rnf141 Antibody

What is RNF141 and why is it important in research?

RNF141, also known as ring finger protein 141, ZFP26, or ZNF230, is a 230 amino acid protein characterized by the presence of a RING-type zinc finger motif. This motif is crucial for its function in the ubiquitination pathway of protein degradation and consists of a conserved cysteine-rich domain capable of binding two zinc atoms, essential for structural integrity and functional activity . RNF141 has emerged as an important research target due to its involvement in cancer progression, particularly colorectal cancer, where it functions as an oncogene by upregulating KRAS activity . Additionally, RNF141 plays a role in reproductive biology, with isoform 1 specifically expressed in the testis and potentially acting as a transcription factor during spermatogenesis . Its absence in azoospermic men highlights its importance in testis development and male fertility, making it a significant target for both cancer and reproductive research .

What are the known isoforms of RNF141 and their tissue distribution?

RNF141 is expressed in two distinct isoforms resulting from alternative splicing, each with a unique tissue distribution pattern:

The specific expression of isoform 1 in the testis suggests a specialized role in reproductive biology, particularly as a transcription factor during spermatogenesis . Its importance is underscored by its absence in azoospermic men, indicating its essential role in testis development and male fertility . In contrast, isoform 2's broader tissue distribution suggests more generalized cellular functions across multiple organ systems, potentially related to protein degradation pathways via its ubiquitination activity .

What detection methods are compatible with RNF141 antibodies?

RNF141 antibodies, such as the mouse monoclonal IgG1 kappa light chain antibody (J-23), are versatile research tools compatible with multiple detection methods:

These methods enable comprehensive characterization of RNF141 expression, localization, and interactions in various experimental contexts . The ability to detect RNF141 across mouse, rat, and human samples makes these antibodies particularly valuable for comparative studies across species .

How should RNF141 antibodies be optimized for immunohistochemistry in cancer tissue analysis?

When optimizing RNF141 antibodies for immunohistochemistry in cancer tissue analysis, researchers should implement a systematic approach:

• Antigen retrieval optimization: Test multiple retrieval methods (heat-induced epitope retrieval with citrate buffer pH 6.0 versus EDTA buffer pH 9.0) to determine which best exposes the RNF141 epitope in formalin-fixed, paraffin-embedded tissues.

• Antibody dilution titration: Perform a dilution series (typically 1:50 to 1:500) to identify the optimal concentration that maximizes specific signal while minimizing background. For RNF141 detection in colorectal cancer tissues, studies have successfully employed dilutions in the 1:100-1:200 range .

• Validation with positive and negative controls: Include known RNF141-expressing tissues (such as testis for isoform 1 or colorectal cancer tissues with confirmed high expression) as positive controls, and use tissues from RNF141 knockdown models or tissues known to lack expression as negative controls .

• Signal detection system selection: Compare DAB (3,3'-diaminobenzidine) versus fluorescent-based detection systems to determine which provides better discrimination between RNF141-positive and negative cells, especially when examining heterogeneous tumor tissues.

• Counterstaining optimization: Adjust hematoxylin counterstaining intensity to provide cellular context without obscuring specific RNF141 staining.

Research has demonstrated that RNF141 is extensively upregulated in colorectal cancer tissues compared to adjacent normal tissues, making proper antibody optimization crucial for accurate assessment of its expression patterns in clinical samples .

What are the recommended protocols for using RNF141 antibodies in protein-protein interaction studies?

For investigating protein-protein interactions involving RNF141, particularly its interaction with KRAS and other potential binding partners, the following methodological approach is recommended:

• Co-immunoprecipitation (Co-IP):

  • Lyse cells in a non-denaturing buffer (typically containing 1% NP-40 or 0.5% Triton X-100, 150mM NaCl, 50mM Tris-HCl pH 7.4, plus protease inhibitors)

  • Pre-clear lysates with Protein A/G beads

  • Incubate pre-cleared lysates with RNF141 antibody (2-5 μg per 500 μg total protein) overnight at 4°C

  • Add Protein A/G beads and incubate for 1-2 hours

  • Wash extensively (at least 5 times) with IP buffer

  • Elute and analyze by Western blotting for potential interacting partners

• Bimolecular Fluorescence Complementation (BiFC):

  • Clone RNF141 and potential interacting proteins (e.g., KRAS) into compatible BiFC vectors

  • Co-transfect into appropriate cell lines

  • Allow 24-48 hours for expression and fluorophore maturation

  • Visualize under fluorescence microscopy

• GST Pull-down Assay:

  • Express GST-RNF141 in a bacterial system and purify using glutathione sepharose

  • Incubate purified GST-RNF141 with cell lysates containing potential binding partners

  • Wash extensively to remove non-specific binding

  • Elute and analyze by Western blotting

These approaches have been successfully employed to demonstrate direct binding between RNF141 and KRAS in colorectal cancer research, revealing that RNF141 promotes KRAS activity by facilitating its enrichment on the plasma membrane . The combination of these complementary techniques provides robust validation of protein-protein interactions, with Co-IP detecting endogenous interactions, BiFC visualizing interactions in living cells, and GST pull-down confirming direct binding.

How can RNF141 antibodies be used to assess subcellular localization changes in response to stimuli?

When studying RNF141 subcellular localization changes in response to various stimuli, a multi-faceted approach using RNF141 antibodies is recommended:

• Immunofluorescence microscopy protocol:

  • Seed cells on coverslips and apply experimental stimuli

  • Fix cells (4% paraformaldehyde, 10 minutes), permeabilize (0.1% Triton X-100, 5 minutes), and block (5% BSA, 1 hour)

  • Incubate with primary RNF141 antibody (typically 1:100-1:200 dilution) overnight at 4°C

  • Apply fluorophore-conjugated secondary antibody (1:500-1:1000) for 1 hour at room temperature

  • Counterstain with DAPI for nuclear visualization

  • Mount and image using confocal microscopy

• Subcellular fractionation with Western blotting:

  • Prepare cytoplasmic, membrane, nuclear, and cytoskeletal fractions using differential centrifugation and specific extraction buffers

  • Validate fraction purity using compartment-specific markers (e.g., GAPDH for cytoplasm, Na+/K+ ATPase for plasma membrane)

  • Analyze equal protein amounts from each fraction by Western blotting with RNF141 antibody

  • Quantify relative RNF141 distribution across fractions under different conditions

• Live-cell imaging with fluorescent protein-tagged RNF141:

  • Generate RNF141-GFP fusion constructs

  • Validate construct functionality by comparing to endogenous RNF141 using the antibody

  • Perform time-lapse imaging during stimulus application

  • Confirm findings with fixed-cell immunofluorescence using RNF141 antibody

Research has shown that RNF141 localization to the plasma membrane is functionally significant, particularly in the context of KRAS activation in colorectal cancer cells . Using these approaches, investigators have demonstrated that RNF141 induces KRAS activation by increasing its enrichment on the plasma membrane, without altering total KRAS expression . This process appears to be facilitated by RNF141's interaction with LYPLA1, highlighting the importance of tracking subcellular localization changes in understanding RNF141's role in cancer progression .

How can specificity of RNF141 antibodies be validated in knockout/knockdown systems?

Rigorous validation of RNF141 antibody specificity is essential for reliable research outcomes. A comprehensive validation protocol using knockout/knockdown systems should include:

• CRISPR/Cas9 knockout validation:

  • Generate complete RNF141 knockout cell lines using CRISPR/Cas9 targeting multiple exons

  • Confirm knockout at the genomic level by sequencing and at the mRNA level by RT-qPCR

  • Compare Western blot results between wild-type and knockout cells using the RNF141 antibody

  • A specific antibody should show complete absence of the target band in knockout cells

• siRNA/shRNA knockdown validation:

  • Transfect cells with RNF141-specific siRNA or transduce with shRNA lentiviral vectors

  • Include scrambled siRNA/shRNA controls

  • Confirm knockdown efficiency at the mRNA level using RT-qPCR

  • Perform Western blot analysis with the RNF141 antibody to verify proportional reduction in protein levels

  • Research has demonstrated successful RNF141 knockdown using lentiviral shRNA (LV-sh-RNF141), resulting in significantly decreased protein expression as verified by Western blot

• Overexpression controls:

  • Generate RNF141 overexpression models using lentiviral vectors (LV-RNF141)

  • Verify increased expression using the RNF141 antibody by Western blot

  • The antibody should show proportional increase in signal intensity corresponding to overexpression levels

• Cross-reactivity assessment:

  • Test the antibody against related RNF family proteins in overexpression systems

  • Examine reactivity in tissues/cells known to lack RNF141 expression

  • Perform peptide competition assays where available

These validation approaches have been successfully implemented in colorectal cancer research, where both knockdown and overexpression systems were used to confirm RNF141 antibody specificity before proceeding with functional studies . The validation demonstrated clear discrimination between different expression levels of RNF141, confirming antibody reliability for subsequent experiments on cancer cell proliferation, apoptosis, migration, and invasion .

What approaches resolve conflicting RNF141 expression data between antibody-based methods?

When encountering discrepancies in RNF141 expression data between different antibody-based detection methods, a systematic troubleshooting approach is required:

• Epitope accessibility analysis:

  • Different antibodies may recognize distinct epitopes that vary in accessibility across methods

  • Map epitope locations of different antibodies and determine if they target regions prone to post-translational modifications or conformational changes

  • Test alternative antigen retrieval protocols for fixed tissues or denaturing conditions for Western blotting

• Method-specific validation:

  • For Western blotting: Compare reducing vs. non-reducing conditions and test multiple lysis buffers

  • For immunohistochemistry: Evaluate multiple fixation protocols and antigen retrieval methods

  • For immunofluorescence: Test different permeabilization reagents and fixation times

  • Always include positive controls (tissues with known high RNF141 expression, such as colorectal cancer samples or testis tissue)

• Complementary non-antibody methods:

  • Correlate protein detection with mRNA levels using RT-qPCR

  • Employ mass spectrometry-based proteomics as an antibody-independent method

  • Research on RNF141 in colorectal cancer successfully employed a multi-method approach, combining real-time PCR, Western blot, and immunohistochemical analysis to comprehensively assess expression patterns

• Isoform-specific considerations:

  • Determine if discrepancies relate to differential detection of RNF141 isoforms

  • Verify which isoforms are expressed in your experimental system

  • Design isoform-specific detection strategies

By systematically addressing these factors, researchers can reconcile conflicting data and establish reliable RNF141 detection protocols across multiple experimental platforms.

How should researchers optimize co-immunoprecipitation protocols for detecting transient RNF141-protein interactions?

Detecting transient or weak interactions between RNF141 and its binding partners requires specialized co-immunoprecipitation (Co-IP) protocols:

• Cross-linking approach:

  • Treat cells with membrane-permeable crosslinkers (e.g., DSP, formaldehyde) at low concentrations (0.5-2%) for short durations (5-15 minutes)

  • Quench the reaction with glycine or Tris

  • Proceed with standard Co-IP protocol using RNF141 antibody

  • Include a reverse crosslinking step before SDS-PAGE analysis

• Detergent optimization:

  • Test multiple detergent types and concentrations to balance solubilization and preservation of protein complexes

  • For RNF141-KRAS interactions, which occur at the plasma membrane, use milder detergents (0.3-0.5% NP-40 or 0.1-0.2% digitonin) rather than stronger options like SDS or deoxycholate

  • Consider detergent-free extraction methods for membrane proteins

• Nucleotide state control (particularly for GTPases like KRAS):

  • Add non-hydrolyzable GTP analogs (GTPγS) to stabilize GTP-bound states

  • Include EDTA/EGTA to promote nucleotide exchange or Mg²⁺ to stabilize nucleotide binding

  • Research has shown that RNF141 specifically increases GTP-bound KRAS, making these considerations particularly relevant

• Proximity-based alternatives:

  • For particularly challenging interactions, consider proximity ligation assay (PLA)

  • This technique can detect protein interactions in situ with high sensitivity

  • Requires two primary antibodies (anti-RNF141 and anti-interacting protein) from different species

  • Results in punctate fluorescent signals where proteins are in close proximity (<40 nm)

• Sequential immunoprecipitation (tandem IP):

  • First IP with RNF141 antibody

  • Elute under mild conditions

  • Perform second IP with antibody against suspected interacting protein

These approaches have proven successful in demonstrating the interaction between RNF141 and KRAS in colorectal cancer research, where standard Co-IP was complemented with immunofluorescence assays, bimolecular fluorescence complementation (BiFC), and GST pull-down assays to provide robust confirmation of the interaction . This multi-method strategy is particularly important for validating transient or context-dependent interactions that might be missed by any single approach.

How can RNF141 antibodies be used to investigate its role in colorectal cancer progression?

RNF141 antibodies serve as essential tools for investigating the protein's involvement in colorectal cancer (CRC) progression through multiple experimental approaches:

• Expression correlation with clinical parameters:

  • Immunohistochemical analysis of patient tumor samples using RNF141 antibodies has revealed significantly higher expression in CRC tissues compared to adjacent normal tissues

  • Expression levels have been correlated with T stage, suggesting a relationship between RNF141 abundance and tumor invasion depth

  • This approach requires careful standardization of staining protocols and scoring systems to ensure reproducibility across patient cohorts

• Functional pathway analysis:

  • RNF141 antibodies enable Western blot detection of key downstream effectors following RNF141 manipulation

  • Knockdown and overexpression studies have revealed that RNF141 affects proliferation markers (PCNA), apoptosis regulators, and cell cycle proteins

  • Specific findings include decreased PCNA expression after RNF141 knockdown and increased PCNA expression following RNF141 overexpression in HCT116, SW480, and DLD-1 colorectal cancer cell lines

• Subcellular localization and protein-protein interactions:

  • Immunofluorescence with RNF141 antibodies has demonstrated co-localization with KRAS at the plasma membrane

  • Co-immunoprecipitation has confirmed direct interaction between RNF141 and KRAS

  • These methods revealed that RNF141 promotes KRAS enrichment at the plasma membrane, enhancing its activity without altering total KRAS expression levels

• Angiogenesis assessment:

  • RNF141 antibodies enable the study of its effect on angiogenic potential

  • Tube formation assays with HUVECs have shown that RNF141 knockdown disrupts tube formation, while its overexpression enhances this process

These applications of RNF141 antibodies have collectively established that RNF141 functions as an oncogene in CRC by upregulating KRAS activity through promoting its enrichment on the plasma membrane, which subsequently drives proliferation, migration, invasion, and angiogenesis while inhibiting apoptosis .

What are the optimal experimental designs for studying RNF141's E3 ligase activity using specific antibodies?

Investigating RNF141's E3 ligase activity requires carefully designed experiments utilizing specific antibodies:

• In vitro ubiquitination assays:

  • Express and purify recombinant RNF141 (wild-type and RING domain mutants)

  • Combine with E1, E2 enzymes, ubiquitin (wild-type, K48-only, K63-only, or tagged versions), ATP, and potential substrates

  • Incubate at 30-37°C for 1-3 hours

  • Analyze by Western blotting using anti-ubiquitin and RNF141-specific antibodies

  • Include controls lacking individual components to verify specificity

• Cellular ubiquitination analysis:

  • Transfect cells with HA/FLAG-tagged ubiquitin and RNF141 (or use endogenous RNF141)

  • Treat with proteasome inhibitors (MG132, 10μM, 4-6 hours) to prevent degradation of ubiquitinated proteins

  • Lyse cells under denaturing conditions (1% SDS with boiling) followed by dilution for immunoprecipitation

  • Immunoprecipitate potential substrates and immunoblot with anti-ubiquitin antibodies

  • Alternatively, immunoprecipitate ubiquitinated proteins using anti-HA/FLAG and immunoblot for substrates

• RING domain mutant comparisons:

  • Design RING domain mutations affecting zinc coordination (typically cysteine to alanine substitutions)

  • Express wild-type and mutant RNF141 in cellular systems

  • Compare ubiquitination activity using the methods above

  • Analyze effects on protein-protein interactions, particularly with KRAS, using co-immunoprecipitation with RNF141 antibodies

• Proximity-dependent ubiquitination detection:

  • Implement BioID or TurboID fusion proteins with RNF141

  • Express in target cells, provide biotin

  • Purify biotinylated proteins and analyze by mass spectrometry

  • Validate identified substrates using RNF141 antibodies in ubiquitination assays

While current research has established RNF141's role as a RING finger protein involved in the ubiquitination pathway , detailed characterization of its specific E3 ligase activity, substrate specificity, and ubiquitin chain preferences in the context of colorectal cancer remains an area for further investigation. The experimental designs outlined above would leverage RNF141 antibodies to fill these knowledge gaps and potentially identify novel therapeutic targets.

How can researchers utilize RNF141 antibodies to develop potential therapeutic approaches for colorectal cancer?

RNF141 antibodies can facilitate the development of therapeutic strategies for colorectal cancer through several research applications:

• Target validation and patient stratification:

  • Evaluate RNF141 expression in large patient cohorts using tissue microarrays and immunohistochemistry

  • Correlate expression levels with clinical outcomes and treatment responses

  • Identify patient subgroups most likely to benefit from RNF141-targeted therapies

  • Current research has already established that RNF141 is extensively upregulated in CRC tissues compared to adjacent normal tissues, providing initial validation of its potential as a therapeutic target

• Therapeutic antibody development pipeline:

  • Generate screening assays using current research-grade RNF141 antibodies to identify epitopes critical for:

    • RNF141-KRAS interaction

    • RNF141 localization to the plasma membrane

    • RNF141's interaction with LYPLA1

  • Develop and test therapeutic antibodies or antibody fragments targeting these epitopes

  • Assess cell penetration methods for targeting intracellular RNF141

• Small molecule inhibitor screening:

  • Develop RNF141 antibody-based competition assays to screen for small molecules that disrupt:

    • RNF141-KRAS binding

    • RNF141 membrane localization

  • Employ fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) assays with labeled antibodies to monitor protein-protein interactions in the presence of candidate inhibitors

• Combination therapy assessment:

  • Use RNF141 antibodies to monitor protein expression and activity during treatment with existing therapies

  • Investigate synergistic effects between RNF141 inhibition and other treatment modalities

  • Since RNF141 promotes KRAS activity, combining RNF141 inhibition with downstream KRAS pathway inhibitors might enhance therapeutic efficacy

• Plug-and-Play antibody strategy adaptation:

  • Explore the potential of adapting innovative antibody-based "Plug-and-Play" strategies, similar to those developed for other targets like SARS-CoV-2 spike protein

  • This could involve engineering RNF141-targeting antibodies with modified Fc regions to enhance cellular penetration or recruitment of immune effectors

  • Alternatively, bispecific antibodies targeting both RNF141 and cell surface markers on colorectal cancer cells could be developed

These approaches leverage the research finding that RNF141 functions as an oncogene by upregulating KRAS activity in colorectal cancer . Given that KRAS mutations are present in approximately 30-50% of colorectal cancers and are associated with resistance to many targeted therapies, targeting the RNF141-KRAS axis represents a potentially valuable therapeutic strategy that could overcome limitations of direct KRAS inhibition .