UBXN2A Antibody, HRP conjugated

What is UBXN2A and what cellular functions does it regulate?

UBXN2A (also known as UBXD4) belongs to the UBXD family of proteins containing the ubiquitin regulatory domain X (UBX). It functions as a p97 adaptor protein primarily localized in the cytosol and nucleus. UBXN2A is involved in several essential cellular processes including protein phosphatase regulation, ubiquitin binding, autophagosome assembly, Golgi organization, membrane fusion, nuclear envelope reassembly, and proteasome-mediated ubiquitin-dependent protein catabolic processes .

Most notably, UBXN2A plays a significant role in regulating the degradation of nicotinic acetylcholine receptors (nAChRs) containing the α3 subunit. It does this by interfering with CHIP-mediated ubiquitination of α3, thereby protecting this receptor subunit from endoplasmic reticulum-associated degradation (ERAD) . This protective function allows for enhanced maturation and trafficking of α3-containing nAChRs to the plasma membrane, which has implications for both normal ganglionic transmission and the mechanisms underlying nicotine addiction.

What are the typical applications for HRP-conjugated UBXN2A antibodies?

HRP-conjugated UBXN2A antibodies are primarily utilized in enzyme-linked immunosorbent assays (ELISA) . Unlike unconjugated UBXN2A antibodies that can be used for Western blotting, flow cytometry, and immunohistochemistry on paraffin-embedded sections , the HRP-conjugated variant offers the advantage of direct detection without requiring secondary antibody incubation steps.

The conjugation of HRP to UBXN2A antibodies enables chromogenic detection through various substrates including diaminobenzidine (DAB), ABTS, TMB, and TMBUS in the presence of hydrogen peroxide . The resulting colored reaction products can be quantified using spectrophotometric methods, providing precise measurement of UBXN2A presence in experimental samples. Direct detection with HRP-conjugated antibodies is particularly valuable in complex protocols where eliminating additional wash and separation steps can significantly reduce protocol time and potential sources of experimental variation.

How does the structure of UBXN2A influence antibody selection and experimental design?

UBXN2A contains distinct functional domains that influence antibody targeting strategies. Available antibodies often target the C-terminal region, specifically amino acids 166-195 . Understanding the domain organization of UBXN2A is crucial when selecting antibodies for specific experimental applications.

The protein contains a UBX domain that mediates interaction with p97/VCP, and researchers studying these interactions should select antibodies that do not interfere with this binding interface. For studies focusing on UBXN2A's interaction with CHIP or HSC70/HSP70 proteins, antibodies targeting regions away from these interaction domains would be preferable. When designing experiments to study UBXN2A's role in α3 nAChR regulation, researchers should consider that UBXN2A can be found in a protein complex containing p97, α3, and CHIP . Therefore, experimental conditions should preserve these protein-protein interactions when relevant to the research question.

How can HRP-conjugated UBXN2A antibodies be optimized for detection of protein-protein interactions in ERAD pathways?

For optimizing detection of UBXN2A interactions within ERAD pathways, researchers should consider several methodological refinements. When investigating UBXN2A's interactions with CHIP, p97, and HSC70/HSP70 in the context of α3 nAChR degradation, proteasome inhibitors such as MG132 (10 μM for 12 hours) can be employed to enhance E3 ligase-substrate interactions . This approach effectively inhibits the proteasome complex at the ERAD level, leading to accumulation of substrates regardless of the status of the p97/E3 ligases complex.

For co-immunoprecipitation studies, treatment with p97 inhibitors like DBeQ (5-10 μM for 3 hours) can provide insights into the dependency of UBXN2A-substrate interactions on p97 function . When designing pulldown experiments to study the physical interaction between UBXN2A and CHIP, expression systems using tagged constructs (such as HA-tagged UBXN2A and His-tagged CHIP) can be particularly effective . For optimal visualization of these interactions, the HRP-conjugated antibodies should be used at concentrations determined through careful titration experiments, typically starting at dilutions around 1:1000 for Western blotting applications .

What strategies can address potential cross-reactivity when using HRP-conjugated UBXN2A antibodies in multiplex immunoassays?

Cross-reactivity presents a significant challenge in multiplex immunoassays involving UBXN2A detection. Since UBXN2A interacts with various proteins in the ubiquitin-proteasome system, including CHIP, p97, HSC70/HSP70, and nAChR subunits , distinguishing specific signals becomes crucial. To address this, researchers can implement several strategies.

First, careful antibody selection is essential - choosing antibodies raised against unique epitopes of UBXN2A (such as the C-terminal region aa 166-195) minimizes potential cross-reactivity . Second, implementing stringent blocking protocols using 3-5% BSA or non-fat dry milk in TBS-T can reduce non-specific binding. Third, incorporating additional washing steps with elevated salt concentrations (up to 500 mM NaCl) can disrupt weak, non-specific interactions while preserving the specific antibody-antigen binding.

For multiplex assays specifically, sequential detection protocols should be considered where the HRP signal from the UBXN2A antibody is developed and quenched before introducing antibodies against potential interaction partners. Additionally, researchers should validate specificity through appropriate controls, including the use of UBXN2A knockdown samples generated using shRNA against UBXN2A (as described in the literature ) to confirm signal specificity.

How can researchers distinguish between different conformational states of UBXN2A using HRP-conjugated antibodies?

Distinguishing between different conformational states of UBXN2A requires sophisticated experimental approaches. UBXN2A may adopt different conformations when interacting with various binding partners within the ERAD pathway. To investigate these conformational states, researchers can employ limited proteolysis experiments before antibody detection.

When UBXN2A is in complex with different partners (p97, CHIP, HSC70/HSP70), certain epitopes may become masked or exposed. By implementing crosslinking strategies prior to immunodetection, these transient protein-protein interactions can be stabilized. For instance, using membrane-permeable crosslinkers like DSP (dithiobis(succinimidyl propionate)) at low concentrations (0.1-0.5 mM) for short durations (10-30 minutes) can preserve protein complexes without significantly altering their native conformation.

Additionally, researchers can leverage domain-specific UBXN2A antibodies targeting distinct regions (WT, ΔC, SEP, Linker, UBX domains as referenced in the literature ) to map conformational changes. Complementary techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) coupled with epitope-specific HRP-conjugated antibody detection can provide high-resolution insights into the structural dynamics of UBXN2A in different functional states.

What buffer conditions are optimal for maintaining HRP activity in UBXN2A antibody conjugates?

Optimal buffer conditions are critical for preserving HRP enzymatic activity in UBXN2A antibody conjugates. The recommended buffer should maintain a pH between 6.5-8.5 for maximum stability . When preparing buffers for diluting HRP-conjugated UBXN2A antibodies, researchers should avoid several components that can inhibit HRP activity or interfere with antibody-antigen binding.

Specifically, buffer compositions should limit or exclude:

• BSA and gelatin (keep below 0.1%)

• Tris (maintain below 50mM)

• Glycerol (keep below 50%)

Additionally, researchers must completely avoid buffers containing thiomersal/thimerosal, merthiolate, sodium azide, glycine, proclin, and nucleophilic components such as primary amines (amino acids, ethanolamine) and thiols (mercaptoethanol, DTT) . These compounds can significantly inhibit HRP activity or interfere with the antibody-antigen interaction.

For optimal long-term stability, specialized stabilizers like LifeXtend™ HRP conjugate stabilizer can protect antibody-HRP conjugates from degradation factors, particularly when working at room temperature .

What are the optimal detection methods for visualizing UBXN2A in different subcellular compartments?

UBXN2A localizes to both the cytosol and nucleus , requiring specific methodological approaches for accurate visualization in different subcellular compartments. For immunohistochemical or immunocytochemical detection of UBXN2A, HRP-conjugated antibodies can be employed with DAB substrate for a permanent, insoluble brown precipitate that allows for detailed morphological assessment.

For nuclear UBXN2A detection, additional permeabilization steps are crucial. A sequential permeabilization protocol is recommended: first with 0.1% Triton X-100 for 10 minutes followed by 0.5% SDS for 5 minutes to ensure nuclear membrane penetration. When studying UBXN2A in the context of the ERAD pathway, co-staining with ER markers (calnexin, PDI) can provide valuable context. For such multiplex detection, tyramide signal amplification (TSA) can be employed to enhance sensitivity of the HRP-conjugated antibody.

For quantitative assessment of UBXN2A distribution between subcellular compartments, subcellular fractionation followed by ELISA or Western blot analysis using HRP-conjugated antibodies at 1:1000 dilution provides reliable results. When studying UBXN2A's role in regulating α3 nAChR degradation, co-localization studies with nAChR subunits using confocal microscopy can reveal spatial relationships critical to understanding the protective function of UBXN2A against ERAD.

How can researchers validate the specificity of HRP-conjugated UBXN2A antibodies in their experimental system?

Validating antibody specificity is essential for generating reliable research data. For HRP-conjugated UBXN2A antibodies, multiple validation approaches should be implemented. First, genetic knockdown or knockout controls provide the most definitive validation. Using shRNA against UBXN2A (as described in previous studies ) to create knockdown cell lines offers an excellent negative control for antibody specificity testing.

Peptide competition assays represent another valuable validation method. Pre-incubating the HRP-conjugated UBXN2A antibody with excess synthetic peptide corresponding to the immunogen (amino acids 166-195 from the C-terminal region ) should abolish specific signals if the antibody is truly specific. The absence of signal reduction with unrelated peptides further confirms specificity.

Cross-species reactivity testing can provide additional validation. Since many UBXN2A antibodies are reactive to both human and mouse UBXN2A , comparing detection patterns across species with known expression patterns of UBXN2A can support specificity claims. Importantly, researchers should include appropriate isotype controls matched to the antibody host species and class.

For quantitative applications like ELISA, standard curves using recombinant UBXN2A protein (such as recombinant UBX domain-containing protein 2A protein, amino acids 15-162 ) should be generated to assess sensitivity and dynamic range. Signal linearity across a dilution series of both the antibody and the target protein provides further validation of specific detection.

How should researchers design experiments to study UBXN2A's role in protecting α3 nAChR from ERAD?

Researchers investigating UBXN2A's protective role against ERAD-mediated degradation of α3 nAChR should implement a multi-faceted experimental approach. Begin with establishing cellular models expressing both α3 nAChR subunits and UBXN2A, such as differentiated PC12 cells, which have been successfully used in previous studies . Modulate UBXN2A levels through overexpression using tagged constructs or knockdown using shRNA against UBXN2A.

To assess α3 ubiquitination levels, immunoprecipitate α3 nAChR subunits and probe for ubiquitin using Western blotting. Compare ubiquitination patterns in conditions of UBXN2A overexpression, knockdown, and control. To enhance detection of ubiquitinated species, treat cells with proteasome inhibitor MG132 (10 μM for 12 hours) to prevent degradation of ubiquitinated proteins .

For studying protein-protein interactions, implement co-immunoprecipitation experiments using HRP-conjugated UBXN2A antibodies to detect UBXN2A in complexes with CHIP, p97, HSC70/HSP70, and α3 nAChR. Cross-validate findings using reverse co-immunoprecipitation with antibodies against interaction partners. To examine the impact of p97 activity on these interactions, include experiments with p97 inhibitors like DBeQ (5-10 μM for 3 hours) .

Finally, assess functional outcomes by measuring surface expression of α3-containing nAChRs using biotinylation assays or electrophysiological recordings under conditions of varied UBXN2A expression, which will provide insights into the physiological significance of UBXN2A's protection against ERAD.

What protocol modifications are necessary when using HRP-conjugated UBXN2A antibodies across different experimental platforms?

When transitioning HRP-conjugated UBXN2A antibodies between different experimental platforms, protocol adjustments are essential to maintain optimal performance. For ELISA applications, the primary consideration is antibody concentration. Typically, concentrations should be determined through titration, starting with a 1:1000 dilution and adjusting based on signal-to-noise ratio .

When adapting to Western blotting, membrane blocking becomes critical. Use 5% non-fat dry milk in TBS-T for 1 hour at room temperature to reduce background. For detection, chemiluminescent substrates offer higher sensitivity than colorimetric alternatives, though the latter may provide more stable signals for densitometric analysis. Exposure times should be optimized to avoid signal saturation.

For immunohistochemistry, antigen retrieval methods dramatically impact epitope accessibility. Heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes is recommended for UBXN2A detection in paraffin-embedded tissues, with antibody dilutions around 1:50 . When moving to flow cytometry, permeabilization conditions must be optimized - 0.1% saponin in PBS with 1% BSA typically provides good results for intracellular UBXN2A detection.

In all applications, incorporate parallel detection with unconjugated antibodies followed by secondary HRP detection as a performance benchmark. This comparison helps identify any sensitivity losses due to the direct HRP conjugation and guides necessary protocol adjustments.