RNF43 Antibody, HRP conjugated

What is RNF43 and why is it important in cancer research?

RNF43 (RING finger protein 43) is a 90 kDa transmembrane E3 ubiquitin ligase belonging to the ZNRF3 family of proteins. It contains a putative 23 amino acid signal sequence, a 174 amino acid extracellular domain, a transmembrane domain, and a cytoplasmic domain with an atypical RING-type zinc finger. RNF43 functions primarily as a negative regulator of the Wnt signaling pathway by ubiquitinating Frizzled Wnt receptors, promoting their turnover and thereby antagonizing Wnt signaling. This regulatory role is critical as RNF43 is expressed in stem cells at the bottom of colon crypts, where it limits the ability of Wnts to induce proliferation .

RNF43's importance in cancer research stems from its frequent mutation in multiple cancers, particularly colorectal malignancies. Deletion or mutation of RNF43 leads to hypersensitivity to Wnts and promotes adenoma formation, making it a significant tumor suppressor gene . Furthermore, while RNF43 mutations generally promote cancer through loss of function, RNF43 has paradoxically been observed to be over-expressed in some cancers, correlating with growth-promoting activity in colorectal and hepatocellular cancer pathogenesis .

What epitopes of RNF43 are most commonly targeted by commercial antibodies?

Commercial RNF43 antibodies target various regions of the protein, with significant variation in the epitopes recognized. Analysis of available antibodies reveals targeting of multiple domains: N-terminal regions (AA 41-140), middle regions (AA 401-500), and C-terminal domains (AA 543-572 and AA 566-697) . The selection of target epitope is critical for experimental design as different domains may be differently exposed depending on protein conformation, post-translational modifications, and protein-protein interactions.

How do HRP-conjugated RNF43 antibodies differ from other conjugated versions?

HRP-conjugated RNF43 antibodies utilize horseradish peroxidase as the reporter enzyme, which catalyzes a reaction producing a colorimetric, chemiluminescent, or fluorescent signal depending on the substrate used. This differs from fluorophore-conjugated antibodies like Alexa Fluor 488-conjugated versions, which emit fluorescence directly when excited with appropriate wavelengths. The primary differences lie in detection method, sensitivity, stability, and application suitability.

What validation steps are essential before using RNF43 antibodies in research?

Given the documented issues with RNF43 antibody specificity, rigorous validation is absolutely critical before conducting experiments. Multiple studies have shown that commercially available RNF43 antibodies detect non-specific bands or staining patterns that persist even in RNF43 knockout cells . Therefore, the following validation steps are essential:

How can researchers optimize western blot protocols for HRP-conjugated RNF43 antibodies?

Optimizing western blot protocols for HRP-conjugated RNF43 antibodies requires special consideration given the extremely low endogenous expression levels of RNF43 and potential non-specific binding issues:

• Protein enrichment: Standard immunoblotting may be insufficient to detect endogenous RNF43. Consider immunoprecipitation prior to western blotting to concentrate the target protein. This approach has been demonstrated to be necessary even in cells with HA-tagged endogenous RNF43 .

• Blocking optimization: Use 5% BSA in TBS-T rather than milk for blocking when working with phospho-sensitive epitopes, as RNF43 function is regulated by serine phosphorylation .

• Extended exposure times: Due to low expression levels, longer exposure times may be necessary, but this increases the risk of detecting non-specific bands. Always run appropriate knockout controls in parallel to distinguish specific from non-specific signals.

• Gradient gels: Use 4-12% gradient gels to better resolve the full-length RNF43 (approximately 90 kDa) from potential truncated forms or non-specific bands.

• Stringent washing: Implement more stringent washing steps (longer and more frequent washes with TBS-T) to reduce background signal that may interfere with detecting low-abundance RNF43.

• Signal enhancement systems: Consider using signal enhancement systems compatible with HRP detection that can improve sensitivity while maintaining signal-to-noise ratio.

Remember that published research has shown that several commercial RNF43 antibodies detect bands at the expected molecular weight even in RNF43 knockout cell lines, indicating these bands are not RNF43 . Thus, appropriate controls are absolutely essential for accurate interpretation.

What controls should be included when performing immunohistochemistry with HRP-conjugated RNF43 antibodies?

When performing immunohistochemistry (IHC) with HRP-conjugated RNF43 antibodies, comprehensive controls are essential to ensure reliable results:

• Genetically defined controls: Include tissue sections from RNF43 knockout models or cell lines prepared as formalin-fixed paraffin-embedded (FFPE) blocks. Research has shown that antibodies like HPA008079, ab84125, and ab217787, commonly used for IHC, produce non-specific staining patterns in RNF43-deficient samples .

• Absorption controls: Pre-incubate the antibody with excess immunizing peptide before application to tissue sections. Specific staining should be abolished while non-specific staining will remain.

• Isotype controls: Include control sections stained with non-specific IgG of the same isotype, host species, and concentration as the RNF43 antibody to identify non-specific binding.

• Known positive and negative tissues: Include tissues with verified high and low/no expression of RNF43 based on transcriptomic data rather than prior antibody-based studies which may be unreliable.

• Titration series: Perform a titration series to determine the optimal antibody concentration that maximizes specific signal while minimizing background.

• Alternative detection methods: Confirm IHC results using RNA-based methods like in situ hybridization to verify RNF43 expression patterns independently of antibody-based detection.

Published research has demonstrated that IHC staining patterns observed with commercial RNF43 antibodies were identical in RNF43-expressing and RNF43-knockout samples, strongly suggesting that previous IHC-based studies of RNF43 expression in tissues may have detected non-specific signals rather than RNF43 protein .

How reliable are subcellular localization studies using RNF43 antibodies?

Current evidence strongly suggests that subcellular localization studies using available RNF43 antibodies are highly unreliable. Comprehensive validation studies have demonstrated that four commonly used commercial antibodies (HPA008079, 8D6, ab84125, and ab217787) all produced non-specific staining patterns in immunofluorescence experiments that were identical in both RNF43-expressing and RNF43-knockout cell lines .

The HPA008079 antibody showed prominent non-specific nuclear staining, 8D6 displayed both nuclear staining and non-specific labeling of cellular protrusions, ab84125 produced punctate staining of perinuclear structures resembling Golgi or endoplasmic reticulum, and ab217787 detected non-specific nuclear structures with diffuse cytoplasmic staining . These patterns were observed regardless of whether RNF43 was present in the cells, conclusively demonstrating that these staining patterns do not represent true RNF43 localization.

These findings call into question previously published reports suggesting nuclear functions for RNF43, such as tethering TCF7L2 to the nuclear membrane or roles in the DNA damage response through γH2AX ubiquitination . Researchers should exercise extreme caution when interpreting published subcellular localization data for RNF43 and should consider alternative approaches such as epitope tagging of endogenous RNF43 followed by tag-specific antibody detection, though even this approach may be challenging due to extremely low endogenous expression levels .

What are the major challenges in detecting endogenous RNF43 with antibodies?

Detecting endogenous RNF43 with antibodies presents several major challenges:

• Extremely low expression levels: Endogenous RNF43 protein is expressed at such low levels that it cannot be detected by simple immunoblotting or immunofluorescent staining, even when endogenously tagged with epitopes like HA. Researchers have found that immunoprecipitation is necessary before detection becomes possible .

• Non-specific binding: Multiple commercial antibodies have been shown to recognize non-RNF43 proteins even in cell lines where RNF43 has been knocked out, creating false positive signals . This non-specificity has been documented across multiple applications including western blotting, immunofluorescence, and immunohistochemistry.

• Epitope accessibility: RNF43's complex membrane topology may limit epitope accessibility, particularly for antibodies targeting the extracellular or transmembrane domains under non-denaturing conditions.

• Post-translational modifications: RNF43 function is regulated by serine phosphorylation, which may affect antibody binding depending on the target epitope and the phosphorylation state of the protein .

• Dynamic regulation: RNF43 levels and localization may be dynamically regulated in response to Wnt signaling and other cellular conditions, potentially complicating detection if samples are not appropriately timed or preserved.

These challenges collectively suggest that researchers should consider alternative approaches for studying RNF43, such as tagged knock-in lines validated by genomic sequencing, or focusing on functional assays and downstream effects rather than direct protein detection .

How can RNF43 phosphorylation status be assessed using antibody-based techniques?

• Phospho-specific antibodies: While phospho-specific antibodies would be ideal, no validated commercial phospho-specific RNF43 antibodies are currently documented in the literature. Development of such antibodies would require careful validation using phospho-mimetic (e.g., serine to aspartate) and phospho-deficient (e.g., serine to alanine) mutants as controls.

• Phosphatase treatment controls: Researchers can treat sample lysates with lambda phosphatase prior to immunoblotting with general RNF43 antibodies. A mobility shift after phosphatase treatment would indicate phosphorylation, though this approach requires antibodies that can reliably detect endogenous RNF43, which is problematic given known specificity issues.

• Phos-tag SDS-PAGE: This technique incorporates Phos-tag molecules into acrylamide gels to specifically retard the migration of phosphorylated proteins. When combined with western blotting using RNF43 antibodies, this can reveal phosphorylated species as higher molecular weight bands compared to unphosphorylated forms.

• Mass spectrometry approaches: Given the limitations of antibody-based detection, phosphorylation analysis by mass spectrometry following immunoprecipitation of epitope-tagged RNF43 may be more reliable than direct antibody detection of phosphorylated forms.

• Functional assays with phospho-mutants: Rather than directly detecting phosphorylation, researchers can compare the function of wild-type RNF43 with phospho-mimetic (3SD) and phospho-deficient (3SA) mutants in assays measuring Frizzled receptor ubiquitination and degradation .

Research has demonstrated that RNF43(3SA) and RNF43(3SD) phospho-mutants behave similarly to wild-type RNF43 in terms of protein-protein interactions, homodimer formation, heterodimer formation with ZNRF3, and subcellular localization, suggesting that phosphorylation primarily regulates catalytic activity rather than these other properties .

What are the most common false positive patterns observed with RNF43 antibodies?

Understanding common false positive patterns is crucial for avoiding misinterpretation of results when working with RNF43 antibodies. Research has documented the following non-specific patterns that persist even in RNF43 knockout cells:

• Western blotting false positives: Multiple bands at various molecular weights, including bands at the expected size for RNF43 (approximately 90 kDa), have been observed with antibodies including HPA008079, 8D6, ab84125, and ab217787 .

• Immunofluorescence false positives:

  • Nuclear staining: HPA008079 and 8D6 antibodies produce strong nuclear staining patterns regardless of RNF43 expression

  • Perinuclear/ER/Golgi-like staining: ab84125 shows punctate staining of structures adjacent to the nucleus

  • Cellular protrusions: 8D6 antibody non-specifically stains cellular protrusions

  • Nuclear structures and diffuse cytoplasmic staining: ab217787 produces these patterns independent of RNF43 expression

• Immunohistochemistry false positives: The same non-specific staining patterns observed in immunofluorescence experiments are also seen in FFPE tissue sections, with nuclear staining being particularly problematic for HPA008079 and 8D6 antibodies .

How should researchers interpret discrepancies between antibody-based detection and mRNA expression data for RNF43?

When facing discrepancies between antibody-based protein detection and mRNA expression data for RNF43, researchers should approach interpretation with the following considerations:

Given that multiple studies have now demonstrated the unreliability of several commercial RNF43 antibodies, researchers should be extremely cautious about concluding that protein expression patterns differ from mRNA expression patterns without multiple lines of supporting evidence .

What alternative approaches can replace antibody-based detection for studying RNF43?

Given the significant challenges with antibody-based detection of endogenous RNF43, researchers should consider these alternative approaches:

• Genome editing with epitope tags: CRISPR/Cas9-mediated knock-in of small epitope tags (HA, FLAG, etc.) at the endogenous RNF43 locus followed by detection with highly specific tag antibodies. This approach has been successfully implemented, though even tagged endogenous RNF43 required immunoprecipitation before detection by western blotting due to extremely low expression levels .

• Fluorescent protein fusions: Knock-in of fluorescent proteins like GFP at the endogenous locus can enable live-cell imaging of RNF43 without antibodies, though the large size of fluorescent proteins may affect protein function and localization.

• Proximity labeling techniques: BioID or APEX2 fusions to RNF43 can identify proximal proteins and indirectly map subcellular localization without relying on direct antibody detection of RNF43.

• Functional readouts: Measuring downstream effects of RNF43 activity, such as Frizzled receptor levels or Wnt pathway activation, can serve as functional proxies for RNF43 expression and activity.

• mRNA detection methods: In situ hybridization or single-cell RNA sequencing can provide spatial information about RNF43 expression at the transcript level, which may be more reliable than protein detection given current antibody limitations.

• Mass spectrometry: For quantitative protein detection, targeted mass spectrometry approaches like selected reaction monitoring (SRM) can detect and quantify specific RNF43 peptides without antibodies.

• Genetic reporter systems: Knock-in of split reporter systems or transcriptional reporters at the endogenous locus can enable functional studies without directly detecting the protein.

These approaches can circumvent the specificity issues plaguing current RNF43 antibodies and provide more reliable data about this important tumor suppressor protein's expression, localization, and function in normal and disease states .

What technical advances might improve RNF43 antibody specificity and sensitivity?

Several technical advances could potentially address the current limitations in RNF43 antibody specificity and sensitivity:

• Novel immunization strategies: Using highly purified full-length RNF43 protein rather than peptide fragments might generate antibodies with improved specificity. Additionally, subtractive immunization techniques, where animals are tolerized to common cross-reactive epitopes before immunization with RNF43, could reduce non-specific binding.

• Recombinant antibody technology: Phage display or yeast display libraries could be screened against RNF43 in the presence of competing proteins to select for highly specific binders. These technologies also allow for affinity maturation to improve sensitivity for low-abundance endogenous RNF43.

• Nanobodies and alternative binding scaffolds: Single-domain antibodies (nanobodies) or non-antibody scaffolds like DARPins or Affibodies might access epitopes that conventional antibodies cannot, potentially offering improved specificity.

• Conformational epitope targeting: Developing antibodies that recognize specific conformational states of RNF43 rather than linear epitopes might improve specificity, particularly if these conformations are unique to RNF43.

• Proximity-dependent detection: Developing split reporter systems where one part is fused to a validated RNF43 interactor and the other to an anti-RNF43 antibody could provide signal only when both components are in close proximity, reducing false positives from non-specific antibody binding.

• Multiplexed verification approaches: Creating antibody panels that target different RNF43 epitopes and analyzing their staining patterns using multiplexed imaging could help distinguish true from false signals through coincidence detection.

These advances would need rigorous validation using the same genetic knockout controls that have revealed the limitations of current antibodies, ensuring that any new reagents truly offer improved specificity for endogenous RNF43 .

How are recent findings on RNF43 antibody specificity impacting interpretation of previous research?

Recent findings demonstrating poor specificity of RNF43 antibodies have significant implications for interpreting previous research:

The scientific community should recognize these limitations when evaluating previous literature on RNF43 and consider employing more stringent controls in future studies, including genetic knockout validation and complementary methodology beyond antibody-based detection .

What promising applications exist for HRP-conjugated RNF43 antibodies despite current limitations?

Despite the significant limitations of current RNF43 antibodies, several promising applications for HRP-conjugated versions could be developed with proper validation:

• Detecting overexpressed RNF43: Although current antibodies fail to reliably detect endogenous RNF43, some can detect overexpressed protein . HRP-conjugated antibodies could therefore be valuable for screening cell lines or tissues engineered to overexpress wild-type or mutant RNF43, particularly in functional studies examining the consequences of RNF43 mutations found in cancer.

• Proximity ligation assays: By combining an RNF43 antibody with antibodies against well-established RNF43 interactors like ZNRF3, FZD receptors, or DVL, proximity ligation assays could provide more specific detection through co-localization requirements, reducing the impact of individual antibody non-specificity.

• Western blotting of immunoprecipitated RNF43: For cells with epitope-tagged endogenous RNF43, HRP-conjugated RNF43 antibodies could provide sensitive detection following immunoprecipitation with tag-specific antibodies, especially when investigating post-translational modifications or protein-protein interactions .

• Screening for modulators of RNF43 stability: In cell-based assays where RNF43 is expressed at higher levels, HRP-conjugated antibodies could help identify compounds or genetic factors that modulate RNF43 protein stability or expression, potentially leading to therapeutic approaches for cancers with RNF43 mutations.

• Multiplex staining with orthogonal validation: Combining HRP-conjugated RNF43 antibodies with orthogonal markers in multiplex staining approaches could help disambiguate true from false signals through correlation analysis with functional or genetic markers of RNF43 pathway activity.