RANBP10 Antibody, Biotin conjugated

What is RANBP10 and why is it significant in cellular function?

RANBP10 (RAN binding protein 10) is a ubiquitously expressed and evolutionarily conserved protein that functions as a RAN-GTP exchange factor (GEF). It plays critical roles in regulating several factors involved in cellular progression and signaling pathways . The protein has a calculated molecular weight of approximately 67 kDa (620 amino acids) and is typically observed at 67-70 kDa in experimental systems . RANBP10 is significant because it interacts with multiple cellular components involved in nucleocytoplasmic transport and signaling pathways. Recent research has demonstrated that RANBP10 shows increased expression in certain cancer types, including glioblastoma, suggesting its potential role in tumor progression through regulation of cell proliferation, migration, and invasion .

What are the known molecular characteristics of the RANBP10 protein?

RANBP10 has several important molecular characteristics relevant to researchers:

The protein contains functional domains that facilitate its role as a RAN-GTP exchange factor and enable interactions with other cellular proteins . Research has shown that RANBP10 can suppress the promoter activity of FBXW7, thereby increasing the protein stability of c-Myc in glioblastoma cells, which potentially contributes to cancer progression .

What is the advantage of using a biotin-conjugated antibody for RANBP10 detection?

Biotin-conjugated antibodies offer several methodological advantages for RANBP10 detection:

The biotin-avidin/streptavidin interaction is one of the strongest non-covalent interactions in nature, making biotin-conjugated antibodies highly sensitive detection tools . This exceptionally strong binding (Kd = 10^-15 M) enables superior signal amplification in various assay formats. The small size of the biotin molecule (244 Da) minimizes interference with antibody-antigen binding, preserving the antibody's specificity and affinity for RANBP10 .

Additionally, biotin-conjugated antibodies can be used with various avidin/streptavidin conjugates (HRP, fluorophores, gold particles), offering flexibility in detection methods. This versatility makes biotin-conjugated RANBP10 antibodies particularly useful in complex experimental setups requiring signal amplification, such as immunohistochemistry of tissues with low RANBP10 expression or multiplex detection systems .

What are the validated applications for biotin-conjugated RANBP10 antibody?

The biotin-conjugated RANBP10 antibody has been validated for ELISA applications according to the manufacturer's specifications . While the biotin-conjugated version has specific validated applications, other RANBP10 antibodies have demonstrated utility in:

• Western Blot (WB): Validated at dilutions of 1:500-1:1000

• Immunohistochemistry (IHC): Validated at dilutions of 1:20-1:200

• Immunofluorescence (IF): Validated in published applications

Researchers should note that application validation is antibody-specific, and when transitioning to using the biotin-conjugated version for applications beyond ELISA, additional optimization and validation may be necessary . The biotin conjugation offers enhanced detection sensitivity through avidin/streptavidin systems, which can be particularly valuable for detecting low-abundance RANBP10 in complex samples.

What are the recommended protocols for using biotin-conjugated RANBP10 antibody in ELISA?

For ELISA applications using biotin-conjugated RANBP10 antibody, researchers should consider the following optimized protocol:

• Coating: Coat ELISA plate wells with capture antibody or antigen (if using a sandwich or competitive ELISA format)

• Blocking: Block non-specific binding sites with appropriate blocking buffer (typically 1-5% BSA in PBS)

• Sample Addition: Add diluted samples containing RANBP10 protein

• Primary Antibody: If using as a detection antibody, add biotin-conjugated RANBP10 antibody at an optimized dilution

• Detection System: Add streptavidin-HRP (or other avidin conjugate) at manufacturer-recommended dilution

• Substrate Development: Add appropriate substrate and measure signal

The biotin-conjugation provides signal amplification through high-affinity binding to streptavidin detection systems. Researchers should titrate the biotin-conjugated antibody to determine optimal concentration for their specific assay conditions . For quantitative applications, inclusion of a standard curve using recombinant RANBP10 protein is recommended.

How can I optimize western blot protocols for RANBP10 detection?

While the biotin-conjugated RANBP10 antibody is primarily validated for ELISA, researchers interested in western blot applications should consider the following optimization steps:

• Sample Preparation: RANBP10 has been successfully detected in various tissue lysates including human skeletal muscle, mouse heart tissue, and cell lines such as MCF-7 and LNCaP .

• Dilution Optimization: Start with dilutions between 1:500-1:1000 for RANBP10 antibodies. For biotin-conjugated versions, initial testing may require a range of dilutions to determine optimal concentration .

• Detection System: When using biotin-conjugated antibodies, employ streptavidin-HRP for signal development. This enhances sensitivity compared to traditional secondary antibodies.

• Expected Band Size: Look for RANBP10 at 67-70 kDa, as demonstrated in published western blot analyses .

For validation, published western blot images show clear RANBP10 detection in human skeletal muscle tissue lysate at antibody concentrations of 1 μg/mL and 2 μg/mL, providing reference data for expected results .

In which tissues and cell types is RANBP10 commonly expressed?

RANBP10 exhibits a broad expression pattern across multiple tissues and cell types. Based on antibody validation studies, RANBP10 has been positively detected in:

This expression pattern indicates RANBP10's importance in multiple tissue types and suggests its involvement in fundamental cellular processes . The detection of RANBP10 in diverse tissues makes the biotin-conjugated antibody a valuable tool for comparative expression studies across different physiological and pathological states.

What is the connection between RANBP10 and cancer pathogenesis?

Recent research has established significant connections between RANBP10 and cancer pathogenesis:

RANBP10 has been found to be overexpressed in glioblastoma (GBM), the most aggressive brain malignancy in adults. High RANBP10 expression correlates with poor survival outcomes in GBM patients . Mechanistically, studies have demonstrated that RANBP10 promotes GBM cell proliferation, migration, invasion, and tumor growth through regulation of the FBXW7–c-Myc axis .

Specifically, RANBP10 suppresses the promoter activity of FBXW7 (a known tumor suppressor), which leads to increased protein stability of c-Myc (an oncogenic transcription factor) in GBM cells. When RANBP10 is downregulated in experimental models, significant inhibition of GBM cell growth and invasiveness occurs .

Additionally, previous studies have indicated RANBP10 overexpression in prostate cancer cells, where it contributes to androgen receptor (AR) activation, suggesting a potential role in hormone-dependent cancers as well .

What storage and handling practices maximize RANBP10 antibody stability and performance?

To ensure optimal performance of biotin-conjugated RANBP10 antibody, researchers should follow these evidence-based storage and handling guidelines:

• Storage Temperature: Store at -20°C or -80°C for long-term preservation of antibody activity .

• Aliquoting: Upon receipt, prepare small working aliquots to avoid repeated freeze-thaw cycles that can compromise antibody quality .

• Buffer Conditions: The antibody is typically supplied in PBS containing preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol, pH 7.4) to maintain integrity .

• Thawing Protocol: Thaw antibody aliquots on ice or at 4°C rather than at room temperature to minimize denaturation.

• Shelf Life: When properly stored, the antibody remains stable for one year from the shipment date .

High temperatures should be avoided during handling as they can accelerate degradation of both the antibody and the biotin conjugate . For assays requiring diluted antibody solutions, prepare fresh dilutions on the day of the experiment whenever possible to maintain optimal binding capacity.

How can I troubleshoot weak or non-specific signals when using biotin-conjugated RANBP10 antibody?

When encountering weak or non-specific signals with biotin-conjugated RANBP10 antibody, consider these methodical troubleshooting approaches:

For Weak Signals:

• Antibody Concentration: Increase the concentration of biotin-conjugated RANBP10 antibody gradually to enhance signal intensity.

• Sample Preparation: Ensure proper protein extraction and denaturation protocols to maximize RANBP10 epitope exposure.

• Incubation Conditions: Extend incubation time or adjust temperature to promote binding efficiency.

• Detection System: Verify the activity of the streptavidin conjugate and consider using more sensitive substrates.

For Non-specific Signals:

• Blocking Optimization: Increase blocking reagent concentration or time to reduce background binding.

• Washing Stringency: Implement more rigorous washing steps to remove unbound antibodies.

• Endogenous Biotin Blocking: In tissue samples, pretreat with avidin/biotin blocking kit to neutralize endogenous biotin that may cause background.

• Cross-Reactivity Assessment: Validate antibody specificity using RANBP10 knockdown or knockout samples as negative controls.

For IHC applications, optimization of antigen retrieval methods is critical; the antibody has shown good results with TE buffer pH 9.0, though citrate buffer pH 6.0 may serve as an alternative .

How can I validate the specificity of biotin-conjugated RANBP10 antibody in my experimental system?

Validating antibody specificity is crucial for generating reliable research data. For biotin-conjugated RANBP10 antibody, implement these validation strategies:

• Positive Control Selection: Use tissues or cell lines with confirmed RANBP10 expression such as MCF-7 cells, human skeletal muscle tissue, or mouse heart tissue, which have shown consistent positive results in published studies .

• Genetic Knockdown/Knockout Controls: Implement RANBP10 knockdown (siRNA/shRNA) or knockout (CRISPR-Cas9) systems to generate negative controls. The absence of signal in these systems strongly supports antibody specificity, as demonstrated in published knockout validation studies .

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide before application to your sample. Signal reduction confirms specific binding to the target epitope.

• Molecular Weight Verification: In western blot applications, confirm that the detected band appears at the expected molecular weight of 67-70 kDa for RANBP10 .

• Multi-antibody Comparison: If available, compare results with alternative RANBP10 antibodies raised against different epitopes to confirm consistent detection patterns.

For biotin-conjugated antibodies specifically, include additional controls to address potential background from endogenous biotin or non-specific streptavidin binding.

How can biotin-conjugated RANBP10 antibody be utilized in drug discovery or therapeutic research?

Biotin-conjugated RANBP10 antibody offers several strategic advantages in drug discovery and therapeutic research:

The antibody can be employed to assess RANBP10 inhibition by candidate drugs targeting the RANBP10-dependent pathways implicated in cancer progression. Given that RANBP10 promotes GBM progression by regulating the FBXW7–c-Myc axis, therapeutic compounds that disrupt this interaction could be identified and quantified using biotin-conjugated RANBP10 antibody in various binding and functional assays .

In therapeutic nanoparticle development, biotin-conjugated RANBP10 antibody can be integrated into avidin-based nanocarrier systems. This approach utilizes the strong non-covalent interaction between avidin and biotin (Kd = 10^-15 M) to create stable drug delivery platforms that potentially target cells overexpressing RANBP10 .

The antibody can also facilitate target validation studies through immunoprecipitation of RANBP10 protein complexes, helping to identify interaction partners that might serve as alternative drug targets in the same pathway. Additionally, it can be used in high-throughput screening assays to identify compounds that modulate RANBP10 expression or function .

What are the considerations for using biotin-conjugated RANBP10 antibody in multiplex detection systems?

When incorporating biotin-conjugated RANBP10 antibody into multiplex detection systems, researchers should address these technical considerations:

• Endogenous Biotin Interference: Biological samples, particularly tissues like liver and kidney, contain endogenous biotin that can cause false positive signals. Implement biotin blocking steps (using streptavidin followed by free biotin) before adding biotin-conjugated antibodies .

• Sequential Detection Strategy: In multiplex fluorescence assays, apply the biotin-conjugated RANBP10 antibody in a sequential manner rather than simultaneously with other primary antibodies to minimize cross-reactivity.

• Spectral Separation: When using multiple fluorophores, ensure adequate spectral separation between the fluorophore conjugated to streptavidin (for RANBP10 detection) and other detection channels to prevent bleed-through.

• Signal Amplification Control: The avidin-biotin system provides significant signal amplification, which may create imbalanced signal intensities in multiplex assays. Titrate the concentration of biotin-conjugated RANBP10 antibody and streptavidin conjugate carefully .

• Validation with Single-plex Controls: Always validate multiplex results by comparing with single-plex detection to ensure that multiplexing does not alter antibody performance or specificity.

These considerations are particularly important when studying RANBP10 in complex disease contexts where multiple biomarkers need to be analyzed simultaneously.

How might RANBP10 antibodies contribute to understanding its role in neurological disorders?

RANBP10 antibodies, including biotin-conjugated variants, can significantly advance research into neurological disorders through several approaches:

RANBP10 has been detected in mouse brain tissue via western blot analysis, suggesting its presence and potential functional importance in neural tissues . The biotin-conjugated RANBP10 antibody can facilitate detailed mapping of protein expression patterns across different brain regions and neural cell types using immunohistochemistry and immunofluorescence techniques.

Recent research has identified connections between RANBP10 and glioblastoma progression, indicating potential roles in brain pathology . The antibody can help investigate whether RANBP10 expression or localization is altered in other neurological conditions, including neurodegenerative diseases where protein transport mechanisms are often dysregulated.

In molecular neuroscience research, the biotin-conjugated antibody can be combined with streptavidin-conjugated quantum dots or fluorophores for high-resolution imaging of RANBP10 dynamics in living neurons. This approach could reveal its potential roles in neuronal development, synaptic plasticity, or response to neurological insults.

Furthermore, using avidin-based nanoparticle technology in conjunction with biotin-conjugated RANBP10 antibodies could potentially develop targeted delivery systems for therapeutic agents across the blood-brain barrier, as similar approaches have shown promise with other receptor-mediated transport systems .

What emerging technologies might enhance the utility of biotin-conjugated RANBP10 antibody?

Several cutting-edge technologies are poised to expand the research applications of biotin-conjugated RANBP10 antibody:

• Proximity Ligation Assays (PLA): The biotin-conjugated antibody can be integrated into PLA to visualize and quantify RANBP10 interactions with binding partners at single-molecule resolution in situ. This would provide unprecedented insights into how RANBP10 functions within protein complexes that regulate cellular processes .

• CRISPR-based Imaging: Combining biotin-conjugated RANBP10 antibody with CRISPR-based genomic tagging systems could enable simultaneous visualization of RANBP10 protein localization and its gene expression regulation.

• Single-Cell Proteomics: The high sensitivity of the avidin-biotin system makes biotin-conjugated RANBP10 antibody ideal for emerging single-cell proteomic technologies, allowing researchers to examine RANBP10 expression heterogeneity within tissues .

• Nanoparticle-based Theranostics: Leveraging the avidin-biotin interaction, biotin-conjugated RANBP10 antibody could be incorporated into multifunctional nanoparticles that simultaneously image and deliver therapeutics to cells with altered RANBP10 expression .

• High-throughput Automated Imaging: Machine learning-enhanced microscopy paired with biotin-conjugated RANBP10 antibody could enable large-scale screening of RANBP10 expression across tissue arrays and cellular models, accelerating biomarker discovery.

These technological integrations would significantly expand our understanding of RANBP10's biological functions and its potential contributions to disease mechanisms.

What key research questions about RANBP10 remain unanswered?

Despite recent advances, several fundamental questions about RANBP10 biology await investigation:

• Regulatory Mechanisms: How is RANBP10 expression regulated at transcriptional and post-translational levels across different tissues and disease states? The biotin-conjugated antibody could help quantify RANBP10 levels under various cellular conditions and regulatory influences.

• Signal Transduction Pathways: Beyond its established role in the FBXW7–c-Myc axis in glioblastoma , what other signaling pathways involve RANBP10? Comprehensive interactome analysis using the biotin-conjugated antibody for immunoprecipitation could identify novel interaction partners.

• Functional Domains: Which domains of RANBP10 are essential for its various cellular functions, and how might these be targeted therapeutically? Domain-specific functional studies using the antibody could map critical regions of the protein.

• Isoform-Specific Functions: Do alternative splice variants of RANBP10 exist with distinct cellular functions? The biotin-conjugated antibody may help identify and characterize potential isoforms.

• Therapeutic Targeting: Given its role in cancer progression , can RANBP10 be effectively targeted for therapeutic intervention? The antibody could be instrumental in developing screening assays for inhibitors and evaluating their efficacy.

• Evolutionary Conservation: How conserved is RANBP10 function across species, and what does this tell us about its fundamental biological importance? Cross-species analysis using the antibody could provide evolutionary insights.

Addressing these questions will significantly advance our understanding of RANBP10 biology and its potential as a therapeutic target or diagnostic marker.