recognition of multiple epitopes

• Consider epitope location - ensure the target epitope is accessible in native protein complexes

• For RNF170-TLR3 interactions, antibodies targeting regions outside the interaction interface are preferable

Experimental controls:

• Input control: Save a portion of lysate before immunoprecipitation

• IgG control: Perform parallel IP with non-specific IgG

• Knockout/knockdown control: Use lysates from RNF170-deficient cells

• Reciprocal Co-IP: Confirm interactions by IP with antibodies against binding partners (e.g., TLR3)

Protocol optimization:

• Adjust antibody amounts (typically 1-5 μg per mg of protein lysate)

• Optimize incubation time and temperature (4-16 hours at 4°C usually optimal)

• Consider crosslinking approaches for transient interactions

• For difficult interactions, mild formaldehyde crosslinking (0.1-0.3%) can help preserve complexes

How can signal-to-noise ratio be improved when using RNF170 antibodies for immunofluorescence?

Optimizing immunofluorescence with RNF170 antibodies requires careful attention to fixation, permeabilization, and detection parameters:

Fixation optimization:

• Compare different fixatives:

  • 4% paraformaldehyde (10-15 minutes) - preserves structure but may reduce epitope accessibility

  • Methanol (-20°C, 10 minutes) - better for some intracellular epitopes

  • Mixture of paraformaldehyde and methanol for dual benefits

• Adjust fixation time to minimize overfixation which can mask epitopes

Permeabilization considerations:

• For transmembrane proteins like RNF170, test different permeabilization agents:

  • 0.1-0.3% Triton X-100 (5-10 minutes)

  • 0.1-0.5% Saponin (10 minutes)

  • 0.1% Digitonin (selective membrane permeabilization)

• Optimize permeabilization time to maximize antibody access while preserving structure

Antibody incubation optimization:

• Dilution range: typically 1:50-1:200 for immunofluorescence

• Extended incubation: overnight at 4°C often yields better results than 1-2 hours at room temperature

• Consider using signal amplification systems for low abundance targets

Background reduction strategies:

• Increase blocking stringency (5% BSA or 10% serum from secondary antibody host species)

• Include 0.1-0.3% Triton X-100 in blocking and antibody diluent buffers

• Extend washing steps (at least 3x10 minutes with gentle agitation)

• Use secondary antibodies pre-adsorbed against other species

• Include knockout or primary antibody omission controls to identify non-specific signals

These optimization strategies are particularly important when attempting to visualize colocalization between RNF170 and interaction partners like TLR3 or organelle markers like KDEL (ER) and EEA1 (early endosomes) .

How can RNF170 antibodies be used to investigate the role of RNF170 in neurological disorders?

RNF170 antibodies provide valuable tools for investigating RNF170's role in neurological conditions:

Tissue expression analysis:

• Use RNF170 antibodies for IHC or IF to examine:

  • Expression patterns in different regions of normal and diseased brains

  • Cellular localization in neurons versus glia

  • Changes in expression or distribution in disease models

• Compare expression across development or disease progression

Mutation impact assessment:
RNF170 mutations have been implicated in autosomal dominant sensory ataxia:

• Generate cell models expressing wild-type or mutant RNF170

• Use RNF170 antibodies to compare:

  • Protein stability and expression levels

  • Subcellular localization

  • Interaction with binding partners (IP-3 receptors)

  • Ubiquitination activity

Biomarker potential evaluation:

• Examine RNF170 levels in cerebrospinal fluid or exosomes using RNF170 antibodies

• Correlate with disease severity or progression

• Compare with other established biomarkers

Therapeutic target validation:

• Use RNF170 antibodies to monitor changes in:

  • RNF170 expression after drug treatment

  • RNF170 subcellular localization

  • RNF170-mediated ubiquitination

• Develop cell-based assays to screen for compounds that modify RNF170 function

What advanced microscopy techniques can be combined with RNF170 antibodies for high-resolution analysis?

Advanced microscopy techniques provide deeper insights into RNF170 biology:

Super-resolution microscopy:

• Structured Illumination Microscopy (SIM):

  • Achieves ~120 nm resolution

  • Ideal for visualizing RNF170 in ER membranes and endosomes

  • Compatible with standard immunofluorescence protocols

• Stimulated Emission Depletion (STED):

  • Achieves ~30-80 nm resolution

  • Requires bright, photostable fluorophores

  • Optimal for visualizing discrete RNF170 clusters

• Single Molecule Localization Microscopy (PALM/STORM):

  • Achieves ~10-30 nm resolution

  • Requires special fluorophores and buffers

  • Enables quantitative analysis of RNF170 molecular distribution

Proximity-based techniques:

• Förster Resonance Energy Transfer (FRET):

  • Detect direct protein interactions at 1-10 nm scale

  • Requires fluorescently tagged proteins or antibodies with compatible fluorophores

  • Can be used to study RNF170-TLR3 interactions in living cells

• Proximity Ligation Assay (PLA):

  • Visualize protein interactions within 40 nm

  • Uses primary antibodies against RNF170 and interaction partners

  • Generates discrete fluorescent spots at interaction sites

Live-cell approaches:

• Fluorescence Recovery After Photobleaching (FRAP):

  • Measure RNF170 mobility in different compartments

  • Requires fluorescently tagged RNF170 validated with antibodies

  • Compare dynamics before and after immune stimulation

• Fluorescence Correlation Spectroscopy (FCS):

  • Measure concentration and diffusion of fluorescently labeled RNF170

  • Detect changes in molecular dynamics upon activation

  • Correlate with functional outcomes

How might RNF170 antibodies contribute to understanding the cross-talk between innate immunity and neuroinflammation?

RNF170 antibodies offer unique opportunities to investigate the intersection of immunity and neurological disorders:

Cell-type specific analysis:

• Perform multi-label immunofluorescence using:

  • RNF170 antibodies

  • Cell-type markers (neurons, microglia, astrocytes)

  • TLR3 or other innate immunity markers

• Compare RNF170 expression and localization across cell types in health and disease

• Examine changes during neuroinflammation

Pathway integration studies:
RNF170 regulates both calcium signaling (via IP3R degradation) and innate immunity (via TLR3 degradation) . To study this intersection:

• Stimulate neurons or glial cells with TLR3 agonists

• Examine calcium dynamics using fluorescent indicators

• Correlate with RNF170 expression and localization using antibodies

• Compare responses in normal and RNF170-deficient cells

Clinical correlation approaches:

• Analyze RNF170 expression in tissues from patients with neuroinflammatory conditions

• Correlate with inflammatory markers and disease severity

• Compare with findings from animal models of neuroinflammation

Intervention studies:

• Monitor changes in RNF170 expression and localization after treatment with:

  • Anti-inflammatory compounds

  • TLR3 antagonists

  • Calcium modulators

• Use RNF170 antibodies to assess treatment effects on protein levels and distribution

• Correlate with functional outcomes and inflammatory marker levels

This research direction could reveal novel therapeutic targets at the intersection of innate immunity and neurological disorders, where RNF170's dual regulatory roles may be particularly significant.