UBAC2 Antibody, Biotin conjugated

What is UBAC2 and what cellular functions has it been implicated in?

UBAC2 (UBA Domain Containing 2) is a highly conserved protein encoded by a gene located on chromosome 13q32.3. The protein contains ubiquitin-associated domains and is primarily localized in the cytoplasm of cells, as demonstrated by immunofluorescence studies . UBAC2 has been implicated in several biological processes, with significant roles in:

• Protein degradation pathways through its ubiquitin-associated domains

• Cell cycle regulation, particularly affecting G0/G1 phase progression

• Interaction with circular RNAs to modulate gene expression

• Post-transcriptional regulation of cell cycle inhibitors like p27

Recent research has demonstrated that UBAC2 shows differential expression across various tissues and can significantly impact cellular proliferation through multiple molecular pathways . Immunofluorescence analysis has confirmed that UBAC2 is predominantly localized in the cytoplasm of bladder cancer cells, suggesting its primary function occurs in this cellular compartment .

What epitopes does the biotin-conjugated UBAC2 antibody recognize?

The biotin-conjugated UBAC2 antibody (such as ABIN7174419) specifically recognizes amino acids 1-157 of the UBAC2 protein . This N-terminal region is critical for many of the protein's functions. The antibody demonstrates high specificity, having been generated from recombinant human Ubiquitin-associated domain-containing protein 2 (1-157AA) as the immunogen .

The epitope recognition of this antibody is distinct from other available UBAC2 antibodies that target different regions:

• Some antibodies target AA 63-91 (N-terminal region)

• Others recognize AA 107-156

• Some bind to AA 220-310 (more C-terminal region)

This specificity for the N-terminal region allows researchers to detect full-length UBAC2 while potentially distinguishing it from truncated forms or specific domains of the protein .

What is the reactivity profile of biotin-conjugated UBAC2 antibody?

The biotin-conjugated UBAC2 antibody (ABIN7174419) has been validated for reactivity with human UBAC2 . This specificity is important for researchers designing experiments with human cell lines or tissues. In contrast, some non-conjugated UBAC2 antibodies show broader reactivity profiles:

• Many UBAC2 antibodies react with both human and mouse samples

• Certain antibodies (like 25122-1-AP) have been additionally tested and validated in more diverse species

• Some products show exceptionally broad cross-reactivity, including dog, guinea pig, horse, rabbit, rat, and monkey samples

For researchers working with non-human models, it's essential to verify the specific cross-reactivity of the biotin-conjugated antibody, as conjugation can sometimes affect epitope recognition and species reactivity .

What are the validated applications for biotin-conjugated UBAC2 antibody?

• ELISA: Direct and sandwich ELISA formats with streptavidin detection systems

• Immunohistochemistry (IHC): Particularly with streptavidin-based detection methods

• Flow cytometry: Utilizing the biotin-streptavidin amplification system for enhanced sensitivity

• Pull-down assays: Leveraging the strong biotin-streptavidin interaction

The unconjugated antibody has been successfully used in Western blotting, immunofluorescence, immunohistochemistry, and RNA immunoprecipitation applications, suggesting potential versatility of the biotin conjugate when paired with appropriate streptavidin detection systems .

How can I validate the specificity of my UBAC2 antibody results?

Validating antibody specificity is crucial for reliable research outcomes. For UBAC2 antibody validation, consider these methodological approaches:

• Knockdown/Knockout Controls:

  • Generate UBAC2 knockdown cells using shRNA (such as shUBAC2-1 and shUBAC2-3 that have demonstrated efficient knockdown)

  • Compare antibody signal between control and knockdown samples

  • Expect significant reduction in signal intensity in knockdown samples

• Molecular Weight Verification:

  • The observed molecular weight of UBAC2 is approximately 35-39 kDa

  • Confirm single band at this molecular weight in Western blots

• Tissue/Cell Type Controls:

  • MCF-7 cells, HEK-293 cells, and mouse kidney tissue show positive Western blot results

  • Human colon cancer tissue and testis tissue show positive IHC staining

• Application-Specific Controls:

  • For RNA immunoprecipitation (RIP), include IgG antibody as negative control

  • For immunofluorescence, include secondary-only controls and counter-staining with DAPI

• Cross-Validation:

  • Use multiple antibodies targeting different epitopes of UBAC2

  • Compare results between antibodies to confirm consistent patterns

How can UBAC2 antibodies be utilized to investigate its role in cancer progression?

Research has revealed that UBAC2 is significantly upregulated in bladder cancer tissues and cell lines, with higher expression correlating with poorer patient survival . Researchers can leverage UBAC2 antibodies to explore cancer pathways through these methodological approaches:

• Expression Analysis in Clinical Samples:

  • Use IHC with UBAC2 antibodies to assess expression across tumor stages and grades

  • Correlate expression levels with patient survival and clinical parameters

  • Compare UBAC2 levels between tumors and adjacent normal tissues

• Cellular Localization Studies:

  • Employ immunofluorescence with UBAC2 antibodies to track subcellular localization

  • Investigate potential translocation under different cellular stresses or treatments

  • Co-localize with known cancer pathway proteins

• Protein Interaction Networks:

  • Utilize biotin-conjugated antibodies for pull-down assays followed by mass spectrometry

  • Identify novel UBAC2-interacting proteins in cancer cells

  • Verify interactions through co-immunoprecipitation with reciprocal antibodies

• Functional Studies:

  • Combine UBAC2 knockdown approaches with antibody detection of downstream targets

  • Monitor changes in cell cycle regulators (particularly p27) after UBAC2 modulation

  • Assess proliferation markers in xenograft models using IHC with UBAC2 antibodies

What is the relationship between UBAC2 and the p27 pathway, and how can this be investigated?

UBAC2 has been identified as a regulator of p27 expression through post-transcriptional mechanisms . This relationship can be methodically investigated using these approaches:

• Protein Expression Analysis:

  • Use Western blotting to monitor p27 levels after UBAC2 knockdown

  • Quantify changes in related cell cycle proteins (CDK2, CDK4, CDK6, cyclin D1, cyclin E)

  • Track temporal dynamics of p27 regulation following UBAC2 modulation

• Transcriptional vs. Post-transcriptional Regulation:

  • Compare p27 mRNA and protein levels using RT-qPCR and Western blotting

  • Employ luciferase reporter assays with p27 3′-UTR and promoter constructs

  • Results from previous studies show UBAC2 knockdown amplifies p27 3′-UTR activity without affecting promoter activity

• Mechanism Dissection:

  • Investigate miRNA involvement using miR-182-5p inhibitors or mimics

  • Perform RNA immunoprecipitation to assess UBAC2 binding to circular RNA BCRC-3

  • Design rescue experiments to determine if BCRC-3 knockdown reverses effects of UBAC2 depletion

• Functional Outcomes:

  • Measure cell cycle distribution using flow cytometry after UBAC2 manipulation

  • Assess cellular proliferation with CCK8 or similar assays

  • Evaluate in vivo tumor growth in xenograft models with modified UBAC2 expression

How can I investigate the interaction between UBAC2 and circular RNA BCRC-3?

The interaction between UBAC2 and circular RNA BCRC-3 represents a novel regulatory mechanism in cancer biology . This interaction can be methodically explored through:

• RNA Immunoprecipitation (RIP):

  • Use UBAC2 antibody (such as 25122-1-AP) for RIP assays

  • Include IgG antibody as negative control

  • Purify RNA from immunoprecipitated complexes using RNeasy Mini Kit

  • Perform RT-PCR to detect BCRC-3 enrichment in UBAC2 pulldowns

• RNA Fluorescence in situ Hybridization (FISH) with IF:

  • Combine FISH for BCRC-3 using Cy3-labeled probes with IF for UBAC2

  • Analyze co-localization using confocal microscopy

  • Quantify spatial overlap between BCRC-3 and UBAC2 signals

• Functional Validation:

  • Design experiments with UBAC2 knockdown, BCRC-3 knockdown, and double knockdown

  • Monitor p27 expression and miR-182-5p activity across these conditions

  • Assess if BCRC-3 knockdown reverses the effects of UBAC2 depletion on p27 levels

• Mechanistic Dissection:

  • Perform competitive binding assays to determine if UBAC2 affects BCRC-3/miR-182-5p interaction

  • Use domain mapping to identify which regions of UBAC2 interact with BCRC-3

  • Employ mutational analysis to confirm binding specificity

What are common issues when working with biotin-conjugated antibodies and how can they be resolved?

Biotin-conjugated antibodies present unique technical challenges that can be methodically addressed:

• High Background in Tissues:

  • Issue: Endogenous biotin in tissues can cause high background

  • Solution: Implement avidin/biotin blocking step before primary antibody incubation

  • Protocol: Incubate with avidin solution for 15 minutes, wash, then biotin solution for 15 minutes

• Signal Amplification Considerations:

  • Issue: Excessive amplification with streptavidin systems leading to non-specific signals

  • Solution: Titrate streptavidin-detection reagent carefully

  • Protocol: Test dilution series from 1:500 to 1:5000 to determine optimal signal-to-noise ratio

• Cross-Reactivity Concerns:

  • Issue: Conjugation can sometimes alter epitope recognition or increase non-specific binding

  • Solution: Include additional blocking steps and validate with known positive/negative controls

  • Controls: Include tissue from UBAC2 knockout models if available, or use tissues known to lack UBAC2 expression

• Storage and Stability Issues:

  • Issue: Biotin conjugates can lose activity over time

  • Solution: Maintain antibody in recommended liquid form at -20°C

  • Best Practice: Aliquot to avoid repeated freeze-thaw cycles and validate regularly with positive controls

How can I optimize immunoprecipitation experiments using UBAC2 antibodies?

Immunoprecipitation with UBAC2 antibodies requires careful optimization for successful protein-protein or protein-RNA interaction studies:

• Antibody Selection and Validation:

  • Choose antibodies proven effective in IP applications (such as 25122-1-AP)

  • Validate antibody performance in your specific cell type before proceeding to IP

  • Consider using biotin-conjugated antibodies with streptavidin beads for clean pulldowns

• Cell Lysis Optimization:

  • Use RIPA lysis buffer as demonstrated in previous UBAC2 studies

  • Include protease inhibitors to prevent protein degradation

  • For RNA-protein interactions, add RNase inhibitors to preserve RNA integrity

• Binding Conditions:

  • Optimize antibody amount (typically 2-5 μg per reaction)

  • Incubate overnight at 4°C with gentle rotation

  • Use approximately 1.5 × 10^7 cells per IP reaction for sufficient protein yield

• Controls and Validation:

  • Always include IgG control antibody for non-specific binding assessment

  • Input samples (5-10% of lysate) should be run alongside IP samples

  • For RNA IP, perform RT-PCR on input and IP samples to calculate enrichment

• Detection Methods:

  • For protein interactions, use Western blotting with antibodies against suspected interacting partners

  • For RNA interactions, purify RNA with RNeasy Mini Kit and analyze by RT-PCR or sequencing

What controls should be included when performing functional studies with UBAC2?

Rigorous controls are essential for reliable UBAC2 functional studies:

• Knockdown/Overexpression Controls:

  • Include at least two different shRNA constructs targeting UBAC2 (such as shUBAC2-1 and shUBAC2-3)

  • Use non-targeting shRNA or empty vector controls

  • Validate knockdown efficiency at both protein and mRNA levels by Western blot and qRT-PCR

• Rescue Experiments:

  • Reintroduce shRNA-resistant UBAC2 to confirm phenotype specificity

  • Consider domain mutants to identify functional regions

  • Include both wild-type and mutant rescue constructs

• Downstream Effector Validation:

  • Monitor multiple cell cycle regulators (p27, CDK2, CDK4, cyclin D1, etc.)

  • Check both protein and mRNA levels to distinguish transcriptional from post-transcriptional effects

  • Perform pathway inhibitor experiments to confirm specificity

• In Vivo Validation:

  • Establish xenograft models with UBAC2-knockdown cells

  • Include appropriate number of animals per group (at least n=5)

  • Confirm maintained knockdown in excised tumors

  • Validate key findings from in vitro experiments in tumor samples

• Molecular Mechanism Controls:

  • For BCRC-3 interaction studies, include BCRC-3 knockdown and co-knockdown with UBAC2

  • For miRNA studies, use miR-182-5p mimics or inhibitors to confirm pathway specificity

  • Employ luciferase reporter assays with wild-type and mutated binding sites

Beyond cancer, what other disease associations with UBAC2 merit investigation?

While bladder cancer has been a primary focus for UBAC2 research, evidence suggests broader disease relevance warranting investigation:

• Autoimmune Conditions:

  • UBAC2 gene polymorphisms have been associated with Behcet's disease

  • Research indicates UBAC2 upregulation may promote Behcet's disease progression

  • Methodological approach: Comparative expression analysis across autoimmune disorders using antibody-based techniques

• Other Cancer Types:

  • UBAC2 antibodies have shown positive staining in colon cancer tissues

  • Expression in multiple cancer cell lines suggests broader oncogenic relevance

  • Research strategy: Pan-cancer IHC analysis with UBAC2 antibodies to identify additional cancer associations

• Metabolic Disorders:

  • UBAC2's role in protein degradation pathways suggests potential involvement in metabolic regulation

  • Research approach: Investigate UBAC2 expression and localization in metabolic tissues under normal and disease conditions

• Developmental Processes:

  • The evolutionary conservation of UBAC2 suggests fundamental biological functions

  • Experimental design: Developmental expression profiling using UBAC2 antibodies across tissue/organ formation stages

How can UBAC2 antibodies be employed in biomarker development for cancer detection?

Given UBAC2's differential expression in cancer tissues and correlation with survival, it holds potential as a biomarker:

• Tissue-Based Diagnostic Applications:

  • Develop IHC scoring systems for UBAC2 expression in tumor biopsies

  • Correlate expression levels with tumor grade, stage, and patient outcomes

  • Methodology: Standardized IHC protocols with biotin-conjugated or other UBAC2 antibodies

• Liquid Biopsy Development:

  • Investigate UBAC2 protein levels in patient serum or exosomes

  • Design ELISA or other immunoassays using biotin-conjugated UBAC2 antibodies

  • Research approach: Compare detection sensitivity between conjugated and unconjugated antibody formats

• Multimarker Panel Integration:

  • Combine UBAC2 with established cancer markers for improved predictive value

  • Assess correlation with BCRC-3 and miR-182-5p levels in patient samples

  • Experimental strategy: Multiplex immunoassays incorporating UBAC2 detection

• Therapeutic Response Monitoring:

  • Track changes in UBAC2 expression during treatment

  • Correlate expression changes with treatment efficacy and resistance development

  • Methodology: Serial sampling with standardized antibody-based detection systems

What novel technological applications could enhance UBAC2 research beyond traditional antibody techniques?

Advancing UBAC2 research may benefit from emerging technologies:

• Proximity Labeling Approaches:

  • Develop UBAC2 fusion proteins with BioID or APEX2 for in situ proximity labeling

  • Identify novel interaction partners in living cells

  • Validate findings using traditional co-IP with UBAC2 antibodies

• Advanced Imaging Techniques:

  • Apply super-resolution microscopy with UBAC2 antibodies for detailed localization studies

  • Utilize live-cell imaging with UBAC2-fluorescent protein fusions

  • Compare subcellular distribution data between fixed-cell antibody staining and live-cell approaches

• Single-Cell Analysis:

  • Employ CyTOF or similar technologies with metal-conjugated UBAC2 antibodies

  • Characterize UBAC2 expression heterogeneity within tumor samples

  • Correlate with other cancer markers at single-cell resolution

• CRISPR-Based Functional Genomics:

  • Generate CRISPR knockouts or knock-ins of UBAC2

  • Create domain-specific mutations to dissect functional regions

  • Validate antibody specificity against these genetic models

  • Combine with antibody-based readouts of downstream pathway effects