UBXN2A Antibody, Biotin conjugated

What is the function of UBXN2A protein and why is it important in cancer research?

UBXN2A is a UBX-domain-containing protein that acts as a potent mortalin inhibitor in cells. Mortalin (also known as mot-2) is an oncoprotein overexpressed in various cancer cells that promotes multiple oncogenic signaling pathways and antagonizes chemotherapy-induced cell death . UBXN2A binds to and occupies mortalin's binding pocket located within the substrate-binding domain (SBD), effectively interrupting mortalin's interaction with its partners, particularly p53 .

Research has demonstrated that UBXN2A expression can significantly decrease cell proliferation and migration of cancer cells, particularly in colon cancer and osteosarcoma models . Furthermore, UBXN2A overexpression has been shown to markedly increase the apoptotic response of cancer cells to chemotherapeutic agents such as 5-fluorouracil (5-FU) . This dual role of UBXN2A—inhibiting mortalin's oncogenic function and enhancing chemosensitivity—makes it a promising target for cancer research and potential therapeutic development.

Key Research Findings on UBXN2A Function:

How does biotin conjugation affect UBXN2A antibody performance in immunoprecipitation experiments?

Biotin conjugation of UBXN2A antibodies provides significant advantages for immunoprecipitation (IP) experiments investigating UBXN2A-protein interactions. The high-affinity binding between biotin and streptavidin creates a strong, specific capture system that facilitates efficient pull-down of UBXN2A and its protein complexes.

When designing immunoprecipitation experiments with biotin-conjugated UBXN2A antibodies, researchers should consider the following methodological aspects:

First, the biotin conjugation allows for a more efficient pull-down compared to traditional antibody-protein A/G approaches. As demonstrated in previous studies, His-tag pull-down assays successfully isolated UBXN2A protein and its binding partners for subsequent analysis . Biotin-conjugated antibodies can improve this process by reducing background and non-specific binding.

Second, when investigating UBXN2A-mortalin interactions specifically, it's essential to design protocols that preserve these protein-protein interactions. The evidence indicates that UBXN2A binds to the substrate-binding domain (SBD) of mortalin, with three amino acids (PRO442, ILE558, and LYS555) being crucial for this interaction . Harsh lysis conditions may disrupt these interactions.

Finally, biotin-conjugated antibodies are particularly valuable for studying the CHIP-mediated proteasomal degradation of mot-2 that is enhanced by UBXN2A. Research has shown that UBXN2A promotes CHIP-dependent ubiquitination of mot-2 , and biotin-conjugated antibodies can help isolate these ubiquitinated complexes efficiently.

What are the optimal conditions for using biotin-conjugated UBXN2A antibody in Western blot analysis?

For optimal Western blot detection of UBXN2A using biotin-conjugated antibodies, several critical parameters should be carefully controlled based on the protein's characteristics and expression patterns observed in research studies.

Buffer Composition and Blocking Conditions:

When preparing samples for UBXN2A Western blotting, it's important to use lysis buffers that preserve protein-protein interactions if you're studying UBXN2A-mortalin complexes. Research has shown that UBXN2A binds to mortalin's substrate-binding domain , and these interactions can be disrupted by harsh detergents.

A recommended blocking solution is 5% non-fat dry milk in TBS-T (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature. This blocking condition has been effectively used in studies examining UBXN2A expression and its effects on downstream proteins .

Detection System Optimization:

When using biotin-conjugated UBXN2A antibodies, a streptavidin-HRP (horseradish peroxidase) conjugate is typically used for detection. The concentration of streptavidin-HRP should be optimized to minimize background while maintaining sensitivity. Based on experimental procedures in mortalin-UBXN2A interaction studies, a 1:5000 to 1:10000 dilution of streptavidin-HRP is generally effective .

For enhanced sensitivity in detecting low levels of endogenous UBXN2A, an amplification step using enhanced chemiluminescence (ECL) substrates is recommended. This approach has been successfully applied in studies examining UBXN2A-mediated effects on apoptosis markers and cell proliferation .

Verification and Controls:

To ensure specificity, include positive controls with known UBXN2A expression (such as HCT-116 or U2OS cells transfected with UBXN2A expression constructs) and negative controls (such as UBXN2A-knockout U2OS cells) . These controls help validate antibody specificity and can serve as references for band intensity comparisons.

How can researchers effectively detect UBXN2A-mortalin interactions using biotin-conjugated antibodies?

Detecting UBXN2A-mortalin interactions requires careful experimental design due to the specific nature of their binding domains. Biotin-conjugated UBXN2A antibodies can be effectively employed in several complementary approaches to investigate these interactions.

Co-Immunoprecipitation (Co-IP) Strategy:

When designing co-IP experiments to study UBXN2A-mortalin interactions, researchers should consider that UBXN2A binds to mortalin's substrate-binding domain (SBD), with PRO442, LYS555, and ILE558 amino acids being crucial for this interaction . A recommended protocol involves:

• Lysing cells in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, and protease inhibitors

• Incubating lysates with biotin-conjugated UBXN2A antibody (5 μg/mg of protein)

• Capturing complexes with streptavidin-coated magnetic beads

• Washing thoroughly to remove non-specific binding

• Eluting and analyzing by Western blot for mortalin

This approach has proven effective in studies examining mortalin-UBXN2A interactions and can reveal whether the interactions are altered under different experimental conditions .

Validation Through Mutagenesis:

To confirm the specificity of detected interactions, a mutagenesis approach targeting key amino acids in mortalin's binding pocket has been successfully employed. Previous research utilized point mutations (P442A, I558A, and K555A) in mortalin's SBD domain to demonstrate the specificity of UBXN2A binding . Researchers can employ similar strategies when using biotin-conjugated antibodies by:

• Generating expression constructs for wild-type and mutant mortalin

• Transfecting cells and performing co-IP with biotin-conjugated UBXN2A antibodies

• Comparing the pull-down efficiency between wild-type and mutant mortalin

This approach provides strong evidence for the specificity of detected interactions and can help identify the precise binding domains involved.

Proximity Ligation Assay (PLA):

For in situ detection of UBXN2A-mortalin interactions in fixed cells or tissues, PLA using biotin-conjugated UBXN2A antibodies paired with anti-mortalin antibodies offers advantages in spatial resolution. This technique can complement biochemical approaches and provide information about the subcellular localization of interactions, which is particularly relevant given that UBXN2A has been shown to affect mortalin's functions in specific cellular compartments .

What role does UBXN2A play in proteasomal degradation, and how can this be studied with biotin-conjugated antibodies?

UBXN2A has been identified as a critical regulator of proteasomal degradation, specifically enhancing CHIP-mediated ubiquitination and subsequent degradation of mortalin/mot-2 . This function represents an important mechanism by which UBXN2A exerts its anti-cancer effects.

Mechanism of UBXN2A-Enhanced Proteasomal Degradation:

Research has demonstrated that UBXN2A promotes the ubiquitination of mot-2 through interaction with the carboxyl terminus of the HSP70-interacting protein (CHIP) E3 ubiquitin ligase . This process involves several steps:

• UBXN2A binds to mot-2 via its SEP domain

• UBXN2A facilitates CHIP-dependent ubiquitination of mot-2

• Ubiquitinated mot-2 is targeted for proteasomal degradation

• Reduced mot-2 levels lead to decreased cell proliferation and increased apoptosis in cancer cells

Experimental Approaches Using Biotin-Conjugated Antibodies:

To investigate UBXN2A's role in proteasomal degradation, researchers can employ biotin-conjugated UBXN2A antibodies in the following experimental designs:

• Ubiquitination Assays: In vitro ubiquitination assays using biotin-conjugated UBXN2A antibodies to immunoprecipitate UBXN2A-mot-2-CHIP complexes, followed by Western blotting for ubiquitin. This approach has been used successfully to demonstrate CHIP-dependent ubiquitination of mot-2 .

• Proteasome Inhibition Studies: Treating cells with proteasome inhibitors (e.g., MG132) while manipulating UBXN2A expression levels and using biotin-conjugated antibodies to track changes in mot-2 protein levels. Previous research has shown that proteasome inhibition can block UBXN2A-mediated reduction in mot-2 levels .

• CHIP Depletion Experiments: Silencing CHIP expression using siRNA while monitoring UBXN2A-mediated effects on mot-2 using biotin-conjugated antibodies. This approach has confirmed that UBXN2A enhances mot-2 degradation in a CHIP-dependent manner .

Research Data on UBXN2A and Proteasomal Degradation:

How does UBXN2A expression affect chemosensitivity, and how can biotin-conjugated antibodies help investigate this phenomenon?

UBXN2A has been shown to significantly enhance cancer cell sensitivity to chemotherapeutic agents, particularly 5-fluorouracil (5-FU) . This chemosensitizing effect represents a promising avenue for cancer treatment strategies, especially for mortalin-enriched tumors such as osteosarcoma.

Mechanism of UBXN2A-Mediated Chemosensitization:

Research has revealed that UBXN2A enhances chemosensitivity through multiple mechanisms:

• UBXN2A binds to and inhibits mortalin, preventing its anti-apoptotic functions

• UBXN2A expression stabilizes p53 by interfering with mortalin-CHIP E3 ubiquitin ligase

• UBXN2A promotes apoptosis pathways in the presence of chemotherapeutic agents

• UBXN2A facilitates the degradation of mortalin, reducing its protective effects against chemotherapy

Studies have demonstrated that silencing UBXN2A in human osteosarcoma U2OS cells significantly reduced the cytotoxic effect of 5-FU, while overexpression of UBXN2A markedly increased the apoptotic response to 5-FU treatment .

Research Applications of Biotin-Conjugated UBXN2A Antibodies:

Biotin-conjugated UBXN2A antibodies are valuable tools for investigating the mechanisms of chemosensitization:

• Tracking UBXN2A Induction: Biotin-conjugated antibodies can monitor UBXN2A expression levels following treatment with UBXN2A enhancers like veratridine (VTD) . This allows researchers to correlate UBXN2A expression with chemosensitivity.

• Apoptosis Pathway Analysis: Immunoprecipitation with biotin-conjugated UBXN2A antibodies can identify key proteins in apoptosis pathways that interact with UBXN2A following chemotherapy treatment.

• In Vivo Imaging: For animal studies, biotin-conjugated antibodies can be used for imaging UBXN2A expression in tumor xenografts treated with chemotherapy, providing visual evidence of UBXN2A's role in treatment response.

Experimental Data on UBXN2A and Chemosensitivity:

What technical considerations are important for immunofluorescence staining using biotin-conjugated UBXN2A antibodies?

Immunofluorescence staining with biotin-conjugated UBXN2A antibodies requires careful optimization to achieve specific subcellular localization data, particularly important given UBXN2A's interactions with mortalin in different cellular compartments.

Fixation and Permeabilization Protocol:

For optimal detection of UBXN2A by immunofluorescence, the following protocol is recommended based on successful approaches in published research:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Block with 5% normal serum (matching secondary antibody host) for 1 hour

• Incubate with biotin-conjugated UBXN2A antibody (1:200 dilution) overnight at 4°C

• Detect with fluorophore-conjugated streptavidin (1:500 dilution) for 1 hour at room temperature

This approach has been effective in studies examining the subcellular distribution of UBXN2A and its colocalization with binding partners such as mortalin .

Subcellular Localization Considerations:

When using biotin-conjugated UBXN2A antibodies for localization studies, researchers should consider that UBXN2A has been shown to affect the distribution of proteins in different cellular compartments. Research has demonstrated that UBXN2A expression can lead to stabilization of p53 in the cytoplasm and its upregulation in the nucleus . Therefore, counterstaining with markers for specific organelles (e.g., mitochondria for mortalin localization) is recommended for comprehensive analysis.

Additionally, subcellular fractionation experiments have revealed that induced UBXN2A decreases the level of mortalin and its chaperone partner, HSP60 . This finding suggests that researchers should examine multiple cellular compartments when studying UBXN2A-mediated effects.

Co-localization Analysis with Binding Partners:

For co-localization studies examining UBXN2A and its binding partners (particularly mortalin), dual immunofluorescence staining is recommended. This approach involves:

• Using biotin-conjugated UBXN2A antibody with fluorophore-conjugated streptavidin

• Combining with a directly labeled antibody against mortalin or other binding partners

• Analyzing co-localization using appropriate imaging software with statistical measures (Pearson's correlation coefficient, Manders' overlap coefficient)

This approach can provide valuable insights into the spatial relationship between UBXN2A and its binding partners under different experimental conditions.

How can researchers optimize flow cytometry protocols using biotin-conjugated UBXN2A antibodies?

Flow cytometry using biotin-conjugated UBXN2A antibodies offers a powerful approach for quantifying UBXN2A expression levels and correlating them with functional outcomes such as apoptosis in large cell populations. Optimizing flow cytometry protocols requires attention to several key parameters.

Sample Preparation and Antibody Titration:

For intracellular staining of UBXN2A, cells must be properly fixed and permeabilized while preserving epitope recognition. Based on successful flow cytometry approaches in UBXN2A research:

• Fix cells with 2% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1% saponin or 0.1% Triton X-100 in PBS for 10 minutes

• Block with 2% BSA in PBS for 30 minutes

• Incubate with titrated concentrations of biotin-conjugated UBXN2A antibody (typically starting with 1:100 and testing serial dilutions)

• Detect with fluorophore-conjugated streptavidin

Antibody titration is critical to determine the optimal concentration that provides the highest signal-to-noise ratio. Previous studies examining UBXN2A-mediated effects on apoptosis have successfully employed flow cytometry to quantify Annexin V levels in response to UBXN2A expression .

Multi-Parameter Analysis Strategy:

To gain comprehensive insights into UBXN2A function, multi-parameter flow cytometry protocols combining UBXN2A staining with functional markers are recommended:

• UBXN2A and Apoptosis Markers: Combine biotin-conjugated UBXN2A antibody with Annexin V and propidium iodide to correlate UBXN2A expression with early and late apoptosis. This approach has been successful in demonstrating that UBXN2A overexpression enhances 5-FU-induced apoptosis in U2OS cells .

• UBXN2A and Cell Cycle Analysis: Combine UBXN2A staining with DNA content analysis using propidium iodide to investigate how UBXN2A affects cell cycle progression, particularly given its anti-proliferative effects in cancer cells .

• UBXN2A and Mortalin Co-expression: Dual staining for UBXN2A and mortalin can provide insights into their relative expression levels and potential correlation with cell death pathways.

Gating Strategy and Controls:

For reliable flow cytometry analysis of UBXN2A expression, implement the following controls and gating strategy:

• Use cells with confirmed UBXN2A overexpression as positive controls (e.g., transiently transfected GFP-UBXN2A cells)

• Include UBXN2A-silenced cells as negative controls (e.g., cells transfected with lentiviral shRNA against UBXN2A)

• Employ FMO (fluorescence minus one) controls to set accurate gates

• Gate on single, viable cells before analyzing UBXN2A expression

• Consider comparing UBXN2A expression levels across different treatment conditions (e.g., with and without 5-FU or VTD)

This comprehensive approach allows for accurate quantification of UBXN2A expression and its correlation with functional cellular outcomes.

What considerations are important for using biotin-conjugated UBXN2A antibodies in chromatin immunoprecipitation (ChIP) experiments?

While UBXN2A is primarily studied for its protein-protein interactions, emerging research suggests potential roles in regulating transcription through indirect mechanisms. Chromatin immunoprecipitation (ChIP) using biotin-conjugated UBXN2A antibodies can provide insights into these processes, with several important methodological considerations.

ChIP Protocol Optimization:

When adapting biotin-conjugated UBXN2A antibodies for ChIP experiments, researchers should consider the following protocol modifications:

• Crosslinking Conditions: Use 1% formaldehyde for 10 minutes at room temperature, as this preserves protein-protein interactions without excessive crosslinking that could mask the UBXN2A epitope.

• Sonication Parameters: Optimize sonication conditions to generate DNA fragments of 200-500 bp, which is ideal for high-resolution mapping of potential UBXN2A-associated chromatin regions.

• Antibody Incubation: Incubate chromatin with biotin-conjugated UBXN2A antibody overnight at 4°C with gentle rotation to ensure maximum binding.

• Streptavidin Bead Selection: Use high-capacity streptavidin magnetic beads for efficient capture of biotin-conjugated antibody complexes.

• Elution Conditions: Consider using biotin competition elution to minimize background, as this specifically disrupts the biotin-streptavidin interaction.

Biological Context for UBXN2A ChIP:

When designing ChIP experiments with UBXN2A antibodies, consider that UBXN2A may indirectly affect transcription through its interactions with mortalin and p53. Research has shown that UBXN2A binding to mortalin leads to stabilization of p53 and its upregulation in the nucleus , which could affect p53-dependent transcriptional programs.

Potential targets for ChIP-qPCR validation following UBXN2A ChIP might include:

• p53-responsive genes involved in cell cycle regulation and apoptosis

• Genes affected by mortalin-dependent transcription factors

• Promoters of genes encoding proteins in the ubiquitin-proteasome pathway

Controls and Validation Approaches:

To ensure the specificity and reliability of ChIP results with biotin-conjugated UBXN2A antibodies:

• Include IgG-biotin control ChIP samples to establish background signal levels

• Perform parallel ChIP experiments with antibodies against known interacting partners (e.g., p53)

• Validate ChIP-Seq findings with targeted ChIP-qPCR on selected genomic regions

• Compare ChIP results between conditions with normal UBXN2A expression and UBXN2A overexpression or knockdown

• Consider sequential ChIP (re-ChIP) to identify genomic regions simultaneously bound by UBXN2A and its interacting partners

How can biotin-conjugated UBXN2A antibodies be utilized in therapeutic development research?

Biotin-conjugated UBXN2A antibodies offer valuable tools for therapeutic development research, particularly in the context of exploiting UBXN2A's anti-cancer properties. Several research avenues can benefit from these antibodies in the drug discovery process.

Target Validation and Screening Assays:

Biotin-conjugated UBXN2A antibodies can be employed in high-throughput screening assays to identify compounds that modulate UBXN2A expression or function:

• Compound Screening: Develop cell-based assays using biotin-conjugated UBXN2A antibodies to identify compounds that induce UBXN2A expression, similar to veratridine (VTD) . This approach could discover novel UBXN2A enhancers with improved pharmacological properties.

• Binding Displacement Assays: Create in vitro assays to screen for compounds that mimic UBXN2A's binding to mortalin, using biotin-conjugated antibodies to detect displacement of UBXN2A from mortalin complexes.

• Proteasomal Degradation Enhancement: Develop assays to identify compounds that enhance UBXN2A-mediated proteasomal degradation of mortalin, measuring changes in ubiquitination and protein levels using biotin-conjugated antibodies .

Mechanism-of-Action Studies:

For compounds identified as potential therapeutics targeting the UBXN2A-mortalin pathway, biotin-conjugated antibodies can help elucidate their mechanism of action:

• Protein Interaction Analysis: Use pull-down assays with biotin-conjugated UBXN2A antibodies to determine how candidate compounds affect UBXN2A's interaction with mortalin and other partners.

• Subcellular Localization Changes: Employ immunofluorescence with biotin-conjugated antibodies to track changes in UBXN2A and mortalin localization following compound treatment.

• Pathway Impact Assessment: Combine biotin-conjugated UBXN2A antibodies with antibodies against downstream effectors (e.g., p53, apoptosis markers) to map pathway alterations induced by candidate compounds.

Combination Therapy Research:

Research has demonstrated that UBXN2A expression enhances the cytotoxic effect of chemotherapeutic agents like 5-FU . Biotin-conjugated UBXN2A antibodies can facilitate research into optimal combination therapy strategies:

• Synergy Assessment: Use flow cytometry with biotin-conjugated UBXN2A antibodies to quantify how UBXN2A levels correlate with apoptotic responses to different chemotherapeutic agents.

• Resistance Mechanism Investigation: Compare UBXN2A expression and function between chemotherapy-sensitive and resistant cell lines using biotin-conjugated antibodies to identify potential resistance mechanisms related to UBXN2A pathway alterations.

• In Vivo Efficacy Monitoring: Employ biotin-conjugated antibodies in immunohistochemistry of tumor xenograft sections to correlate UBXN2A expression with treatment response in animal models.

Translational Research Applications: