UBE2C Recombinant Monoclonal Antibody

What is UBE2C and why is it significant for research?

UBE2C (Ubiquitin-conjugating enzyme E2C), also known as UBCH10, belongs to the ubiquitin-conjugating enzyme family and plays a crucial role in the E3 anaphase-promoting complex (APC/C). This enzyme participates in controlling multiple stages of the cell cycle, including the inactivation of the mitotic spindle assembly checkpoint. UBE2C facilitates ubiquitin-dependent proteasomal degradation by initiating K11-linked ubiquitin chains on APC/C substrates . The significance of UBE2C lies in its differential expression pattern between normal and cancerous tissues. Research studies show that UBE2C expression is low in normal tissues but dramatically upregulated in tumors derived from tissues such as lung, breast, and prostate . This expression pattern makes UBE2C a valuable research target for understanding tumorigenesis and identifying potential therapeutic interventions.

What types of UBE2C antibodies are available for research?

Researchers have access to several UBE2C antibodies with varying characteristics suited for different experimental applications. Currently available options include:

• Mouse monoclonal antibodies (e.g., 66087-1-Ig from Proteintech) with IgG1 isotype

• Rabbit polyclonal antibodies (e.g., #14234 from Cell Signaling Technology)

• Mouse monoclonal antibodies with IgG2b isotype (e.g., CPTC-UBE2C-1)

Each antibody offers specific advantages depending on the experimental design and research questions. For instance, mouse monoclonal antibodies provide high specificity and reproducibility for targeted epitopes, while rabbit antibodies may offer broader epitope recognition. The selection should be based on the intended application, species reactivity requirements, and the specific epitope of interest .

What is the molecular weight and structure of UBE2C that antibodies target?

UBE2C is a relatively small protein with a calculated molecular weight of approximately 20 kDa, consisting of 179 amino acids . This molecular weight is consistently observed across different antibody validation studies, as confirmed by western blot analyses from multiple manufacturers . The protein's structure allows it to function as a key component in ubiquitin-mediated protein degradation pathways. Understanding the molecular characteristics of UBE2C is essential for proper antibody selection and experimental design, particularly when validating antibody specificity through molecular weight confirmation on western blots. Researchers should note that post-translational modifications may occasionally cause slight shifts in the observed molecular weight compared to the calculated value.

How should UBE2C antibodies be optimized for Western Blot experiments?

For optimal Western Blot results with UBE2C antibodies, researchers should follow these methodological guidelines:

• Dilution optimization: Begin with the manufacturer's recommended range (1:1000-1:4000 for antibody 66087-1-Ig or 1:1000 for antibody #14234) and perform a dilution series to determine optimal conditions for your specific sample type .

• Sample preparation: UBE2C has been successfully detected in various cell lines including HeLa, HEK-293, HepG2, U2OS, A549, HSC-T6, and NIH/3T3 cells . For protein extraction, use a lysis buffer containing protease inhibitors to prevent degradation.

• Blocking conditions: A 5% non-fat milk or BSA solution in TBST is typically effective for reducing background signal.

• Expected results: UBE2C should appear as a distinct band at approximately 20 kDa . Validation can be performed using positive control cell lines like HeLa cells, which consistently express detectable levels of UBE2C.

• Quantitative analysis: For comparative studies, normalize UBE2C expression to appropriate housekeeping proteins such as β-actin or GAPDH.

When troubleshooting, common issues include non-specific binding (requiring increased antibody dilution or more stringent washing) and weak signal (potentially resolved through increased antibody concentration or enhanced chemiluminescence detection methods).

What are the optimal conditions for immunohistochemistry (IHC) using UBE2C antibodies?

Successful immunohistochemistry with UBE2C antibodies requires careful attention to methodology:

• Tissue preparation: Both frozen and formalin-fixed paraffin-embedded (FFPE) tissues can be used. For FFPE samples, antigen retrieval is critical.

• Antigen retrieval: Recommended methods include TE buffer (pH 9.0) or citrate buffer (pH 6.0) . The selection between these methods can significantly impact staining quality and should be optimized for each tissue type.

• Antibody dilution: Start with 1:200-1:800 for antibody 66087-1-Ig, adjusting based on signal intensity and background levels .

• Detection systems: Both chromogenic (DAB) and fluorescent detection systems can be employed depending on research needs.

• Positive controls: Human lung cancer tissue and human colon cancer tissue have been verified as positive controls for UBE2C antibodies . The imaging mass cytometry on colon cancer tissue using CPTC-UBE2C-1 antibody has also demonstrated positive results .

For result interpretation, UBE2C typically shows nuclear and cytoplasmic staining patterns in positive samples. Signal intensity often correlates with the proliferative status of the tissue, with higher expression observed in more aggressive tumors, particularly those with poor differentiation and advanced pathological stages .

How can immunofluorescence (IF) protocols be optimized for UBE2C visualization?

For immunofluorescence applications, consider these methodological recommendations:

• Cell preparation: Both fixed cultured cells and tissue sections can be used. For cultured cells, 4% paraformaldehyde fixation for 15-20 minutes at room temperature is typically effective.

• Permeabilization: A brief treatment with 0.1-0.5% Triton X-100 in PBS facilitates antibody access to intracellular targets.

• Antibody dilution: Use 1:400-1:1600 dilution for antibody 66087-1-Ig as a starting point . Titration is recommended for each cell type.

• Co-localization studies: UBE2C antibodies can be combined with markers for specific cellular compartments or cell cycle phases to provide contextual information about protein function and localization.

• Imaging parameters: Confocal microscopy with appropriate laser settings for the selected fluorophore will provide optimal visualization of UBE2C localization patterns.

HeLa cells serve as reliable positive controls for IF applications with UBE2C antibodies . When interpreting results, researchers should look for predominantly nuclear staining with some cytoplasmic distribution, particularly in actively dividing cells. The staining pattern may vary with cell cycle stage, reflecting UBE2C's dynamic role in cell division processes.

How can UBE2C antibodies be utilized in cancer biomarker research?

UBE2C antibodies are valuable tools for investigating UBE2C's potential as a cancer biomarker through several methodological approaches:

• Tissue microarray (TMA) analysis: UBE2C antibodies can be applied to TMAs containing multiple patient samples to evaluate expression patterns across different cancer types and stages. This approach facilitates correlation of UBE2C expression with clinicopathological parameters.

• Prognostic studies: Research has demonstrated that high UBE2C expression correlates with shorter disease-specific survival in tongue squamous cell carcinoma (TSCC) patients, particularly those with poor cell differentiation and advanced pathological stages . Similar methodologies can be applied to other cancer types.

• Expression correlation analysis: Researchers can combine UBE2C immunostaining with other molecular markers to establish expression correlation patterns and potential functional relationships in carcinogenesis.

• Multi-parameter flow cytometry: UBE2C antibodies can be incorporated into flow cytometry panels to quantitatively assess expression levels in various cell populations and correlate with other cellular parameters.

The methodological approach should include appropriate statistical analyses to validate the significance of UBE2C expression in relation to patient outcomes. For example, multivariate analysis adjusting for clinical variables can help establish the independent prognostic value of UBE2C expression, as demonstrated in studies on squamous cell carcinoma .

What considerations are important when using UBE2C antibodies in co-immunoprecipitation (Co-IP)?

Co-immunoprecipitation with UBE2C antibodies requires careful methodological planning:

• Antibody selection: Choose antibodies validated for Co-IP applications, such as antibody 66087-1-Ig, which has been cited in published Co-IP studies .

• Lysis conditions: Use non-denaturing lysis buffers that preserve protein-protein interactions. A typical formulation includes 150 mM NaCl, 1% NP-40 or Triton X-100, 50 mM Tris pH 8.0, and protease inhibitors.

• Precipitation protocol: Pre-clear lysates with protein A/G beads before antibody addition to reduce non-specific binding. Incubate with UBE2C antibody overnight at 4°C, followed by protein A/G bead capture.

• Controls: Include isotype-matched control antibodies to identify non-specific interactions. Additionally, performing reciprocal Co-IPs (pulling down with antibodies against suspected interacting partners) strengthens the validity of identified interactions.

• Detection methods: Western blotting of Co-IP samples should include both input controls and IP samples to verify successful precipitation and specific interactions.

When investigating UBE2C interactions, researchers should focus on components of the APC/C complex and potential substrates relevant to cell cycle regulation. The transient nature of enzyme-substrate interactions may necessitate the use of crosslinking approaches or proteasome inhibitors to stabilize these interactions prior to Co-IP.

How can UBE2C antibodies be employed in chromatin immunoprecipitation (ChIP) experiments?

While UBE2C itself is not a transcription factor, researchers investigating mechanisms of UBE2C regulation may employ ChIP methodologies to study transcription factors controlling UBE2C expression:

This integrative approach allows researchers to connect transcriptional regulation of UBE2C with its protein expression patterns and functional consequences in cellular processes, particularly in cancer contexts where UBE2C overexpression has been linked to genomic amplification.

UBE2C in Cancer Research

Validating UBE2C antibody specificity through knockout (KO) or knockdown (KD) approaches is critical for ensuring reliable research outcomes:

• siRNA/shRNA knockdown validation:

  • Transfect cells with UBE2C-targeting siRNA or shRNA constructs alongside appropriate controls

  • Confirm knockdown efficiency at mRNA level via qRT-PCR

  • Use UBE2C antibodies in Western blot to demonstrate reduced protein levels (typically 70-90% reduction in effective knockdowns)

  • Apply the same antibody in parallel applications (IHC, IF) to confirm signal reduction across platforms

• CRISPR-Cas9 knockout validation:

  • Generate UBE2C knockout cell lines using CRISPR-Cas9 technology

  • Confirm successful editing through sequencing

  • Demonstrate complete absence of UBE2C protein using antibodies in Western blot

  • Validate antibody specificity by comparing wild-type and knockout cells in all intended applications

• Rescue experiments:

  • Reintroduce UBE2C expression in knockdown or knockout systems

  • Confirm restored expression using antibodies

  • This approach helps distinguish between specific and non-specific antibody signals

Published studies have employed knockdown and knockout approaches for UBE2C validation, as noted in the scientific literature . These methodologies provide the strongest evidence for antibody specificity and should be incorporated whenever possible, especially when investigating novel aspects of UBE2C biology or when employing antibodies in new experimental systems.

How can researchers correlate UBE2C expression with patient survival and clinical parameters?

Methodological approaches for correlating UBE2C expression with clinical outcomes involve several key steps:

• Patient cohort selection:

  • Define clear inclusion/exclusion criteria

  • Collect comprehensive clinicopathological data

  • Ensure adequate sample size with appropriate statistical power

  • Include samples representing various disease stages and outcomes

• UBE2C detection protocols:

  • Standardize IHC protocols with validated antibody dilutions (1:200-1:800)

  • Implement quantitative scoring systems (e.g., H-score or percentage of positive cells)

  • Use digital pathology tools for objective quantification when possible

  • Perform blinded scoring by at least two independent observers

• Statistical analysis methodologies:

  • Apply Kaplan-Meier survival analysis with log-rank tests to compare high vs. low UBE2C expression groups

  • Perform univariate and multivariate Cox regression analyses to assess independent prognostic value

  • Calculate adjusted hazard ratios (AHR) with 95% confidence intervals

  • Control for confounding variables such as age, sex, and clinical stage

• Data presentation:

  • Present findings in clear tables showing UBE2C expression in relation to various clinical parameters

  • Include statistical measures such as AHR (95% CI) and p-values

This approach has been effectively applied in studies of squamous cell carcinoma, where high UBE2C expression was correlated with shorter disease-specific survival, particularly in male patients with tongue SCC (AHR 1.86, 95% CI 1.20-2.89, p=0.006) . The methodology can be adapted for other cancer types to establish the prognostic significance of UBE2C expression across various malignancies.

What are common technical challenges when working with UBE2C antibodies and their solutions?

Researchers frequently encounter several technical challenges when working with UBE2C antibodies across different applications:

• High background in immunohistochemistry:

  • Problem: Non-specific staining obscuring specific UBE2C signals

  • Solutions:

    • Increase antibody dilution (test range from 1:400-1:800 for initial optimization)

    • Extend blocking time (60 minutes with 5-10% normal serum from the same species as secondary antibody)

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

    • Include thorough washing steps (minimum 3x5 minutes with TBST)

• Inconsistent Western blot results:

  • Problem: Variable band intensity or multiple bands

  • Solutions:

    • Ensure consistent sample loading (use BCA or Bradford assay)

    • Optimize transfer conditions for 20 kDa proteins

    • Test different antibody concentrations (1:1000-1:4000)

    • Include positive control samples (HeLa, HEK-293, HepG2 cells)

    • Verify specificity with knockdown controls

• Weak signal in immunofluorescence:

  • Problem: Low detection sensitivity in IF applications

  • Solutions:

    • Use lower antibody dilution (start at 1:400)

    • Optimize fixation protocol (test 4% PFA vs. methanol fixation)

    • Increase antibody incubation time (overnight at 4°C)

    • Employ signal amplification methods (tyramide signal amplification)

    • Use confocal microscopy with appropriate laser settings

• Inconsistent results across different tissue types:

  • Problem: Variation in staining patterns between tissues

  • Solutions:

    • Customize protocols for each tissue type

    • Adjust antibody concentration based on target expression levels

    • Include tissue-specific positive and negative controls

    • Validate with alternative detection methods (WB, qRT-PCR)

Systematic optimization of these parameters will significantly improve the reliability and reproducibility of experiments using UBE2C antibodies across different research applications.

How should researchers design controls for UBE2C antibody experiments?

Robust experimental design for UBE2C antibody applications requires comprehensive control strategies:

• Positive controls:

  • Cell lines: HeLa, HEK-293, HepG2, U2OS, A549, HSC-T6, and NIH/3T3 cells have confirmed UBE2C expression

  • Tissues: Human lung cancer and colon cancer tissues show reliable UBE2C expression

  • Recombinant protein: Purified UBE2C protein can serve as a definitive positive control

• Negative controls:

  • Antibody controls: Isotype-matched non-specific antibodies at equivalent concentrations

  • Secondary-only controls: Omit primary antibody to assess non-specific binding of secondary antibody

  • Blocking peptide controls: Pre-incubation of antibody with immunizing peptide to verify specificity

  • Biological controls: Tissues or cells with confirmed low UBE2C expression (most normal tissues show low expression levels)

• Knockdown/knockout controls:

  • siRNA/shRNA knockdown: Partial reduction of UBE2C expression

  • CRISPR-Cas9 knockout: Complete elimination of UBE2C expression

  • These controls provide the strongest validation of antibody specificity

• Dilution series controls:

  • Prepare standard curves with recombinant protein or positive control lysates

  • Test multiple antibody dilutions to identify optimal signal-to-noise ratio

  • Document linear range of detection for quantitative applications

• Cross-validation controls:

  • Employ multiple antibodies targeting different UBE2C epitopes

  • Compare results across different detection methods (WB, IHC, IF)

  • Correlate protein detection with mRNA expression data

Implementation of these control strategies ensures reliable and interpretable results when working with UBE2C antibodies in research applications.

What are the future directions for UBE2C antibody applications in research?

The evolving landscape of UBE2C research presents several promising directions for antibody applications:

• Single-cell analysis methodologies: Integration of UBE2C antibodies into single-cell proteomics workflows will enable more precise characterization of cellular heterogeneity in complex tissues, particularly important for understanding UBE2C's role in tumor microenvironments. Mass cytometry techniques, such as those already validated with CPTC-UBE2C-1 antibody, demonstrate the feasibility of this approach .

• Therapeutic monitoring applications: As inhibition of UBE2C activity shows therapeutic potential , antibodies will become increasingly valuable for monitoring treatment response in preclinical and clinical studies. Developing standardized protocols for quantitative assessment of UBE2C expression before and after therapeutic interventions will be critical.

• Multiplexed imaging approaches: Combining UBE2C antibodies with other markers in multiplexed imaging systems will provide deeper insights into the spatial relationships between UBE2C expression and other molecular and cellular features in tissues. This approach will be particularly valuable for understanding UBE2C's context-dependent functions.

• Liquid biopsy applications: Exploring the potential for detecting UBE2C in circulating tumor cells or extracellular vesicles may provide new opportunities for non-invasive monitoring of cancers with known UBE2C upregulation.

• Integration with multi-omics data: Correlating UBE2C protein expression with genomic, transcriptomic, and other proteomic data will enhance our understanding of the regulatory networks controlling UBE2C expression and function, potentially revealing new therapeutic targets and biomarkers.