RNF7 Antibody

What is RNF7 and why is it important in cancer research?

RNF7 (RING finger protein-7), also known as SAG (sensitive to apoptosis gene), ROC2 (regulator of cullins 2), or Rbx2 (RING-box 2), is an evolutionarily conserved protein with a molecular weight of 12.6 kDa that functions as a component of E3 ubiquitin ligases. RNF7 is critically important in cancer research due to its overexpression in multiple human cancers and its demonstrated roles in tumor proliferation and progression . In pancreatic cancer, RNF7 facilitates tumorigenesis by activating the PI3K/Akt signaling pathway, promoting cell proliferation, migration, and invasion . Similarly, in prostate cancer, RNF7 supports tumor progression through the ERK1/2 pathway activation, with its knockdown resulting in attenuated proliferation and enhanced sensitivity to treatments like cisplatin . As an antioxidant protein, RNF7 also protects cancer cells from apoptosis caused by oxidation, further contributing to cancer cell survival . These multifaceted roles make RNF7 a valuable target for understanding cancer biology and developing potential therapeutic strategies.

What are the recommended applications for RNF7 antibodies in experimental research?

RNF7 antibodies have demonstrated efficacy across multiple experimental applications essential for comprehensive protein characterization. Based on validated testing, the primary applications include Western Blot (WB) at dilutions of 1:500-1:1000, Immunoprecipitation (IP) using 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate, Immunohistochemistry (IHC), and Immunofluorescence (IF/ICC) at dilutions of 1:200-1:800 . Additionally, RNF7 antibodies can be employed for ELISA assays. When designing experiments, researchers should note that these antibodies have been tested for reactivity with human, mouse, and rat samples, with positive Western Blot detection specifically confirmed in human liver, heart, skeletal muscle, and testis tissues . For optimization in specific experimental systems, titration is strongly recommended as results may be sample-dependent. The versatility across these applications makes RNF7 antibodies valuable tools for investigating protein expression, localization, and interactions in both normal and pathological contexts.

How should RNF7 antibody samples be properly stored and handled for optimal performance?

For optimal performance of RNF7 antibodies, proper storage and handling protocols must be strictly followed. The recommended storage conditions for RNF7 antibodies include maintaining them at -20°C in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, the antibodies remain stable for one year after shipment. Importantly, aliquoting is unnecessary for -20°C storage, which simplifies laboratory management. For the 20 μl size preparations, it should be noted that they contain 0.1% BSA as a stabilizer . When handling the antibody for experiments, minimize freeze-thaw cycles by allowing only the required amount to reach room temperature while keeping the stock frozen. Prior to use, gently mix the antibody solution by inversion rather than vortexing to prevent protein denaturation. For long-term storage beyond one year, consider storage at -80°C, particularly if the antibody will be used for sensitive applications like immunofluorescence where signal integrity is critical.

How can RNF7 antibodies be optimized for immunohistochemical detection of expression levels in cancer tissues?

Optimizing RNF7 antibodies for immunohistochemical detection in cancer tissues requires several methodological considerations. Start with tissue-specific antigen retrieval protocols—for RNF7 detection in pancreatic cancer tissues, heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes has demonstrated superior results . The optimal primary antibody dilution range is 1:50-1:500, but this should be determined experimentally for each tissue type . For signal amplification, employ a polymer-based detection system rather than avidin-biotin methods to reduce background signal, which is particularly important when analyzing the typically modest expression differences between normal and cancerous tissues. Counterstaining with hematoxylin for 30 seconds provides adequate nuclear contrast without obscuring RNF7 cytoplasmic and nuclear signals . Always include appropriate positive controls (human testis or skeletal muscle tissue, which naturally express high RNF7 levels) and negative controls (primary antibody omission and IgG isotype controls) . For quantification, use a standardized scoring system that incorporates both staining intensity (0-3+) and percentage of positive cells, as was successfully employed in the pancreatic cancer studies where RNF7 overexpression correlated with poor patient survival .

What strategies are effective for silencing RNF7 expression in experimental cancer models?

Effective RNF7 silencing in experimental cancer models can be achieved through several validated approaches. RNA interference using shRNA delivered via retroviral or lentiviral vectors has demonstrated high efficiency, with published studies showing reduction of RNF7 expression to 11-26% of control levels in prostate cancer cell lines . When designing an RNF7 knockdown experiment, researchers should test multiple shRNA constructs targeting different regions of the RNF7 transcript, as silencing efficiency can vary significantly—for example, shRNF7-2 demonstrated superior knockdown compared to shRNF7-1 in both DU145 and PC3 prostate cancer cell lines (11.2-12.1% versus 24.9-26.2% remaining expression, respectively) . For long-term studies, stable cell lines should be established through antibiotic selection and periodically validated for maintained RNF7 suppression. CRISPR-Cas9 gene editing represents an alternative approach for complete RNF7 knockout, though careful sgRNA design is essential given RNF7's relatively small size (113 amino acids) . For in vivo models, inducible knockdown systems are recommended to avoid potential developmental effects, particularly when studying tumors in which RNF7 significantly affects growth kinetics—as evidenced by the dramatically reduced tumor volume and weight observed in RNF7-silenced xenograft models .

How can researchers effectively investigate RNF7's role in different signaling pathways using available antibodies?

Investigating RNF7's role in signaling pathways requires a multi-methodological approach centered around high-quality antibodies. For PI3K/Akt pathway analysis in pancreatic cancer, researchers should implement co-immunoprecipitation assays using RNF7 antibodies (0.5-4.0 μg per 1.0-3.0 mg lysate) followed by immunoblotting for pathway components like phospho-Akt and phospho-mTOR . Western blot analysis should include pathway inhibitors (such as perifosine at 5 μM concentration) to establish causality through rescue experiments, as demonstrated in studies showing RNF7 overexpression increased phospho-Akt and phospho-mTOR levels, while perifosine treatment reversed these effects . For investigating ERK1/2 pathway connections, time-course experiments following epidermal growth factor (EGF) stimulation (5-60 minutes) have proven effective for detecting the impact of RNF7 knockdown on ERK phosphorylation status . Immunofluorescence co-localization studies using RNF7 antibodies at 1:200-1:800 dilution can reveal subcellular interactions between RNF7 and signaling components . For comprehensive pathway mapping, researchers should combine these approaches with quantitative proteomic analysis of RNF7-interacting proteins under various cellular conditions, always including controls for antibody specificity to ensure observed effects are attributable to RNF7-specific interactions rather than experimental artifacts.

What are common issues in RNF7 antibody-based Western blotting and how can they be resolved?

When performing Western blotting with RNF7 antibodies, several technical challenges may arise. The relatively small size of RNF7 (13 kDa observed molecular weight) requires careful gel preparation—use 15% polyacrylamide gels with extended running times to achieve adequate separation from the dye front . Non-specific bands can appear, particularly in muscle tissue samples where RNF7 is highly expressed; this can be mitigated by increasing blocking time to 2 hours with 5% non-fat milk and including 0.1% Tween-20 in wash buffers . If signal intensity is weak, consider using PVDF membranes instead of nitrocellulose for better protein retention, and optimize primary antibody concentration within the 1:500-1:1000 range . For tissues with low RNF7 expression, increase protein loading to 50-100 μg per lane and employ enhanced chemiluminescence detection systems. Inconsistent results between experiments often stem from variable transfer efficiency; standardize transfer conditions using pre-chilled buffers and monitor with reversible protein stains like Ponceau S. When analyzing post-translational modifications of RNF7, include phosphatase inhibitors in lysis buffers and consider specialized membranes for detecting ubiquitination patterns. Always validate antibody specificity using positive controls (human testis or skeletal muscle tissue) and negative controls (lysates from RNF7 knockdown cells) .

What methodological approaches help distinguish between specific and non-specific binding in RNF7 immunoprecipitation experiments?

To distinguish between specific and non-specific binding in RNF7 immunoprecipitation (IP) experiments, researchers should implement multiple control strategies and optimization techniques. First, always include a negative control using non-immune IgG of the same species and concentration as the RNF7 antibody to identify proteins that bind non-specifically to immunoglobulins or beads . For RNF7 IP, use the recommended antibody amount (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) and optimize the antibody-to-lysate ratio for each experimental system . Pre-clearing lysates with protein A/G beads for 1 hour at 4°C before adding the RNF7 antibody can significantly reduce non-specific binding. When analyzing RNF7 interactions with signaling proteins like those in the PI3K/Akt or ERK1/2 pathways, include controls with pathway inhibitors (e.g., perifosine) or activators to confirm specificity of observed interactions . For reverse confirmation, perform reciprocal IPs using antibodies against suspected RNF7 interacting partners. Additional validation can be achieved through competition assays with recombinant RNF7 protein added to the immunoprecipitation reaction. When investigating ubiquitination targets, include deubiquitinase inhibitors in lysis buffers and consider using denaturing conditions to disrupt non-covalent interactions. Finally, mass spectrometry analysis of immunoprecipitated complexes can provide unbiased identification of true interacting partners versus common contaminants in IP experiments.

How should conflicting data about RNF7's role in apoptosis across different tissue types be interpreted?

The apparently conflicting reports regarding RNF7's role in apoptosis across different tissue types represent an important research complexity requiring nuanced interpretation. In prostate cancer cells, RNF7 knockdown led to increased apoptosis through accumulation of tumor suppressive proteins including NOXA, suggesting an anti-apoptotic function . Conversely, in skeletal muscle cells, RNF7 appears to promote apoptosis while inhibiting autophagy . These divergent findings should be interpreted within a tissue-specific context rather than as contradictions. Different cell types express unique profiles of RNF7 interacting partners and substrates for its E3 ubiquitin ligase activity, potentially explaining its opposing effects. Researchers should analyze these differences by systematically comparing RNF7 binding partners across tissue types using immunoprecipitation followed by mass spectrometry. The subcellular localization of RNF7—cytoplasmic and nuclear—may also vary between tissues, influencing its functional impact . Additionally, the activation status of relevant signaling pathways (PI3K/Akt in pancreatic cancer, ERK1/2 in prostate cancer) likely determines whether RNF7 promotes or inhibits apoptosis . When designing experiments to resolve these discrepancies, researchers should include comprehensive controls, use multiple cell lines representing each tissue type, and employ both gain- and loss-of-function approaches to fully characterize the tissue-specific determinants of RNF7's role in cell death regulation.

How can researchers quantitatively assess changes in RNF7 activity rather than just expression levels?

Quantitatively assessing RNF7 activity, as distinct from mere expression levels, requires specialized methodological approaches focused on its E3 ubiquitin ligase function. Researchers should implement in vitro ubiquitination assays using immunoprecipitated RNF7 from cell lysates, purified E1 and E2 enzymes, ubiquitin, ATP, and known or suspected substrate proteins to measure ubiquitin transfer rates . For cellular systems, pulse-chase experiments tracking the half-life of known RNF7 substrates (such as p21, p27, and NOXA in prostate cancer cells) provide a functional readout of RNF7 activity . Quantitative mass spectrometry using stable isotope labeling can identify changes in the ubiquitinome following RNF7 manipulation, revealing both the breadth and specificity of its activity. Phosphorylation status of pathway components like Akt, mTOR, and ERK1/2 serves as an indirect measure of RNF7 activity; for instance, in pancreatic cancer models, RNF7 overexpression increased phospho-Akt and phospho-mTOR levels, indicating enhanced pathway activation . Reporter systems using luciferase fused to known RNF7 substrates can provide real-time activity measurements in living cells. When implementing these approaches, appropriate controls must include catalytically inactive RNF7 mutants and proteasome inhibitors to distinguish between ubiquitination and degradation effects. By focusing on these functional readouts rather than simple expression analysis, researchers can gain deeper insights into the biological significance of RNF7 in both normal and pathological contexts.

What novel experimental approaches could advance our understanding of RNF7's role in cancer progression?

Advancing our understanding of RNF7's role in cancer progression requires innovative experimental approaches beyond conventional methods. Single-cell proteomics combined with spatial transcriptomics could reveal cell-type specific variations in RNF7 expression and activity within heterogeneous tumor microenvironments, providing insights into cellular interactions that drive progression. CRISPR-Cas9 screens targeting RNF7 interacting partners identified through proximity labeling methods (BioID or APEX) would systematically map the functional importance of these interactions in various cancer contexts . Patient-derived organoids with modulated RNF7 expression could serve as more physiologically relevant models than traditional cell lines for studying effects on tumorigenesis and drug response. For in vivo studies, developing conditional tissue-specific RNF7 knockout mouse models would allow temporal control of RNF7 deletion at different cancer stages. Implementation of proteolysis-targeting chimeras (PROTACs) specifically degrading RNF7 could provide pharmacological validation of RNF7 as a therapeutic target, complementing genetic approaches. High-throughput drug screens in cells with varying RNF7 expression levels might identify synthetic lethal interactions for precision medicine applications. Additionally, investigating post-translational modifications of RNF7 itself using phospho-specific and ubiquitin-specific antibodies could reveal regulatory mechanisms controlling its activity in cancer cells. Each of these approaches would benefit from the application of validated RNF7 antibodies for detection, quantification, and functional characterization .

How might RNF7 antibodies be utilized in translational research for cancer therapeutics?

RNF7 antibodies present several promising applications in translational cancer research toward therapeutic development. For companion diagnostics, immunohistochemical detection of RNF7 using standardized protocols could stratify patients for clinical trials targeting RNF7-dependent pathways, given the established correlation between high RNF7 expression and poor prognosis in cancers like pancreatic and prostate malignancies . Additionally, these antibodies can verify target engagement in preclinical studies of RNF7 inhibitors by monitoring changes in RNF7 protein levels or post-translational modifications. For developing antibody-drug conjugates (ADCs), anti-RNF7 antibodies with verified specificity could be explored as delivery vehicles for cytotoxic payloads, particularly in cancers with high RNF7 expression. In pharmacodynamic studies, measuring changes in downstream effectors like p21, p27, NOXA, phospho-Akt, and phospho-ERK using corresponding antibodies provides critical biomarkers for assessing RNF7-targeting therapies . Flow cytometry with RNF7 antibodies could identify circulating tumor cells with high RNF7 expression as potential indicators of treatment response or resistance. Importantly, when developing antibody-based therapeutics targeting the RNF7 pathway, researchers must account for RNF7's dual localization in both cytoplasmic and nuclear compartments, which may necessitate different delivery strategies . Each of these translational applications requires rigorous validation of antibody specificity and optimal protocols for the specific clinical or preclinical context.

What technical developments in antibody technology could enhance future RNF7 research applications?

Future RNF7 research would benefit substantially from several technical advancements in antibody technology. Development of conformation-specific antibodies that selectively recognize active versus inactive RNF7 conformations would enable direct measurement of functional states rather than mere protein levels. Similarly, antibodies specific to post-translationally modified forms of RNF7 (phosphorylated, ubiquitinated) would facilitate research into regulatory mechanisms controlling its activity . For improved detection sensitivity in tissues with low RNF7 expression, signal amplification technologies such as tyramide signal amplification or quantum dot-conjugated secondary antibodies could enhance detection limits while maintaining specificity. Single-domain antibodies (nanobodies) against RNF7 would enable live-cell imaging of endogenous RNF7 dynamics and interactions when coupled with fluorescent proteins. For therapeutic applications, bispecific antibodies targeting both RNF7 and components of relevant signaling pathways (PI3K/Akt, ERK1/2) could provide synergistic inhibition of cancer progression . Additionally, intrabody development (antibodies designed for intracellular expression) could allow functional inhibition of RNF7 in specific subcellular compartments. To address reproducibility challenges, recombinant antibody technology with defined sequences would ensure batch-to-batch consistency compared to traditional polyclonal antibodies. Finally, antibody engineering to enable penetration of the blood-brain barrier would support investigation of RNF7's roles in brain tumors and neurological disorders, expanding the scope of RNF7 research beyond the currently studied cancer types.