UBAC2 Antibody

What is UBAC2 and why is it significant in cellular research?

UBAC2 (Ubiquitin-Associated Domain-Containing Protein 2) is a protein that plays critical roles in several cellular pathways including ER-phagy (selective autophagy of endoplasmic reticulum fragments) and regulation of inflammatory responses. Research has identified UBAC2 as a phospho-regulated ER-phagy receptor that can suppress the unfolded protein response and sterile inflammation . The protein is particularly significant because:

• It functions as an ER-phagy receptor to maintain optimal immunity by balancing ER-phagy and inflammatory responses

• It has been implicated in several disease pathways including Behçet's disease and bladder cancer

• As an ER resident protein, it undergoes autophagic degradation upon binding GABARAP through its LC3-interacting region (LIR)

For accurate detection and study of this protein, researchers should select antibodies validated for their specific application and sample type.

What applications are UBAC2 antibodies validated for?

UBAC2 antibodies have been validated for multiple research applications with varying recommended dilutions:

When planning experiments, researchers should perform antibody titration in their specific testing system as reactivity can vary between human and mouse samples .

How should UBAC2 antibodies be stored and handled for optimal performance?

For maximum stability and performance of UBAC2 antibodies:

• Store at -20°C. Most preparations are stable for one year after shipment

• For long-term storage, aliquot the antibody to avoid repeated freeze/thaw cycles which can degrade antibody performance

• Most commercial UBAC2 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some preparations contain 0.1% BSA for additional stability in smaller (20μl) sizes

• Always centrifuge briefly before opening the vial to ensure solution homogeneity

• Allow antibody to reach room temperature before use in experimental procedures

Proper storage and handling will help maintain antibody specificity and sensitivity throughout your research project.

What is the molecular weight of UBAC2 and how does this impact Western blot analysis?

Understanding the expected molecular weight of UBAC2 is crucial for proper interpretation of Western blot results:

• The calculated molecular weight based on amino acid sequence is 39 kDa (344 amino acids)

• The observed molecular weight in Western blot applications is typically 35 kDa

• This discrepancy between calculated and observed weights can be attributed to post-translational modifications or protein folding

When performing Western blot analysis:

• Use appropriate molecular weight markers covering the 30-45 kDa range

• Exercise caution when interpreting bands, as the 4 kDa difference between calculated and observed weights could lead to misidentification

• Consider using positive controls (MCF-7 cells, HEK-293 cells, or mouse kidney tissue) which have been validated for UBAC2 detection

How does UBAC2 phosphorylation affect its function in ER-phagy, and how can researchers study this mechanism?

UBAC2 undergoes critical phosphorylation that regulates its function in ER-phagy. To investigate this mechanism:

• Phosphorylation site identification: Research has shown that MARK2 (microtubule affinity-regulating kinase 2) phosphorylates UBAC2 at serine (S) 223, which promotes its dimerization .

• Functional significance: This phosphorylation is a key regulatory step because:

  • Dimerized UBAC2 interacts more strongly with GABARAP

  • Enhanced GABARAP interaction facilitates selective degradation of the ER

  • This process restrains inflammatory responses and acute ulcerative colitis in mouse models

• Methodological approach to study phosphorylation:

  • Use phospho-specific antibodies if available

  • Employ PhastID (BPL-assisted biotin identification) to capture endogenous proteins on the endoplasmic reticulum membrane as demonstrated in published research

  • Use kinase inhibitors specific to MARK2 to confirm the pathway

  • Generate phospho-mimetic (S223D) and phospho-deficient (S223A) mutants to study functional impacts

• Experimental controls: Include bafilomycin A1 (autolysosome inhibitor) and MG132 or carfilzomib (proteasome inhibitors) to distinguish between degradation pathways, as research has shown UBAC2 degradation is blocked by bafilomycin A1 but not by proteasome inhibitors .

What are the key considerations when using UBAC2 antibodies in RNA immunoprecipitation (RIP) assays?

RNA immunoprecipitation (RIP) assays with UBAC2 antibodies require careful optimization due to the unique interactions of UBAC2 with circular RNAs. Based on published research using UBAC2 in RIP assays :

• Cell preparation:

  • Use approximately 1.5 × 10^7 cells per assay for adequate protein yield

  • Ensure complete cell lysis to access ER-associated UBAC2

• Antibody selection and controls:

  • Use a validated UBAC2 antibody (e.g., Cat. No. 25122-1-AP from Proteintech Group)

  • Always include an IgG antibody control to assess non-specific binding

  • Use Protein A/G magnetic beads for immunoprecipitation

• RNA purification and analysis:

  • Purify RNA complexes using RNeasy Mini Kit or equivalent

  • Include RNase inhibitors throughout the procedure

  • Perform RT-PCR for target RNA detection (particularly relevant for circular RNAs like BCRC-3 which has been shown to interact with UBAC2)

• Validation methods:

  • Confirm findings with RNA fluorescence in situ hybridization using labeled probes (e.g., Cy3-labeled BCRC-3 probe)

  • Use confocal microscopy to detect co-localization signals (Nikon A1Si Laser Scanning Confocal Microscope or equivalent)

This approach has successfully demonstrated that UBAC2 can bind to BCRC-3 and affect its interaction with miR-182-5p, ultimately impacting p27 expression .

How do genetic variants of UBAC2 affect antibody detection and what are the implications for disease research?

Genetic variants of UBAC2 present challenges for antibody-based detection and have significant implications for disease research:

• Known genetic variants:

  • SNP rs7999348 (A/G) has been identified as having an independent genetic effect in the UBAC2 gene associated with Behçet's disease

  • The minor "G" allele in rs7999348 is associated with increased risk of Behçet's disease

• Impact on antibody detection:

  • Variations near functional epitopes may alter antibody binding efficiency

  • When working with patient samples, consider potential allelic variants that might affect antibody recognition

  • UBAC2 transcript variants show differential expression based on genotype; variant 1 (NM_001144072.1) shows significantly increased expression in individuals homozygous for the risk "G" allele compared to protective "A" allele carriers

• Methodological approaches:

  • Perform genotyping of key SNPs (e.g., rs7999348) when studying disease associations

  • Use multiple antibodies targeting different epitopes to ensure robust detection

  • Include controls with known genotypes when possible

  • Consider transcript-specific detection methods for comprehensive analysis

• Experimental design considerations:

  • In studies of Behçet's disease, stratify samples by UBAC2 genotype

  • Use conditional haplotype analysis to identify independent genetic effects

  • Assess allele-specific gene expression using appropriate databases (e.g., GENe Expression VARiation database)

These considerations are essential for accurate interpretation of antibody-based experiments in the context of UBAC2-associated diseases.

What techniques can researchers use to study UBAC2's role in ER-phagy flux, and what controls are necessary?

To effectively study UBAC2's role in ER-phagy flux, researchers should employ multiple complementary techniques:

• ER-phagy reporter systems:

  • Use the RFP-GFP-RAMP4 reporter system to monitor ER-phagy flux

  • Track the production of RFP fragments under starvation-induced autophagy or ER stress conditions

  • Compare results between wild-type and UBAC2 knockout cells to determine UBAC2-dependent effects

• Microscopy-based approaches:

  • Employ fluorescence microscopy to quantify red puncta formation as a measure of ER-phagy

  • Use confocal analysis to assess co-localization of UBAC2 with autophagy markers (particularly GABARAP)

  • Apply immunofluorescence with specific UBAC2 antibodies to visualize subcellular localization (cytoplasmic/ER membrane)

• Genetic manipulation strategies:

  • Generate UBAC2 knockout cells using CRISPR-Cas9 (validated in HeLa, THP-1, and HT-29 cells)

  • Create LIR motif mutants (e.g., W275A and L278A) to assess the impact of disrupting the GABARAP interaction

  • Develop phospho-site mutants to analyze the role of MARK2-mediated phosphorylation

• Essential controls:

  • Autophagy inducers: starvation media and thapsigargin (TG) for ER stress induction

  • Autophagy inhibitors: bafilomycin A1 to block autolysosome function

  • Proteasome inhibitors: MG132 or carfilzomib to distinguish between degradation pathways

  • Wild-type cells as baseline controls for knockout experiments

  • LIR motif mutants as negative controls for GABARAP interaction studies

These methodologies have successfully demonstrated that UBAC2 depletion attenuates ER-phagy flux under both starvation-induced autophagy and ER stress conditions .

How can researchers effectively use UBAC2 antibodies to investigate its role in cancer progression, particularly in bladder cancer?

To investigate UBAC2's role in cancer progression using antibody-based approaches:

• Expression analysis in clinical samples:

  • Perform immunohistochemistry (IHC) on tumor tissues and adjacent normal tissues using validated UBAC2 antibodies at 1:3000-1:12000 dilution

  • Calculate the integrated optical density per stained area (IOD/area) using image analysis software (e.g., Image-Pro Plus version 6.0)

  • Compare expression levels across different grades and pathological stages

  • Correlate expression with clinical outcomes using Kaplan-Meier survival analysis

• Cellular localization studies:

  • Use immunofluorescence (IF) analysis with UBAC2 antibodies at 1:50-1:500 dilution

  • Employ co-staining with organelle markers to confirm ER localization

  • Analyze using confocal microscopy for high-resolution imaging

• Functional studies:

  • Generate stable UBAC2 knockdown cancer cell lines using shRNA

  • Validate knockdown efficiency by Western blot and qRT-PCR

  • Assess effects on proliferation, cell cycle progression, and tumor growth

  • Use antibodies against proliferation markers (e.g., Ki-67) in xenograft models

• Molecular interaction analysis:

  • Perform RNA immunoprecipitation (RIP) with UBAC2 antibodies to identify RNA binding partners

  • Investigate interactions with circular RNAs (e.g., BCRC-3) and miRNAs

  • Analyze downstream effects on cell cycle regulators like p27

Research has demonstrated that UBAC2 is significantly upregulated in bladder cancer tissues and cell lines, and higher expression correlates with lower survival rates. UBAC2 knockdown inhibits cancer cell proliferation both in vitro and in vivo by increasing p27 expression through a mechanism involving circular RNA BCRC-3 and miR-182-5p .

What are the critical parameters for optimizing Western blot protocols with UBAC2 antibodies?

For optimal Western blot results with UBAC2 antibodies, researchers should consider the following parameters:

• Sample preparation:

  • Include positive controls such as MCF-7 cells, HEK-293 cells, or mouse kidney tissue which have been validated for UBAC2 detection

  • Use appropriate lysis buffers that effectively solubilize membrane proteins, as UBAC2 is an ER membrane protein

  • Add protease inhibitors to prevent degradation during sample preparation

• Gel selection and transfer conditions:

  • Use 10-12% polyacrylamide gels for optimal resolution around the 35-39 kDa range

  • Consider using gradient gels (4-20%) if analyzing multiple proteins of different sizes

  • Optimize transfer conditions for membrane proteins (longer transfer times or semi-dry transfer methods)

• Antibody dilution and incubation:

  • Use recommended dilutions: 1:500-1:1000 or 1:500-1:2000 for UBAC2 antibodies

  • Optimize primary antibody incubation (typically overnight at 4°C)

  • Use 5% non-fat dry milk or BSA in TBST as blocking buffer

  • Consider titrating antibody concentration for your specific sample type

• Detection and imaging:

  • Use appropriate secondary antibodies compatible with your detection system

  • For low abundance samples, consider using more sensitive detection methods (ECL plus or chemiluminescent substrates)

  • Optimize exposure times to avoid saturation while maintaining sensitivity

• Troubleshooting common issues:

  • High background: Increase blocking time or washing steps

  • No signal: Check primary antibody concentration, consider antigen retrieval

  • Multiple bands: Verify specificity with knockout/knockdown controls

  • Unexpected molecular weight: Consider post-translational modifications or transcript variants

Following these optimization steps will help ensure specific and sensitive detection of UBAC2 in Western blot applications.

How can researchers verify the specificity of UBAC2 antibodies in their experimental systems?

Verifying antibody specificity is crucial for accurate data interpretation. For UBAC2 antibodies, consider these verification approaches:

• Genetic validation approaches:

  • Use CRISPR-Cas9 UBAC2 knockout cell lines as negative controls

  • Employ siRNA or shRNA knockdown of UBAC2 to demonstrate reduced signal

  • Perform antibody testing on cells overexpressing UBAC2 to confirm increased signal

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide

  • Compare results with and without peptide pre-absorption

  • Signal reduction confirms binding to the intended epitope

• Cross-validation with multiple antibodies:

  • Test antibodies from different suppliers targeting different UBAC2 epitopes

  • Compare detection patterns across applications (WB, IHC, IF)

  • Consistent results across antibodies increase confidence in specificity

• Analysis of expected expression patterns:

  • Verify subcellular localization (UBAC2 is primarily localized in the cytoplasm/ER membrane)

  • Confirm expected molecular weight (observed: 35 kDa; calculated: 39 kDa)

  • Examine tissue distribution patterns against published data

• Validation across applications:

  • Verify that results in Western blot correspond with immunofluorescence findings

  • Confirm that protein detection correlates with mRNA expression data

  • Assess whether functional outcomes match with expected UBAC2 biology

These approaches collectively provide strong evidence for antibody specificity and help avoid misinterpretation of experimental results.

What special considerations are needed when using UBAC2 antibodies for immunohistochemistry in different tissue types?

Immunohistochemistry (IHC) with UBAC2 antibodies requires tissue-specific optimization:

• Antigen retrieval optimization:

  • For UBAC2 antibodies, suggested antigen retrieval methods include:

    • TE buffer pH 9.0 (primary recommendation)

    • Citrate buffer pH 6.0 (alternative method)

  • Optimization is particularly important for formalin-fixed paraffin-embedded (FFPE) tissues

• Tissue-specific considerations:

  • Positive control tissues: human colon cancer tissue and human testis tissue have been validated for UBAC2 IHC

  • For cancer studies: compare tumor tissue with adjacent normal tissue

  • Note tissue-specific expression patterns to avoid misinterpretation

• Dilution optimization:

  • Recommended dilution range for IHC is 1:3000-1:12000

  • Wide dilution range indicates need for careful titration

  • Optimize based on specific tissue type and fixation method

• Detection systems:

  • Use appropriate detection systems based on tissue autofluorescence concerns

  • For tissues with high background, consider non-fluorescent detection methods

  • For co-localization studies, select compatible fluorophores

• Quantification approaches:

  • Calculate integrated optical density per stained area (IOD/area) using image analysis software

  • Evaluate at 400-fold magnification

  • Analyze 4-6 representative staining fields per section

• Tissue-specific validation:

  • In bladder cancer studies, use established cell lines (EJ, UMUC3, T24) as references

  • For inflammatory disease research, compare expression in inflammatory vs. normal tissues

  • Validate findings with alternative methods (Western blot, qRT-PCR)

Following these tissue-specific considerations will enhance the reliability and reproducibility of UBAC2 IHC results across different experimental conditions.

How can UBAC2 antibodies be used to study the protein's role in inflammatory responses and potential therapeutic applications?

Recent research has revealed UBAC2's significant role in inflammatory regulation, opening new avenues for therapeutic exploration:

• Inflammatory pathway investigation:

  • Use UBAC2 antibodies to study its interaction with ER stress and the unfolded protein response (UPR)

  • Investigate how UBAC2-mediated ER-phagy impacts inflammatory signaling

  • Monitor changes in UBAC2 expression and localization during inflammatory challenges

  • Research has demonstrated that UBAC2 restrains inflammatory responses and acute ulcerative colitis via ER-phagy

• Disease model applications:

  • Apply UBAC2 antibodies in models of ulcerative colitis to track protein expression

  • Study UBAC2 phosphorylation state in response to inflammatory stimuli

  • Investigate UBAC2 interactions with MARK2 kinase during inflammation

  • Research shows UBAC2 deficiency results in inflammatory responses through disruption of ER homeostasis

• Therapeutic target validation:

  • Use antibodies to validate UBAC2 as a druggable target in inflammatory diseases

  • Screen for compounds that modulate UBAC2 phosphorylation or dimerization

  • Monitor therapeutic efficacy through changes in UBAC2-mediated ER-phagy

  • Recent findings indicate that ER-phagy directed by the MARK2-UBAC2 axis may provide targets for inflammatory disease treatment

• Experimental approaches:

  • Employ phospho-specific antibodies to monitor UBAC2 S223 phosphorylation state

  • Use co-immunoprecipitation to study UBAC2 dimerization under different conditions

  • Apply GABARAP interaction assays to assess ER-phagy activation

  • Combine with genetic models (UBAC2 variants from inflammatory diseases or LIR motif mutations) to correlate with in vivo inflammation

This emerging research direction suggests UBAC2 antibodies will be valuable tools for developing novel therapeutic strategies targeting inflammatory disorders.

What are the latest methodological advances in using UBAC2 antibodies to study its interaction with circular RNAs in cancer research?

The discovery of UBAC2's interaction with circular RNAs represents a new frontier in cancer research methodology:

• RNA-protein interaction detection:

  • RNA immunoprecipitation (RIP) with UBAC2 antibodies has successfully demonstrated binding to circular RNA BCRC-3

  • Protocol optimization includes using approximately 1.5 × 10^7 cells, specific UBAC2 antibodies (e.g., Cat. No. 25122-1-AP), and Protein A/G magnetic beads

  • RNA purification with RNeasy Mini Kit followed by RT-PCR provides quantitative assessment of bound circular RNAs

• Visualization techniques:

  • RNA fluorescence in situ hybridization with Cy3-labeled BCRC-3 probe combined with UBAC2 immunofluorescence enables co-localization studies

  • Confocal microscopy (e.g., Nikon A1Si Laser Scanning Confocal Microscope) provides high-resolution imaging of RNA-protein interactions

  • These techniques have revealed that UBAC2 binds to BCRC-3, affecting its interaction with miR-182-5p

• Functional validation approaches:

  • Luciferase reporter assays with p27 3′-UTR have demonstrated that UBAC2 knockdown amplifies reporter activity

  • Combined knockdown experiments (UBAC2 and BCRC-3) help establish the functional relationship between these molecules

  • Flow cytometry for cell cycle analysis (using propidium iodide staining and ModFit LT software) connects molecular interactions to cellular phenotypes

• Translational research applications:

  • Analysis of UBAC2 and circular RNA expression in patient samples correlates with survival outcomes

  • Kaplan-Meier survival analysis of bladder cancer patients revealed that higher UBAC2 expression correlates with worse survival probability

  • This methodological framework provides a basis for investigating similar mechanisms in other cancer types