UBE2E2 Antibody

What are the validated applications for UBE2E2 antibodies?

UBE2E2 antibodies have been successfully validated for multiple applications in research settings. Western blot (WB) applications typically use dilutions between 1:500-1:10000 depending on the specific antibody. Immunohistochemistry (IHC) typically requires dilutions of 1:20-1:200, while immunofluorescence/immunocytochemistry (IF/ICC) applications use 1:10-1:100 dilutions . When performing ELISA, specific binding protocols should be followed according to manufacturer recommendations. The antibody has been demonstrated to produce positive Western blot results with human thyroid gland tissue, human brain tissue, and mouse embryo tissue samples .

How should I validate the specificity of a UBE2E2 antibody?

Validating antibody specificity is essential to ensure reliable experimental results. For UBE2E2 antibodies, a multi-step approach is recommended:

• Knockdown/knockout validation: Compare antibody signals between normal cells and UBE2E2-knockdown or knockout cells. For example, UBE2E2-knockout cell lines created using CRISPR/Cas9 technology (targeting exon 2 of human UBE2E2) can serve as negative controls .

• Site-directed mutagenesis: Validate binding specificity by testing against mutant forms of UBE2E2. Mutations in the active site cysteine (Cys139) have been shown to impair both function and cellular distribution of UBE2E2 .

• Cross-reactivity assessment: Test the antibody against related E2 enzymes, particularly those in the UBE2E family (UBE2E1, UBE2E3) to ensure specificity.

• Multiple tissue validation: Confirm detection in multiple tissues known to express UBE2E2, such as human pancreas, brain tissue, and cell lines like HeLa, LNCaP, 293T, and Jurkat cells .

What is the difference between polyclonal and monoclonal UBE2E2 antibodies?

The choice between polyclonal and monoclonal UBE2E2 antibodies depends on your specific experimental needs:

• Polyclonal UBE2E2 antibodies (e.g., 11844-1-AP): These recognize multiple epitopes on the UBE2E2 protein, typically providing higher sensitivity but potentially lower specificity. They're generated in rabbits using UBE2E2 fusion proteins as immunogens. These antibodies are suitable for researchers seeking to maximize detection in applications like Western blotting or IHC across multiple species (human, mouse, rat) .

• Monoclonal UBE2E2 antibodies (e.g., EPR13003(B), CPTC-UBE2E2-3): These recognize a single epitope, offering higher specificity at potentially lower sensitivity. Monoclonal antibodies like EPR13003(B) are recombinant antibodies generated with defined epitope recognition, providing consistent lot-to-lot reproducibility. Mouse monoclonal antibodies like CPTC-UBE2E2-3 have been validated through indirect ELISA showing high binding affinity .

The optimal choice depends on your application, with monoclonals preferred for highly specific detection and polyclonals for maximum sensitivity across species.

What are the optimal conditions for Western blotting with UBE2E2 antibodies?

For optimal Western blot results with UBE2E2 antibodies, follow these evidence-based protocols:

For antibodies like EPR13003(B), a higher dilution (1:10000) has been validated with HeLa, LNCaP, 293T, and Jurkat cell lysates loaded at 10 μg per lane . When troubleshooting, remember that UBE2E2 can undergo post-translational modifications, potentially causing slight shifts in molecular weight.

How can I design experiments to study UBE2E2's role in ubiquitination pathways?

To investigate UBE2E2's role in ubiquitination pathways, consider the following experimental approaches:

• In vitro ubiquitination assays: Reconstruct the ubiquitination cascade using recombinant UBE2E2, E1 enzyme, ubiquitin, and potential substrate proteins. This allows monitoring of 'Lys-11'-, 'Lys-48'-, and 'Lys-63'-linked polyubiquitination activities catalyzed by UBE2E2 .

• Proteofection studies: Introduce recombinant UBE2E2 directly into cells to observe effects on specific substrate levels. This approach has been used to demonstrate UBE2E2's role in downregulating VEGFR2 levels in endothelial cells .

• Cycloheximide chase assays: Combine UBE2E2 knockdown with cycloheximide treatment to assess protein stability and turnover rates of potential substrates, as demonstrated with VEGFR2 .

• Structure-based mutagenesis: Generate UBE2E2 mutants with alterations at the active site cysteine (Cys139) to study the impact on enzyme function and cellular distribution .

• Co-immunoprecipitation: Use UBE2E2 antibodies to identify novel E3 ligase partners and substrate proteins. Previous studies have used this approach to demonstrate direct interactions between UBE2E2 and proteins like Nrf2 .

These approaches can be combined to build a comprehensive understanding of UBE2E2's specific roles in different ubiquitination pathways.

What controls should I include when using UBE2E2 antibodies in immunoprecipitation experiments?

For reliable immunoprecipitation (IP) experiments with UBE2E2 antibodies, include these essential controls:

• Input control: Reserve 5-10% of pre-IP lysate to confirm the presence of target proteins.

• Isotype control: Use matched IgG (rabbit IgG for rabbit anti-UBE2E2 antibodies) to assess non-specific binding.

• Knockout/knockdown control: Include lysates from UBE2E2 knockout or knockdown cells to validate antibody specificity. CRISPR/Cas9-generated UBE2E2 knockout cells targeting exon 2 have been successfully used as negative controls .

• Pre-clearing control: Compare pre-cleared versus non-pre-cleared lysates to assess improvement in specificity.

• Reciprocal IP: For protein-protein interaction studies, confirm interactions by performing IP with antibodies against suspected interaction partners (e.g., Nrf2 for UBE2E2-Nrf2 interactions) .

• Post-IP supernatant: Analyze to confirm depletion of the target protein.

• Denaturing IP control: For studying ubiquitination, include samples with denaturation steps (1% SDS, 95°C) before IP to disrupt protein complexes and ensure detection of covalent ubiquitin modifications.

The N-terminal region of UBE2E2 (residues 1-52) has been identified as critical for protein interactions, so consider this when designing experiments and interpreting results .

How can UBE2E2 antibodies be used to investigate its role in disease pathways?

UBE2E2 antibodies can provide valuable insights into various disease mechanisms:

• Type 2 Diabetes Research: UBE2E2 has been implicated in maintaining normal insulin biosynthesis, secretion, and signaling in pancreatic β cells . Researchers can use UBE2E2 antibodies to:

  • Assess protein expression in pancreatic tissue from diabetic vs. healthy subjects

  • Study co-localization with insulin production machinery

  • Investigate associations with glucose-stimulated insulin response pathways

  • Examine how the C risk allele of rs7612463 affects UBE2E2 expression and function

• Cancer Research: UBE2E2 promotes ovarian cancer metastasis through the UBE2E2-Nrf2-p62-Snail signaling axis . Antibodies can be used to:

  • Examine expression patterns in tumor vs. normal tissue (UBE2E2 is upregulated in ovarian cancer)

  • Monitor epithelial-mesenchymal transition (EMT) processes

  • Study interactions with Nrf2 and effects on p62 accumulation

  • Assess the impact of UBE2E2 inhibition on cancer cell migration and metastasis

• Neurodegenerative Disease Research: UBE2E2 interacts with TDP-43, which forms protein aggregates in amyotrophic lateral sclerosis and frontotemporal dementia . Researchers can:

  • Study co-localization with TDP-43 aggregates

  • Investigate UBE2E2's role in protein ubiquitination and clearance mechanisms

  • Examine effects on neurotoxicity in cellular and animal models

For each application, include appropriate controls and consider using complementary techniques such as RNA interference, overexpression studies, and functional assays to validate findings.

What approaches can be used to investigate UBE2E2 interactions with specific E3 ligases?

Understanding UBE2E2's interactions with E3 ligases is crucial for elucidating its specific roles in ubiquitin-mediated pathways. Several approaches can be employed:

• Yeast two-hybrid (Y2H) screening: This has been validated for E2/E3-RING interactions with >94% of E2/E3-RING interactions reported being novel . When designing Y2H experiments:

  • Consider the inherent challenges of modeling complexes with distant E2 family members

  • Be aware that not all complexes with favorable free-energy values (ΔGint) are detected in Y2H assays

  • Use appropriate controls to validate interactions

• Structure-based mutagenesis: Based on X-ray and NMR structures of E2/E3-RING complexes, target conserved amino acids within E3-RING proteins required for E2 binding but not structural integrity .

• In silico prediction and validation: Computational approaches like the Hi-map and IntNet databases can predict E2/E3 interactions, though validation rates vary (28.6% and 11.6% respectively) . Higher confidence predictions are more frequently verified.

• Bioluminescence resonance energy transfer (BRET): This technique allows real-time monitoring of protein-protein interactions in living cells, suitable for detecting dynamic E2/E3 interactions.

• Proximity ligation assay (PLA): This method can visualize endogenous protein-protein interactions with high specificity and sensitivity, ideal for studying UBE2E2 interactions with E3 ligases in their native cellular environment.

When validating predicted interactions, consider that Interolog data from other Y2H studies has shown a validation rate of >84%, suggesting the reliability of this approach for E2/E3 interaction studies .

How can I investigate UBE2E2's role in specific cellular pathways using antibody-based approaches?

UBE2E2's involvement in diverse cellular pathways can be investigated using various antibody-based techniques:

• Antioxidant response pathway: UBE2E2 promotes p62 accumulation and increases Nrf2-ARE system activity . Investigate this pathway using:

  • Co-immunoprecipitation with Nrf2 antibodies (N-terminal of UBE2E2, residues 1-52, is required for interaction)

  • Immunofluorescence co-localization studies with p62 and Nrf2

  • ChIP assays to examine Nrf2 binding to ARE elements in the presence/absence of UBE2E2

• Protein degradation pathways: UBE2E2 influences VEGFR2 levels and turnover in endothelial cells . Study this using:

  • Cycloheximide chase assays combined with UBE2E2 knockdown

  • Immunoblotting to monitor mature VEGFR2 levels

  • Ubiquitination assays to detect UBE2E2-mediated ubiquitination of client proteins

• EMT signaling axis: UBE2E2 activates the Snail signaling pathway by inhibiting ubiquitin-mediated degradation of Snail . Examine this using:

  • Western blot analysis of Snail levels after UBE2E2 manipulation

  • Co-localization studies to visualize interactions

  • Combined knockdown experiments to establish pathway directionality

• TDP-43 aggregation: UBE2E2 influences TDP-43 ubiquitination, aggregation, and neurotoxic properties . Investigate using:

  • Immunofluorescence to detect co-localization with TDP-43 aggregates

  • Biochemical fractionation to separate soluble and insoluble protein fractions

  • Ubiquitination assays to detect UBE2E2-mediated modification of TDP-43

For all these approaches, appropriate knockdown/knockout controls are essential. CRISPR/Cas9 technology targeting exon 2 of human UBE2E2 has been successfully used to create knockout cell lines for validation purposes .

How can I troubleshoot non-specific binding or weak signals with UBE2E2 antibodies?

When facing challenges with UBE2E2 antibody performance, consider these evidence-based troubleshooting approaches:

For validation, compare results with different UBE2E2 antibodies (e.g., CPTC-UBE2E2-2 and CPTC-UBE2E2-3) or using recombinant UBE2E2 as a positive control .

How can I distinguish between UBE2E2 and other UBE2E family members in my experiments?

Distinguishing between highly similar UBE2E family members requires careful experimental design:

• Antibody selection strategies:

  • Choose antibodies raised against unique regions of UBE2E2

  • Validate specificity using knockdown/knockout experiments for each family member

  • Test cross-reactivity with recombinant UBE2E1, UBE2E2, and UBE2E3 proteins

• Western blot optimization:

  • Use higher percentage gels (15-18%) to better separate similarly sized UBE2E proteins

  • Run longer separation times to distinguish subtle size differences

  • Include positive controls for each family member

  • Consider 2D gel electrophoresis to separate based on both size and isoelectric point

• Functional discrimination:

  • In knockdown experiments, only UBE2E1 and UBE2E2 knockdown significantly increased VEGFR2 levels (3-fold), while UBE2E3 or UBE2E4 knockdown had minimal effects

  • This functional difference can be used to distinguish between family members

• qRT-PCR for validation:

  • Design primers specific to unique regions of each UBE2E family member

  • Confirm specificity using known positive and negative samples

  • Use as complementary validation for protein-level experiments

When designing experiments, note that while UBE2E family members share high sequence homology, they can have distinct functions. For example, in endothelial cells, only UBE2E1 and UBE2E2 (not UBE2E3 or UBE2E4) significantly affect VEGFR2 levels .

What are the considerations for using UBE2E2 antibodies in multicolor immunofluorescence studies?

When designing multicolor immunofluorescence experiments involving UBE2E2 antibodies, consider these technical aspects:

• Antibody compatibility:

  • For co-staining with UBE2E2, select antibodies raised in different host species (e.g., mouse anti-UBE2E2 with rabbit antibodies against other targets)

  • For same-species antibodies, use directly conjugated primary antibodies or sequential staining protocols

• Subcellular localization considerations:

  • UBE2E2 localizes to both cytoplasm and nucleus

  • When studying UBE2E2 with Nrf2, note that their interaction occurs through the N-terminal of UBE2E2 (residues 1-52)

  • For TDP-43 co-localization studies, focus on cytoplasmic aggregates in disease models

• Optimal fixation and permeabilization:

  Target Combination	Recommended Fixation	Permeabilization
  UBE2E2 + Nrf2	4% PFA, 15 min, RT	0.1% Triton X-100, 10 min
  UBE2E2 + p62	4% PFA, 15 min, RT	0.2% Triton X-100, 5 min
  UBE2E2 + TDP-43	Methanol, 10 min, -20°C	Not required

• Signal amplification strategies:

  • For low abundance targets, consider tyramide signal amplification (TSA)

  • For co-localization studies with vesicular markers (e.g., EEA1 or GM130), standard fluorophore-conjugated secondaries (Alexa Fluor 488/568) at 1:2000 dilution are sufficient

• Controls and validation:

  • Include single-color controls to assess bleed-through

  • Use appropriate blocking to minimize non-specific binding (5-10% serum from secondary antibody host species)

  • For subcellular fractionation validation, complement IF studies with biochemical approaches

HepG2 cells have been successfully used for IF/ICC with UBE2E2 antibodies and can serve as a positive control for protocol optimization .

How can UBE2E2 antibodies be used to investigate its role in type 2 diabetes pathogenesis?

UBE2E2 has established connections to type 2 diabetes (T2D) through genetic and functional studies. Here's how to leverage UBE2E2 antibodies for diabetes research:

• Genetic risk correlation studies:

  • Use UBE2E2 antibodies to compare protein expression levels in tissues from individuals with different genotypes of the rs7612463 risk allele

  • The C risk allele of rs7612463 has been associated with impaired β-cell function and decreased glucose-stimulated insulin response

  • Measure UBE2E2 expression in pancreatic islets across genotypes to establish genotype-expression correlations

• Insulin secretion pathway investigations:

  • Examine UBE2E2 localization in pancreatic β-cells under normal and diabetic conditions

  • Perform co-immunoprecipitation to identify UBE2E2 interaction partners in insulin-secreting cells

  • Investigate how UBE2E2 expression levels correlate with markers of β-cell function such as:

    • Insulinogenic index (IGI)

    • BIGTT-acute insulin response (BIGTT-AIR)

    • Corrected insulin response (CIR)

• Mechanistic studies:

  • Use UBE2E2 antibodies to monitor protein levels before and after glucose stimulation

  • Investigate UBE2E2's role in insulin biosynthesis through pulse-chase experiments

  • Examine post-translational modifications of UBE2E2 in response to hyperglycemia

• Therapeutic target validation:

  • Test whether modulating UBE2E2 expression/activity can improve insulin secretion in cellular or animal models

  • Use immunohistochemistry to monitor changes in UBE2E2 distribution following treatment with anti-diabetic agents

Research has demonstrated that UBE2E2 plays a pivotal role in maintaining normal insulin biosynthesis, secretion, and signaling in pancreatic β cells, making it a valuable target for understanding T2D pathogenesis .

What methodological approaches can be used to study UBE2E2's role in cancer progression?

UBE2E2 has been implicated in ovarian cancer progression through several mechanisms. Here are methodological approaches using UBE2E2 antibodies to investigate its role in cancer:

• Expression profiling in clinical samples:

  • Use immunohistochemistry to compare UBE2E2 expression in:

    • Cancer vs. normal tissue (UBE2E2 is highly expressed in ovarian cancer but weakly expressed in normal tissue)

    • Primary tumors vs. metastatic sites

    • Different cancer stages

  • Recommended antibody dilution of 1:20-1:200 for IHC applications

• Mechanistic pathway analysis:

  • UBE2E2-Nrf2-p62-Snail signaling axis:

    • Co-immunoprecipitation to confirm direct interaction between UBE2E2 and Nrf2

    • Immunofluorescence to demonstrate co-localization

    • Western blot to monitor effects on p62 accumulation and Snail stability

    • Functional assays to assess epithelial-mesenchymal transition (EMT) markers

• Structure-function relationship studies:

  • Generate mutant forms of UBE2E2:

    • Active site cysteine mutations (Cys139) affect both function and cellular distribution

    • N-terminal truncations (residues 1-52) disrupt interaction with Nrf2

  • Use antibodies to track subcellular localization and interactions of these mutants

• In vivo validation:

  • Use UBE2E2 antibodies for tissue analysis in xenograft models

  • Compare tumors derived from UBE2E2-knockout vs. wild-type cancer cells

  • Assess metastatic potential through immunohistochemical staining of target organs

• Therapeutic development assessment:

  • Monitor changes in UBE2E2 expression and pathway activation following treatment with:

    • Ubiquitination inhibitors

    • Nrf2 pathway modulators

    • EMT inhibitors

Methodologically, combining IHC (1:20-1:200 dilution), Western blot (1:500-1:1000 dilution), and immunofluorescence (1:10-1:100 dilution) provides a comprehensive view of UBE2E2's role in cancer progression .

How can UBE2E2 antibodies contribute to understanding neurodegenerative diseases?

UBE2E2 plays a role in neurodegenerative conditions through its interaction with TDP-43, a protein that forms aggregates in diseases like amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here's how UBE2E2 antibodies can advance this research:

• Pathological aggregate characterization:

  • Use immunohistochemistry to examine co-localization of UBE2E2 with TDP-43 in:

    • Post-mortem brain and spinal cord tissues from ALS/FTD patients

    • Animal models of TDP-43 proteinopathies

    • Cellular models with induced TDP-43 aggregation

  • Apply UBE2E2 antibodies to determine if it is sequestered in pathological inclusions

• Ubiquitination pathway analysis:

  • Perform co-immunoprecipitation to detect UBE2E2-TDP-43 complexes

  • Use ubiquitin antibodies in conjunction with UBE2E2 and TDP-43 antibodies to:

    • Characterize ubiquitin chain types (Lys-11, Lys-48, or Lys-63 linked)

    • Map ubiquitination sites on TDP-43

    • Monitor temporal dynamics of ubiquitination in disease progression

• Mechanistic intervention studies:

  • Manipulate UBE2E2 levels (knockdown/overexpression) and assess effects on:

    • TDP-43 aggregation

    • Neuronal toxicity

    • Protein clearance mechanisms

  • Use UBE2E2 antibodies to confirm manipulation efficacy

• Biomarker development:

  • Evaluate UBE2E2 levels in accessible biofluids (CSF, blood) in correlation with:

    • Disease status

    • Disease progression

    • Response to experimental therapies

• Therapeutic target validation:

  • Use UBE2E2 antibodies to monitor effects of:

    • Proteasome modulators

    • Autophagy enhancers

    • Small molecule inhibitors of protein aggregation