UBXN2B Antibody

What is UBXN2B and what cellular functions should researchers consider when designing experiments?

UBXN2B (UBX domain-containing protein 2B), also known as NSFL1 cofactor p37 or p97 cofactor p37, is a 331 amino acid protein with a calculated molecular weight of 37 kDa that contains one UBX domain and one SEP domain . This protein serves multiple critical cellular functions that should inform experimental design:

• Functions as an adapter protein required for Golgi and endoplasmic reticulum biogenesis

• Maintains Golgi and endoplasmic reticulum integrity during interphase

• Facilitates reassembly of these organelles at the end of mitosis

• Regulates spindle orientation during mitosis by limiting cortical NuMA levels

• Acts as a cofactor of the p97 AAA ATPase

When designing experiments targeting UBXN2B, researchers should consider its context-dependent localization and function throughout the cell cycle, particularly during mitosis when its regulatory role becomes especially prominent.

What applications are UBXN2B antibodies validated for, and what are the recommended dilution ranges?

Current commercially available UBXN2B antibodies have been validated for multiple research applications with specific dilution recommendations:

It is crucial to note that optimal dilutions may vary based on sample type, fixation method, and detection system. Researchers should perform titration experiments to determine the optimal dilution for their specific experimental setup .

What species reactivity is documented for available UBXN2B antibodies?

Different UBXN2B antibodies show varying species reactivity patterns:

*Indicates predicted reactivity based on sequence homology (69% for mouse, 75% for rat)

When working with non-human samples, researchers should select antibodies with documented reactivity for the relevant species or perform preliminary validation experiments to confirm cross-reactivity.

How should researchers optimize UBXN2B antibody usage in immunohistochemistry?

For optimal IHC results with UBXN2B antibodies, researchers should consider the following methodological recommendations:

• Antigen retrieval: For formalin-fixed, paraffin-embedded (FFPE) tissues, using TE buffer (pH 9.0) is suggested. Alternatively, citrate buffer (pH 6.0) may be used as noted in the Proteintech protocol .

• Antibody dilution: Begin with the recommended range (e.g., 1:400-1:1600 for Proteintech 25141-1-AP or 1:200-1:500 for HPA045278) and optimize through titration .

• Detection system: Choose an appropriate detection system based on the host species of the primary antibody. For example, rabbit antibodies like HPA045278 or 25141-1-AP would require anti-rabbit detection systems.

• Positive controls: Human ovary cancer tissue has been validated as a positive control for the Proteintech antibody . Researchers should include appropriate positive control tissues in their experiments.

• Protocol considerations: Follow antibody-specific protocols when available. For example, Proteintech offers downloadable IHC protocols specifically optimized for their UBXN2B antibody (25141-1-AP) .

When analyzing IHC results, consider that UBXN2B expression may vary between tumor and non-tumor tissues, as suggested by research on triple negative breast cancers .

What are the critical considerations for using UBXN2B antibodies in immunofluorescence studies?

For successful immunofluorescence experiments targeting UBXN2B, researchers should optimize:

• Fixation methods:

  • For standard IF: 4% formaldehyde with 0.1% Triton X-100 has been used successfully

  • Alternative method: Methanol fixation at -20°C for 4 minutes

• Permeabilization: Use of 0.5% saponin in 20 mM Pipes buffer (pH 6.8) with 5% BSA, 137 mM NaCl, and 2.7 mM KCl has been documented for successful UBXN2B detection .

• Antibody dilution: Begin with the recommended range (e.g., 1:200-1:800 for Proteintech antibody) and optimize through titration experiments .

• Validated positive controls: Neuro-2a cells have been documented as positive controls for IF detection of UBXN2B .

• Image acquisition parameters: For quantitative analysis of UBXN2B, particularly at the cell cortex, researchers should consider using line scan approaches (10-pixel-wide × 6-µm-long line scans overlapping the cortex) as described by Lee et al. .

For studies focusing on UBXN2B's role in spindle orientation, co-staining with markers like NuMA, LGN, and tubulin is recommended to analyze its functional interactions .

How can researchers validate the specificity of their UBXN2B antibody?

To ensure reliable and reproducible results, researchers should validate UBXN2B antibody specificity through multiple approaches:

• Western blot analysis: Confirm a single band at the expected molecular weight of 37 kDa in appropriate positive control samples .

• siRNA knockdown controls: Include samples treated with UBXN2B-specific siRNA to demonstrate reduced signal intensity, as performed in spindle orientation studies .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before staining to block specific binding.

• Multiple antibody validation: Use antibodies raised against different epitopes of UBXN2B to confirm staining patterns. For example, compare results from antibodies targeting the N-terminal region (AA 2-250, ABIN7174595) vs. those targeting other regions .

• Overexpression controls: In cell models amenable to transfection, include samples overexpressing tagged UBXN2B and confirm co-localization of antibody signal with the tag.

• Cross-reactivity testing: If working with multiple species, validate antibody specificity in each species separately, as cross-reactivity patterns may vary .

Proper antibody validation is particularly important when studying UBXN2B in cancer contexts, where expression changes may be subtle or tissue-specific .

How can researchers design experiments to study UBXN2B's role in spindle orientation during mitosis?

To investigate UBXN2B's function in spindle orientation, researchers can implement these advanced experimental approaches:

• Live-cell imaging: Use time-lapse microscopy to track spindle dynamics in cells with manipulated UBXN2B levels. This method can capture dynamic spindle rotations that occur in p37/UBXN2B-depleted cells .

• Spindle angle quantification: Calculate spindle angles by obtaining x, y, and z coordinates of spindle poles (using γ- or α-tubulin staining) and applying trigonometric functions to determine the angle relative to the growth surface .

• Cortical protein quantification: Measure levels of cortical proteins by overlaying a 3-pixel-wide line on the cortex of metaphase cells and obtaining mean intensity values. For comprehensive analysis, generate intensity profiles around the entire cortex .

• Protein depletion strategies:

  • siRNA knockdown of UBXN2B/p37

  • Co-depletion of UBXN2B with interacting partners (e.g., Repo-Man)

  • Treatment with specific inhibitors (e.g., calyculin A to inhibit phosphatase activity)

• Key experimental controls:

  • Comparison with depletion of known spindle orientation regulators (Gαi, LGN, NuMA)

  • Rescue experiments with siRNA-resistant UBXN2B constructs

  • Controls for Cdk1 activity (MPM2 staining) to rule out cell cycle effects

Researchers should analyze multiple parameters, including spindle rotation frequency, metaphase duration, and cortical localization of NuMA, LGN, and Gαi to comprehensively understand UBXN2B's role .

What approaches can researchers use to study UBXN2B's interactions with the p97 AAA ATPase and associated complexes?

To investigate the functional interactions between UBXN2B and p97 AAA ATPase, researchers can employ these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-UBXN2B antibodies to pull down protein complexes

  • Probe for p97 and other suspected interacting partners (e.g., VCPIP1, USO1, GOLGA2, BET1L)

  • Include appropriate controls (IgG control, UBXN2B-depleted lysates)

• Proximity ligation assays (PLA):

  • Detect and quantify protein-protein interactions in situ

  • Useful for studying interactions in different cell cycle stages

  • Can reveal spatial distribution of UBXN2B-p97 complexes

• Membrane fusion assays:

  • Assess the functional impact of UBXN2B-p97 interaction on membrane fusion activity

  • Include conditions with and without USO1-GOLGA2 tethering and BET1L

• Domain mapping experiments:

  • Create deletion mutants lacking specific domains (UBX domain, SEP domain)

  • Assess impact on p97 binding and functional outcomes

  • Identify critical residues for interaction

• Subcellular fractionation:

  • Separate cellular compartments (cytosol, membranes, organelles)

  • Analyze distribution of UBXN2B-p97 complexes across fractions

  • Examine changes in distribution during cell cycle progression

These approaches can help elucidate how UBXN2B contributes to the diverse functions of p97 complexes in Golgi and ER biogenesis, maintenance, and reassembly .

How can researchers analyze UBXN2B's involvement in regulating NuMA levels at the cell cortex?

To investigate UBXN2B's role in regulating cortical NuMA, researchers should implement these specialized approaches:

• Quantitative immunofluorescence analysis:

  • Use line profile analysis (10-pixel-wide × 6-µm-long) overlapping the cortex to measure NuMA intensity

  • Generate cortical intensity profiles around the entire cell perimeter using ImageJ

  • Calculate the ratio of cortical to cytoplasmic NuMA to normalize for expression differences

• Phosphorylation state analysis:

  • Assess NuMA phosphorylation status using phospho-specific antibodies

  • Use phosphatase inhibitors (calyculin A at 50 nM for 10 minutes) to block dephosphorylation

  • Analyze the impact of Aurora A inhibition (MLN8237 at 20 nM for 24h) on cortical NuMA levels

• Phosphatase pathway investigation:

  • Study PP1 localization using GFP-PP1α-expressing cells

  • Measure PP1 at kinetochores by determining the ratio between GFP signal intensities at kinetochores normalized to cytoplasmic signal

  • Analyze the effect of Repo-Man depletion on cortical NuMA levels

• Co-depletion experiments:

  • Perform single and double depletions of UBXN2B with PP1 regulatory subunits (Repo-Man, Sds-22)

  • Compare cortical NuMA levels across different depletion conditions

  • Include rescue experiments with siRNA-resistant constructs

• Temporal analysis:

  • Compare the regulation of cortical NuMA in metaphase versus anaphase cells

  • Track NuMA recruitment to the cortex during mitotic progression using live-cell imaging

These methodological approaches can help elucidate the mechanistic details of how UBXN2B regulates cortical NuMA levels via the PP1/Repo-Man pathway and impacts spindle orientation .

What are the recommended approaches for investigating UBXN2B expression and function in cancer research?

For researchers studying UBXN2B in cancer contexts, particularly triple negative breast cancer, the following methodological approaches are recommended:

• Comparative expression analysis:

  • Analyze UBXN2B at both mRNA and protein levels across tumor and matched non-tumor tissues

  • Be aware that protein and mRNA expression patterns may show opposite trends, as observed in some cancer studies

  • Use multiple antibodies and detection methods to confirm expression patterns

• Co-expression analysis with functional partners:

  • Analyze correlation between UBXN2B and its functional partners (VCPIP1, p97/VCP)

  • Consider analyzing together with MYBL1, which has been studied in conjunction with UBXN2B in breast cancer research

• Cell line models:

  • Compare UBXN2B expression and function across panels of cancer cell lines

  • Include both hormone receptor-positive and triple-negative breast cancer cell lines if studying breast cancer

• Functional assays:

  • Assess the impact of UBXN2B manipulation on cancer cell proliferation, migration, and invasion

  • Investigate potential roles in ER stress responses, which connect to UBXN2B's known functions in ER biogenesis and maintenance

• UBXN2B antibody application in cancer tissue:

  • For IHC of cancer tissues, human ovary cancer tissue has been validated as a positive control with the Proteintech antibody

  • Consider antigen retrieval optimization, as cancer tissues may require different conditions than normal tissues

These approaches can help elucidate UBXN2B's potential roles in cancer development and progression, particularly in relation to its functions in cellular organization and division .

How can researchers address common problems with UBXN2B antibody applications?

When troubleshooting UBXN2B antibody applications, researchers should consider these common issues and solutions:

• Weak or no signal in Western blots:

  • Increase antibody concentration (start at the upper end of recommended range)

  • Optimize protein loading (37 kDa proteins typically transfer efficiently)

  • Try enhanced detection systems (e.g., enhanced chemiluminescence)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Verify sample preparation methods preserve UBXN2B integrity

• High background in immunofluorescence:

  • Increase blocking time and concentration (5% BSA in PBS)

  • Reduce primary antibody concentration

  • Try alternative fixation methods (compare formaldehyde vs. methanol fixation)

  • Implement additional washing steps with 0.1% Tween-20 in PBS

• Variable staining patterns in IHC:

  • Optimize antigen retrieval (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Standardize fixation time for tissue samples

  • Include positive control tissues (e.g., human ovary cancer tissue)

  • Try signal amplification methods if tissue expression is low

• Inconsistent immunoprecipitation results:

  • Optimize lysis conditions (150 mM KCl, 50 mM Tris pH 7.4, 5 mM MgCl₂, 5% glycerol, 1% Triton X-100)

  • Add phosphatase inhibitors to preserve phosphorylation states

  • Compare different antibodies that recognize distinct epitopes

  • Increase incubation time for antibody-antigen binding

• Cell-cycle dependent variations:

  • Synchronize cells when studying mitotic functions of UBXN2B

  • Compare results in interphase vs. mitotic cells

  • Consider fixation methods that preserve cell cycle-specific structures

For each application, researchers should implement systematic optimization strategies and maintain detailed records of experimental conditions to identify critical variables affecting antibody performance.

What experimental controls should researchers include when using UBXN2B antibodies?

To ensure reliable and interpretable results with UBXN2B antibodies, researchers should implement these essential controls:

• Antibody validation controls:

  • Negative control: Primary antibody omission

  • Isotype control: Non-specific IgG matching the host species and concentration

  • Knockdown control: siRNA depletion of UBXN2B (reduces signal intensity)

  • Peptide competition: Pre-incubation with immunizing peptide (blocks specific binding)

• Sample-specific controls:

  • Positive tissue controls: Human ovary cancer tissue for IHC

  • Positive cell controls: Neuro-2a cells for IF/ICC and WB

  • Loading controls: Housekeeping proteins (e.g., GAPDH, β-actin) for Western blots

• Application-specific controls:

  • For spindle orientation studies: Include Gαi, LGN, or NuMA depletion controls

  • For phosphorylation studies: Include phosphatase inhibitor (calyculin A) controls

  • For cell cycle studies: Include mitotic markers (e.g., phospho-histone H3)

• Technical controls:

  • Antibody titration series to determine optimal concentration

  • Multiple fixation methods comparison (particularly for IF applications)

  • Replicate samples to assess technical variability

• Comparative controls:

  • Multiple antibodies against different UBXN2B epitopes

  • Different detection methods (e.g., fluorescent vs. chromogenic for IHC)

  • Cross-validation with orthogonal techniques (e.g., RNA expression data)

Proper implementation of these controls will strengthen data interpretation and improve reproducibility of UBXN2B antibody-based experiments .

How should researchers interpret conflicting results from different UBXN2B antibodies?

When faced with discrepancies between different UBXN2B antibodies, researchers should systematically analyze potential sources of variation and resolve conflicts through these approaches:

• Epitope mapping analysis:

  • Compare the epitope regions recognized by each antibody

  • Antibodies targeting different domains may detect distinct subpopulations of UBXN2B

  • Example: Compare results from antibodies targeting AA 2-250 region (ABIN7174595) versus antibodies targeting other regions

• Post-translational modification considerations:

  • Assess whether antibodies are sensitive to phosphorylation states

  • UBXN2B function is regulated by phosphorylation/dephosphorylation pathways involving PP1

  • Test phosphatase treatment of samples before antibody application

• Validation in knockout/knockdown systems:

  • Apply all antibodies to samples with UBXN2B depletion by siRNA

  • Antibodies detecting non-specific bands/signals will show residual staining

  • Quantify signal reduction for each antibody to assess specificity

• Application-specific optimization:

  • Different antibodies may perform optimally in different applications

  • For example, rabbit polyclonal 25141-1-AP shows broad application range

  • Monoclonal antibodies (MA5-25432, MA5-25449) may show higher specificity but narrower application range

• Batch-to-batch variation assessment:

  • Request lot-specific validation data from manufacturers

  • Consider antibody validation services for critical experiments

  • Maintain records of antibody lot numbers used in experiments

When publishing results, researchers should clearly report which antibody was used (including catalog number and lot), detailing the validation steps performed and acknowledging any limitations in interpretation based on antibody characteristics.

What emerging methodologies could enhance UBXN2B antibody applications in research?

Several cutting-edge technologies offer significant potential to advance UBXN2B research beyond traditional antibody applications:

• Proximity-based labeling techniques:

  • BioID or TurboID fusion with UBXN2B to identify proximal interaction partners

  • APEX2-UBXN2B fusions for ultrastructural localization via electron microscopy

  • These approaches could reveal cell cycle-specific or compartment-specific interactomes

• Super-resolution microscopy applications:

  • STORM or PALM imaging to resolve nanoscale distribution of UBXN2B at the cell cortex

  • Structured illumination microscopy (SIM) to improve visualization of UBXN2B at mitotic structures

  • These techniques could reveal previously undetectable organizational patterns

• Antibody-based protein degradation:

  • PROTAC or dTAG approaches linked to anti-UBXN2B antibodies or fragments

  • Enables rapid, temporal control of UBXN2B degradation to study acute loss phenotypes

  • Avoids compensatory mechanisms associated with genetic knockdown

• Live-cell antibody applications:

  • Cell-permeable antibody fragments (nanobodies) against UBXN2B

  • Development of genetically encoded intrabodies targeting UBXN2B

  • Would enable real-time tracking of endogenous UBXN2B during cell division

• Single-cell antibody-based techniques:

  • Combining UBXN2B antibody staining with mass cytometry (CyTOF)

  • Integration with spatial transcriptomics to correlate protein localization with gene expression

  • Could reveal heterogeneity in UBXN2B expression and function within tissues

These methodological advances would complement existing UBXN2B antibody applications and potentially reveal new aspects of its function in cellular organization, division, and disease contexts.

How might UBXN2B research contribute to our understanding of cancer progression and potential therapeutic approaches?

The emerging role of UBXN2B in cancer biology, particularly in triple negative breast cancer , suggests several important research directions:

• Prognostic biomarker development:

  • Evaluate correlation between UBXN2B expression patterns and clinical outcomes

  • Develop standardized IHC scoring systems for UBXN2B in tumor tissues

  • Investigate discrepancies between mRNA and protein expression patterns observed in some cancers

• Functional impact on cancer cell biology:

  • Investigate how UBXN2B's role in spindle orientation affects chromosomal stability in cancer cells

  • Examine potential contributions to therapy resistance through ER stress response pathways

  • Study impact on cancer cell division, invasion, and metastatic potential

• Therapeutic target assessment:

  • Evaluate UBXN2B as a potential therapeutic target, particularly in cancers with dysregulated expression

  • Develop screening assays using validated UBXN2B antibodies to identify small molecule modulators

  • Investigate synthetic lethal interactions with existing cancer therapeutics

• Pathway integration analysis:

  • Study how UBXN2B integrates with other cancer-relevant pathways

  • Examine relationships with VCPIP1 and MYBL1, which have been studied alongside UBXN2B in cancer

  • Investigate connections to p97/VCP inhibitors currently in clinical development

• Cancer-specific isoform analysis:

  • Use isoform-specific antibodies to determine if cancer cells express variant forms of UBXN2B

  • Assess functional differences between potential UBXN2B isoforms in normal vs. cancer cells

  • Develop tools to selectively target cancer-relevant forms

These research directions could not only advance our fundamental understanding of cancer biology but also potentially reveal new therapeutic strategies for cancers where UBXN2B plays a significant role.