ARIH2 Antibody

What is ARIH2 and what are its alternative names in research literature?

ARIH2 (ariadne homolog 2) is also known as ARI2 or TRIAD1 in scientific literature. It belongs to the RBR (RING-between-RING) family and ariadne subfamily of E3 ligases. The protein contains one IBR-type zinc finger and two RING-type zinc fingers, which are essential for its function. ARIH2 is a 493 amino acid protein with a calculated molecular weight of 58 kDa, though it is typically observed at approximately 59 kDa in experimental conditions . This protein plays a significant role in mediating both 'Lys-48' and 'Lys-63'-linked polyubiquitination processes that lead to proteasomal degradation of modified proteins .

What cellular functions has ARIH2 been implicated in based on recent research?

Recent research has revealed that ARIH2 plays crucial roles in several cellular processes beyond its basic ubiquitination function. ARIH2 has been shown to significantly influence cell proliferation, particularly in gastric cancer cells where high ARIH2 expression correlates with poor prognosis in patients . Additionally, ARIH2 has been identified as a regulatory factor in immune responses, inflammatory diseases, and skeletal muscle development . In cancer biology, ARIH2 has demonstrated important functions in acute myeloid leukemia and non-small-cell lung cancer, suggesting its involvement in multiple cancer types . Mechanistically, ARIH2 interacts with p21 and induces its ubiquitination, marking it for degradation and thereby affecting cell cycle progression .

How does ARIH2 influence DNA damage responses in cancer cells?

Research indicates that ARIH2 plays a significant role in DNA damage response pathways. When ARIH2 is knocked down in gastric cancer cells, there is a notable increase in DNA damage levels, as measured by γ-H2AX immunofluorescence assays . This increased DNA damage correlates with induction of cell apoptosis and enhanced chemosensitivity when cancer cells are treated with 5-fluorouracil . The relationship between ARIH2 and DNA damage appears to be connected to its regulation of p21, which is a key cell cycle regulator and mediator of DNA damage responses. By targeting and controlling p21 levels through ubiquitination, ARIH2 indirectly influences how cells respond to DNA damage events and chemotherapeutic agents .

What criteria should researchers consider when selecting an ARIH2 antibody for their experiments?

When selecting an ARIH2 antibody, researchers should consider several critical factors. First, verify the specific epitope or region the antibody recognizes—some antibodies target the N-terminal region, C-terminal region, or specific amino acid sequences (e.g., AA 43-360 or AA 1-493) . Second, ensure the antibody has been validated for your specific application (WB, IHC, IP, IF/ICC, or ELISA) as performance can vary significantly between applications . Third, confirm species reactivity—many ARIH2 antibodies have been validated for human, mouse, and rat samples, but cross-reactivity with other species varies by antibody . Finally, review validation data including images of Western blots or immunostaining to ensure the antibody detects the expected molecular weight (approximately 59 kDa) with minimal background or non-specific binding .

What validation experiments should be performed to confirm ARIH2 antibody specificity?

To confirm ARIH2 antibody specificity, researchers should implement a multi-step validation approach. First, perform Western blotting across multiple relevant cell lines or tissues known to express ARIH2 (such as HEK-293, HL-60, or mouse brain tissue) to confirm detection at the expected molecular weight of approximately 59 kDa . Second, include a positive control sample with confirmed ARIH2 expression and a negative control using ARIH2 knockdown (KD) or knockout (KO) samples to verify specificity . Third, conduct peptide competition assays where the antibody is pre-incubated with the immunizing peptide before application, which should eliminate specific binding if the antibody is truly specific. Fourth, perform immunoprecipitation followed by mass spectrometry to confirm that the antibody is pulling down ARIH2 and not cross-reacting with similar proteins. Finally, for immunohistochemistry or immunofluorescence applications, compare staining patterns with multiple ARIH2 antibodies targeting different epitopes to confirm consistent localization patterns .

What are the recommended storage conditions for maintaining ARIH2 antibody activity?

For optimal preservation of ARIH2 antibody activity, storage at -20°C is recommended by most manufacturers. These antibodies are typically stable for one year after shipment when stored properly . Importantly, repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and reduce specific binding capacity . For antibodies provided in liquid form, they are generally supplied in a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability . When working with the antibody, it's best to aliquot the stock solution into smaller volumes upon receipt to minimize freeze-thaw cycles. According to manufacturer guidelines, aliquoting is not necessary for -20°C storage, but it remains a best practice to preserve antibody quality for long-term use . Once reconstituted or diluted for use, ARIH2 antibodies typically remain stable for at least two weeks when stored at 2-4°C .

What are the optimal conditions for using ARIH2 antibodies in Western blotting?

For optimal Western blotting results with ARIH2 antibodies, the recommended dilution range is 1:1000-1:4000, though this should be titrated for each specific experimental system . Sample preparation should include the use of appropriate lysis buffers containing protease inhibitors to prevent degradation of ARIH2. When running SDS-PAGE, sufficient separation time should be allowed as ARIH2 has an observed molecular weight of approximately 59 kDa . For immunoblotting, transfer to PVDF membranes is commonly used, followed by blocking with appropriate blocking agents. Primary antibody incubation should be performed overnight at 4°C for best results . After washing, compatible secondary antibodies should be applied for approximately 2 hours at room temperature. Signal detection can be performed using chemiluminescence or fluorescence-based systems, with a Chemiscope 6000 imaging system being one validated detection method . When analyzing results, researchers should expect a band at approximately 59 kDa, though post-translational modifications may result in slight variations in apparent molecular weight.

What protocol should be followed for immunoprecipitation experiments using ARIH2 antibodies?

For immunoprecipitation experiments with ARIH2 antibodies, the following protocol has been validated: First, lyse cells (such as HeLa cells, which have been confirmed as suitable for ARIH2 IP) in IP lysis buffer containing protease inhibitors . Use 0.5-4.0 μg of ARIH2 antibody for 1.0-3.0 mg of total protein lysate . Incubate the protein extract with the ARIH2 antibody overnight at 4°C with gentle rotation. Add 50 μl of protein A+G agarose beads to the protein-antibody mixture and incubate at 4°C for 4 hours . After incubation, wash the beads thoroughly with pre-cooled PBS buffer to remove non-specific binding proteins and impurities. Elute the immunoprecipitated proteins by adding 40 μl of 1× loading buffer and heat-denaturing the samples . Analyze the immunoprecipitated products by SDS-PAGE followed by Western blotting to confirm successful IP of ARIH2. For co-IP experiments investigating ARIH2 interaction partners (such as p21), additional antibodies against the target protein should be used for detection after ARIH2 pull-down .

How should immunohistochemistry be performed with ARIH2 antibodies on tissue samples?

For immunohistochemistry with ARIH2 antibodies, the following methodology has been validated: Begin by cutting paraffin-embedded tissues into 5 mm thick sections, followed by deparaffinization and hydration steps . Perform antigen retrieval by placing the sections in citrate buffer (pH 6.0) and heating to 95°C for 20 minutes in a microwave oven . Quench endogenous peroxidase activity using an appropriate quenching solution, then block non-specific binding with normal goat serum . Dilute the ARIH2 antibody according to manufacturer's recommendations (typically in the range of 1:20-1:200 for IHC applications) and apply to the sections for overnight incubation at 4°C . After incubation with the primary antibody, apply a compatible horseradish peroxidase-linked secondary antibody and incubate according to the manufacturer's protocol. Develop the signal using DAB (3,3'-diaminobenzidine) substrate, which allows visualization of ARIH2 expression as a brown precipitate . Counterstain with hematoxylin to visualize tissue architecture, then dehydrate, clear, and mount the sections. When analyzing results, compare ARIH2 staining patterns with those of known markers such as Ki67 for proliferation, especially in cancer tissue studies .

What is the recommended protocol for immunofluorescence detection of ARIH2 in cell cultures?

For immunofluorescence detection of ARIH2 in cultured cells, the following protocol is recommended: First, culture cells on appropriate coverslips or chamber slides. Fix cells using 4% paraformaldehyde or another suitable fixative, followed by permeabilization with 0.1-0.5% Triton X-100 in PBS . Block non-specific binding with appropriate blocking buffer (typically containing BSA or normal serum). Dilute the ARIH2 antibody according to manufacturer's recommendations, which is generally in the range of 1:20-1:200 for IF/ICC applications . Incubate cells with the diluted primary antibody overnight at 4°C in a humidified chamber. After thorough washing with PBS, apply fluorophore-conjugated secondary antibodies specific to the host species of the ARIH2 antibody (typically at 1:2000 dilution) . For nuclear visualization, counterstain with DNA-binding dyes such as Hoechst 33342 (1:2000) . Mount slides using anti-fade mounting medium and visualize using confocal microscopy, such as an Olympus FV1000 confocal fluorescence microscope, which has been validated for ARIH2 immunofluorescence detection . Positive IF/ICC signal has been specifically validated in HEK-293 cells, making these cells a good positive control for establishing the technique .

How can ARIH2 antibodies be used to investigate the relationship between ARIH2 and p21 in cancer research?

ARIH2 antibodies can be strategically employed to investigate the ARIH2-p21 relationship in cancer research through several advanced approaches. First, co-immunoprecipitation experiments using ARIH2 antibodies can pull down ARIH2 protein complexes, followed by Western blotting with p21 antibodies to confirm their physical interaction . This approach has successfully demonstrated that ARIH2 directly interacts with p21 in gastric cancer cells . Second, researchers can perform ubiquitination assays by immunoprecipitating p21 followed by Western blotting with ubiquitin antibodies in cells with manipulated ARIH2 expression levels, allowing quantification of how ARIH2 affects p21 ubiquitination . Third, dual immunofluorescence staining with ARIH2 and p21 antibodies can reveal their co-localization patterns within cells. Fourth, chromatin immunoprecipitation (ChIP) assays can determine if ARIH2 indirectly affects p21 expression through transcriptional regulation. Finally, combining ARIH2 antibodies with proximity ligation assays (PLA) offers a powerful method to visualize and quantify ARIH2-p21 interactions in situ, providing spatial information about where these interactions occur within the cell .

What methodologies are recommended for studying ARIH2's role in DNA damage responses using ARIH2 antibodies?

To study ARIH2's role in DNA damage responses, several methodologies incorporating ARIH2 antibodies are recommended. First, immunofluorescence assays using γ-H2AX antibodies (a marker of DNA double-strand breaks) can be performed in cells with ARIH2 knockdown or overexpression to quantify changes in DNA damage levels . This approach has successfully demonstrated increased DNA damage after ARIH2 knockdown in gastric cancer cells . Second, immunoblotting for DNA damage response proteins (such as ATM, ATR, Chk1, Chk2) in ARIH2-manipulated cells can reveal how ARIH2 affects DNA damage signaling pathways. Third, chromatin immunoprecipitation using ARIH2 antibodies can determine if ARIH2 is recruited to sites of DNA damage. Fourth, comet assays combined with ARIH2 immunostaining can correlate ARIH2 expression levels with DNA damage in individual cells. Fifth, for investigating chemosensitivity, researchers should combine ARIH2 antibody-based detection methods with cell viability assays after treatment with DNA-damaging agents such as 5-fluorouracil, as this approach has revealed that ARIH2 knockdown enhances chemosensitivity in gastric cancer cells both in vitro and in vivo .

What protocols can be used to investigate ARIH2-mediated ubiquitination of target proteins?

To investigate ARIH2-mediated ubiquitination of target proteins, researchers can implement several specialized protocols using ARIH2 antibodies. First, in vivo ubiquitination assays can be performed by co-transfecting cells with tagged ARIH2, tagged ubiquitin, and the potential target protein (such as p21), followed by immunoprecipitation under denaturing conditions and Western blotting to detect ubiquitinated forms . Second, in vitro ubiquitination assays using purified components (E1, E2, ARIH2 as E3, ubiquitin, and substrate) can definitively establish ARIH2's direct ubiquitination activity toward specific targets . Third, site-directed mutagenesis of key residues in ARIH2 (particularly within its RBR domain) or in the target protein can identify critical sites for the ubiquitination process, as demonstrated with the K161 residue of p21 and the K48 residue of ubiquitin being crucial for ARIH2-mediated p21 ubiquitination . Fourth, mass spectrometry analysis of immunoprecipitated proteins can identify ubiquitination sites and ubiquitin chain linkage types. Fifth, cycloheximide chase assays combined with ARIH2 antibody detection can measure how ARIH2 affects the half-life of target proteins, providing functional validation of the ubiquitination-mediated regulation .

How can ARIH2 antibodies be used to evaluate ARIH2 as a potential therapeutic target in cancer?

ARIH2 antibodies can be instrumental in evaluating ARIH2 as a potential therapeutic target in cancer through several approaches. First, immunohistochemistry using ARIH2 antibodies on tissue microarrays containing multiple patient samples can correlate ARIH2 expression levels with clinical outcomes, helping establish ARIH2 as a prognostic biomarker . Research has already demonstrated that high ARIH2 expression correlates with poor prognosis in gastric cancer patients . Second, in xenograft tumor models, ARIH2 antibodies can be used to confirm successful ARIH2 knockdown or inhibition and to monitor changes in ARIH2 expression during tumor development and in response to treatments . Third, immunohistochemical staining for proliferation markers (like Ki67) and ARIH2 in the same tumor sections can reveal correlations between ARIH2 expression and tumor proliferation rates . Fourth, combining ARIH2 antibody-based detection with apoptosis assays after treatment with chemotherapeutic agents can assess how ARIH2 inhibition affects chemosensitivity, as research has shown that ARIH2 knockdown enhanced sensitivity to 5-fluorouracil in gastric cancer cells . Finally, ARIH2 antibodies can be used to screen for compounds that disrupt ARIH2 interactions with target proteins or inhibit its E3 ligase activity, potentially identifying novel therapeutic approaches targeting ARIH2 function .

What are common issues encountered in Western blotting with ARIH2 antibodies and how can they be resolved?

Common issues in Western blotting with ARIH2 antibodies include multiple bands, weak signal, high background, and inconsistent results. For multiple bands, researchers should first verify if they represent physiologically relevant isoforms or post-translationally modified forms of ARIH2. If they appear to be non-specific, optimization steps include increasing antibody dilution (recommended range 1:1000-1:4000) , using fresh antibody aliquots, and implementing more stringent washing conditions. For weak signal issues, researchers can try reducing the antibody dilution, extending primary antibody incubation time (overnight at 4°C is recommended) , using enhanced chemiluminescence reagents, or increasing protein loading (validated cell lines like HEK-293 or HL-60 can be used as positive controls) . High background can be addressed by improving blocking conditions, increasing the number and duration of washes, and ensuring that secondary antibody concentrations are not excessive. For inconsistent results, standardize sample preparation methods, use freshly prepared buffers, and establish consistent transfer conditions. Additionally, verify the molecular weight of observed bands against the expected size of approximately 59 kDa for ARIH2 .

How should researchers troubleshoot immunohistochemistry experiments with ARIH2 antibodies?

When troubleshooting immunohistochemistry experiments with ARIH2 antibodies, researchers should systematically address several common issues. For weak or absent staining, optimize antigen retrieval by ensuring appropriate buffer choice (citrate buffer pH 6.0 has been validated) and adequate heating time (20 minutes at 95°C has shown success) . Antibody concentration should be titrated within the recommended range of 1:20-1:200 for IHC applications . For high background or non-specific staining, enhance blocking steps using normal goat serum , increase washing duration and frequency, and ensure endogenous peroxidase quenching is effective. If tissue morphology is compromised, adjust fixation times and handling procedures to preserve tissue architecture while maintaining antigenicity. For inconsistent staining across serial sections, standardize all protocol steps including incubation times and temperatures. Importantly, include appropriate positive controls (tissues known to express ARIH2) and negative controls (omitting primary antibody or using ARIH2-knockout tissues) with each experiment. When counterstaining with hematoxylin, adjust timing to ensure nuclear details remain visible without obscuring ARIH2 immunoreactivity . Finally, compare results with alternative detection systems if problems persist.

What factors might affect the reproducibility of immunofluorescence experiments with ARIH2 antibodies?

Several factors can affect the reproducibility of immunofluorescence experiments with ARIH2 antibodies. First, fixation and permeabilization conditions significantly impact epitope accessibility—overflxation can mask antibody binding sites, while inadequate permeabilization can prevent antibody entry into relevant cellular compartments. Second, antibody concentration must be carefully optimized within the recommended 1:20-1:200 range for IF/ICC applications , as both excessive and insufficient antibody can lead to poor results. Third, the specificity of the secondary antibody is crucial—cross-reactivity with other cellular components can generate misleading signals, so appropriate controls are essential. Fourth, photobleaching during extended imaging sessions can reduce signal intensity, requiring anti-fade mounting media and optimized imaging protocols. Fifth, cell density and growth conditions affect ARIH2 expression levels, with HEK-293 cells being validated as reliable positive controls for ARIH2 IF/ICC experiments . Sixth, batch-to-batch variations in antibodies can impact results, necessitating validation of new lots against previous successful experiments. Finally, microscope settings including exposure time, gain, and laser power should be standardized and documented to ensure reproducible imaging conditions across experiments.

What are the critical parameters for successful co-immunoprecipitation of ARIH2 and its interacting partners?

Successful co-immunoprecipitation of ARIH2 and its interacting partners depends on several critical parameters. First, cell lysis conditions are crucial—use IP lysis buffer that preserves protein-protein interactions while efficiently extracting ARIH2 complexes . Avoid harsh detergents that might disrupt weak or transient interactions. Second, antibody selection is vital—use 0.5-4.0 μg of ARIH2 antibody per 1.0-3.0 mg of total protein lysate, as recommended by validated protocols . Third, incubation conditions significantly impact success—overnight incubation at 4°C with the primary antibody followed by 4 hours with protein A+G agarose beads has been validated for ARIH2 IP . Fourth, washing stringency must balance removing non-specific binding while preserving genuine interactions—pre-cooled PBS buffer has been successfully used for this purpose . Fifth, appropriate negative controls are essential, including IgG-matched isotype controls and, when possible, ARIH2 knockdown samples. Sixth, for detecting specific partners like p21, reverse co-IP (immunoprecipitating with p21 antibody and probing for ARIH2) should be performed to confirm bidirectional interaction . Seventh, sample preparation for subsequent analysis is critical—using 40 μl of 1× loading buffer for elution has been validated for ARIH2 co-IP experiments . Finally, for detecting ubiquitinated proteins in the complex, consider including deubiquitinase inhibitors in all buffers to preserve ubiquitination status.

What emerging techniques might enhance ARIH2 antibody applications in cancer research?

Several emerging techniques promise to enhance ARIH2 antibody applications in cancer research. First, proximity ligation assays (PLA) combined with ARIH2 antibodies can visualize and quantify protein-protein interactions between ARIH2 and binding partners like p21 with single-molecule sensitivity within intact cells. Second, super-resolution microscopy techniques such as STORM or PALM using fluorophore-conjugated ARIH2 antibodies can provide nanoscale localization of ARIH2 within cellular compartments, potentially revealing previously undetected organizational patterns. Third, CRISPR-Cas9 engineered cell lines expressing tagged endogenous ARIH2 can be used with anti-tag antibodies to avoid potential artifacts of overexpression systems. Fourth, live-cell imaging with cell-permeable ARIH2 nanobodies could track ARIH2 dynamics in real-time during cell cycle progression or in response to DNA damage. Fifth, combining single-cell RNA sequencing with ARIH2 immunophenotyping can correlate ARIH2 protein levels with transcriptional states at the single-cell level in heterogeneous tumors. Finally, mass cytometry (CyTOF) incorporating ARIH2 antibodies can simultaneously analyze ARIH2 expression alongside dozens of other cancer-related proteins across large populations of cells, potentially revealing novel correlations between ARIH2 and other cancer pathways .

How might advances in antibody engineering improve detection of ARIH2 in challenging experimental contexts?

Advances in antibody engineering offer promising solutions for detecting ARIH2 in challenging experimental contexts. First, the development of recombinant antibody fragments (Fab, scFv) specific to ARIH2 could provide better tissue penetration in whole-mount preparations or thick tissue sections. Second, site-specific modification of ARIH2 antibodies with smaller fluorophores or quantum dots could reduce steric hindrance issues when accessing densely packed epitopes or protein complexes. Third, bivalent antibodies designed to simultaneously recognize two different epitopes on ARIH2 could enhance specificity and avidity, particularly valuable when working with low-expressing samples. Fourth, engineering antibodies with reduced cross-reactivity to closely related RBR family members would improve specificity for ARIH2 detection. Fifth, developing conformationally sensitive antibodies could distinguish between active and inactive states of ARIH2, offering insights into its functional status rather than merely detecting its presence. Sixth, antibodies optimized for harsh conditions (extreme pH, high temperature, high detergent) could enable ARIH2 detection in previously challenging sample types. Finally, creating bispecific antibodies that simultaneously recognize ARIH2 and one of its key interaction partners (such as p21) could enable selective detection of functional ARIH2 complexes rather than total ARIH2 protein .

What potential clinical applications exist for ARIH2 antibodies in cancer diagnostics and treatment?

ARIH2 antibodies hold significant potential for clinical applications in cancer diagnostics and treatment. For diagnostics, immunohistochemistry with ARIH2 antibodies could be developed into a prognostic marker for gastric cancer patients, as research has already demonstrated that high ARIH2 expression correlates with poor prognosis . ARIH2 antibody-based immunoassays could be standardized for inclusion in diagnostic panels to help stratify patients for treatment decisions. In liquid biopsy approaches, detecting ARIH2 in circulating tumor cells using highly sensitive ARIH2 antibodies might provide non-invasive monitoring of disease progression or treatment response. For therapeutic applications, ARIH2 antibodies could be used to screen for compounds that disrupt ARIH2-mediated ubiquitination of p21 or other targets, potentially identifying novel drug candidates. Antibody-drug conjugates targeting ARIH2 might selectively deliver cytotoxic agents to cancer cells with high ARIH2 expression. Additionally, ARIH2 antibodies could help monitor pharmacodynamic responses to treatments targeting the ubiquitin-proteasome system. Finally, as companion diagnostics, ARIH2 antibody-based assays could identify patients likely to respond to therapies targeting ARIH2 or its signaling pathway, similar to existing companion diagnostic approaches for targeted therapies .

How can ARIH2 antibodies contribute to understanding the relationship between ARIH2 and chemoresistance mechanisms?

ARIH2 antibodies can make significant contributions to understanding chemoresistance mechanisms through multiple research approaches. First, immunohistochemistry comparing ARIH2 expression in paired patient samples before and after chemotherapy failure can reveal whether ARIH2 upregulation correlates with acquired resistance. Second, combining ARIH2 antibody-based protein quantification with cell viability assays following treatment with various chemotherapeutic agents can establish direct relationships between ARIH2 expression levels and drug sensitivity profiles . Third, immunofluorescence co-localization studies with ARIH2 antibodies and markers of drug efflux pumps or anti-apoptotic proteins can reveal potential mechanistic connections. Fourth, ARIH2 antibodies can enable chromatin immunoprecipitation sequencing (ChIP-seq) to identify genes whose expression might be indirectly regulated by ARIH2 and contribute to chemoresistance. Fifth, protein-interaction studies using ARIH2 antibodies for co-immunoprecipitation followed by mass spectrometry can identify novel ARIH2 binding partners in resistant versus sensitive cells. Sixth, phospho-specific ARIH2 antibodies (if developed) could determine whether post-translational modifications of ARIH2 change during resistance development. Research has already established that ARIH2 knockdown enhances sensitivity to 5-fluorouracil in gastric cancer models, providing a foundation for these investigations into the role of ARIH2 in chemoresistance mechanisms .