UBE2I Antibody

What is UBE2I and why is it important in cellular processes?

UBE2I (Ubiquitin-Conjugating Enzyme E2I), also known as UBC9, is the sole E2 SUMO-conjugating enzyme that catalyzes the attachment of SUMO (Small Ubiquitin-like Modifier) proteins to target proteins. This post-translational modification affects protein stability, localization, and interactions with other proteins . UBE2I mediates SUMOylation of various proteins including p53/TP53, FOXL2, KAT5, and ERCC6, making it essential for nuclear architecture, chromosome segregation, and transcription-coupled nucleotide excision repair . In contrast to most ubiquitin-conjugating enzymes that function with E3 ligases, UBE2I can interact directly with protein substrates and may play a role in substrate recognition .

What applications are UBE2I antibodies typically used for?

UBE2I antibodies are validated for multiple applications:

The choice of application should be guided by specific experimental goals and the validated reactivity of each antibody product .

How should I optimize Western blot conditions for UBE2I detection?

For optimal Western blot detection of UBE2I:

• Sample preparation: Use RIPA or Western Blot Buffer Group 1 for cell lysis under reducing conditions .

• Protein loading: 20-30 μg of total protein is typically sufficient.

• Gel selection: Use 12-15% SDS-PAGE gels to adequately resolve the 18 kDa UBE2I protein .

• Transfer conditions: PVDF membranes are recommended with standard transfer conditions .

• Antibody dilution: Primary antibody dilutions range from 1:1000-1:4000 for polyclonal antibodies and up to 1:50000 for high-affinity monoclonal antibodies .

• Detection: Both HRP-conjugated and fluorescently-labeled secondary antibodies work well; expected band should appear at approximately 18 kDa .

Note that in heart and skeletal muscle extracts, some UBE2I antibodies may recognize a 38 kDa protein band not visible in other tissues .

What are the recommended protocols for immunofluorescence staining of UBE2I?

For successful immunofluorescence detection of UBE2I:

• Fixation: Fix cells using 4% formaldehyde/paraformaldehyde for 10-15 minutes at room temperature or use immersion fixation .

• Permeabilization: Use 0.1% PBS-Triton X-100 at room temperature for 15 minutes .

• Blocking: Block with 1-5% BSA or normal serum from the species of the secondary antibody.

• Primary antibody incubation: Use UBE2I antibody at a concentration of 8 μg/mL or at recommended dilution (typically 1:50-1:200) for 1-3 hours at room temperature or overnight at 4°C .

• Secondary antibody: Use appropriate fluorophore-conjugated secondary antibodies (e.g., NorthernLights™ 557-conjugated) .

• Counterstaining: DAPI is commonly used for nuclear counterstaining, which is helpful since UBE2I mainly localizes to the nucleus .

How can I distinguish specific from non-specific bands when probing for UBE2I?

To distinguish specific UBE2I bands:

• Size verification: The UBE2I protein has a calculated molecular weight of 18 kDa, and this should be the primary band observed in most cell types .

• Positive controls: Include validated positive control samples such as HEK-293, U937, or Jurkat cell lysates .

• Tissue-specific considerations: Be aware that UBE2I expression varies by tissue; it's highly expressed in spleen and lung, moderately in kidney and liver, and low in brain. In heart and skeletal muscle, a 38 kDa band may appear instead of the expected 18 kDa band .

• Knockout/knockdown validation: When possible, compare with UBE2I knockout or knockdown samples to confirm specificity.

• Peptide competition: Consider using peptide competition assays where pre-incubation of the antibody with the immunogenic peptide should abolish the specific band.

What is the expected subcellular localization pattern for UBE2I?

UBE2I primarily exhibits nuclear localization with specific enrichment patterns:

• Nuclear speckles: UBE2I significantly colocalizes with SFRS2, a component of nuclear speckles critical for mRNA processing .

• Nucleoplasmic distribution: While UBE2I is present throughout the nucleoplasm, it often appears in concentrated bodies or speckles .

• Limited SUMO colocalization: UBE2I-containing bodies do not completely colocalize with SUMO1 or SUMO2/3, which are primarily localized to the nuclear membrane and nucleoplasm .

• Developmental changes: In mouse oocytes, UBE2I shows specific localization patterns related to transcriptional activity, with changes in UBE2I-containing bodies correlating with BrUTP incorporation .

When performing immunofluorescence studies, these localization patterns can serve as internal controls for antibody specificity .

How does UBE2I expression and function differ across cell types and tissues?

UBE2I shows distinct expression patterns across tissues:

• High expression: Spleen and lung tissues show high levels of UBE2I protein .

• Moderate expression: Kidney and liver tissues display moderate levels .

• Low expression: Brain tissue exhibits low UBE2I levels .

• Specialized detection: In heart and skeletal muscle, the typical 18 kDa UBE2I band may be barely visible or absent, while a 38 kDa protein band is detected instead .

• Developmental regulation: In oocytes and early embryonic development, UBE2I protein levels remain relatively constant despite pronounced changes in mRNA abundance, suggesting high protein stability .

These differences should be considered when designing experiments targeting UBE2I in different systems or when comparing across tissue types .

What are the implications of UBE2I dysregulation in disease models?

Research has identified UBE2I's involvement in several pathological conditions:

• Cancer: UBE2I dysregulation contributes to cancer pathogenesis by altering the SUMOylation of tumor suppressors and oncogenes .

  • In cholangiocarcinoma, UBE2I knockdown inhibits tumorigenesis and enhances chemosensitivity via modulating p27kip1 nuclear localization .

• Metabolic disorders: Conditional knockout of Ube2i in adipocytes (Ube2i a-KO) in mice results in:

  • Lipoatrophy - progressive loss of white adipose tissue (WAT) beginning in adolescence .

  • Metabolic consequences including hyperinsulinemia, hepatic steatosis, and inflammation .

  • Compromised adipocyte function and diminished WAT expansion during postnatal growth .

• Stem cell regulation: UBE2I-dependent SUMOylation reduces levels of the stem cell marker Nanog, implicating it in embryonic stem cell pluripotency maintenance .

These findings highlight UBE2I as a potential therapeutic target across multiple disease contexts .

What strategies can I use if I'm experiencing weak or no UBE2I signal in Western blots?

If you're encountering weak or absent UBE2I signals:

• Sample preparation optimization:

  • Ensure complete cell lysis using appropriate buffers

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if studying phosphorylated forms

• Antibody selection and handling:

  • Verify antibody compatibility with your sample species (human, mouse, rat)

  • Ensure antibody storage according to manufacturer recommendations (typically -20°C with 50% glycerol)

  • Consider trying different antibody clones if available

• Protocol adjustments:

  • Increase protein loading (30-50 μg)

  • Try longer primary antibody incubation (overnight at 4°C)

  • Use more sensitive detection methods (enhanced chemiluminescence)

  • For tissue-specific issues, note that heart and skeletal muscle may show a 38 kDa band instead of the expected 18 kDa band

• Controls:

  • Include positive control lysates such as HEK-293, U937, or Jurkat cells

  • Consider using recombinant UBE2I protein as a positive control

How can I address background issues when using UBE2I antibodies for immunostaining?

To reduce background and improve specificity in immunostaining:

• Fixation optimization:

  • Test different fixation methods (paraformaldehyde vs. methanol)

  • Adjust fixation duration (10-20 minutes at room temperature)

• Blocking improvements:

  • Increase blocking time (1-2 hours)

  • Use 3-5% BSA or normal serum from the secondary antibody species

  • Include 0.1-0.3% Triton X-100 in blocking buffer

• Antibody considerations:

  • Further dilute primary antibody (test a range around recommended dilutions)

  • Reduce secondary antibody concentration

  • Extend washing steps (4-6 washes of 5-10 minutes each)

  • Use highly cross-adsorbed secondary antibodies

• Tissue-specific approaches:

  • For tissue sections, consider antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0 as recommended for UBE2I antibodies)

  • Autofluorescence quenching may be necessary for certain tissues

• Controls to include:

  • Secondary-only controls to assess non-specific binding

  • Isotype controls to evaluate background from primary antibody

How should I design experiments to study UBE2I-mediated SUMOylation of specific target proteins?

To investigate UBE2I-mediated SUMOylation of specific targets:

• Experimental approaches:

  • Co-immunoprecipitation: Use anti-UBE2I antibodies to pull down UBE2I and detect interacting proteins

  • Proximity ligation assays: Detect in situ protein-protein interactions between UBE2I and potential targets

  • SUMO modification detection: Use anti-SUMO antibodies to detect SUMOylated forms of your protein of interest

• Controls and validations:

  • UBE2I knockdown/knockout: Validate the specificity of SUMOylation using siRNA, shRNA, or CRISPR-Cas9 approaches

  • Catalytically inactive UBE2I: Compare with wild-type UBE2I overexpression to distinguish catalytic from non-catalytic functions

  • SUMO-site mutations: Create mutants of putative SUMO acceptor lysines in target proteins

• Analytical considerations:

  • SUMOylated proteins often represent a small fraction of the total protein pool

  • SUMO-targeted ubiquitin ligases may lead to degradation of SUMOylated proteins

  • Post-translational modifications of UBE2I itself (auto-SUMOylation at Lys14, Ser71 phosphorylation) can alter its activity and target recognition

These approaches can help establish both the physical interaction and functional relationship between UBE2I and potential SUMO targets.

What experimental controls should I include when studying UBE2I in developmental contexts?

When investigating UBE2I in developmental systems:

• Expression profiling controls:

  • Temporal controls: Assess both UBE2I mRNA and protein levels throughout developmental stages, as they may show distinct patterns (e.g., in oocytes, UBE2I protein levels remain constant despite mRNA changes)

  • Spatial controls: Compare expression across different tissues or cell types within the same developmental stage

• Functional assessment controls:

  • Partial vs. complete knockout: Compare conditional/tissue-specific knockouts with partial knockdowns to distinguish dose-dependent effects

  • Rescue experiments: Test if phenotypes can be rescued by wild-type UBE2I but not catalytically inactive mutants

  • Downstream target analysis: Examine key SUMOylation targets relevant to developmental processes (e.g., Nanog for stem cell studies)

• Technical considerations:

  • Developmental stage verification: Use established markers to confirm precise developmental timing

  • Tissue-specific expression patterns: Note that UBE2I shows different expression levels across tissues

  • Protein stability: UBE2I is highly stable; RNAi approaches may effectively reduce mRNA but not protein levels in short-term experiments

These controls help distinguish UBE2I-specific developmental effects from secondary consequences and technical artifacts.

What are the relative advantages of monoclonal versus polyclonal antibodies for UBE2I detection?

When selecting between monoclonal and polyclonal antibodies, consider your specific experimental needs, required applications, and whether epitope accessibility might be affected by experimental conditions .

How do different immunodetection methods compare for studying UBE2I localization and function?

Each method provides complementary information about UBE2I biology; combining multiple approaches provides the most comprehensive understanding .

What new roles for UBE2I have been discovered in metabolic regulation?

Recent research has revealed critical roles for UBE2I in metabolic homeostasis:

• Adipocyte-specific functions: Conditional deletion of Ube2i in adipocytes (Ube2i a-KO) leads to:

  • Progressive lipoatrophy beginning in early adolescence

  • Compromise of white adipose tissue (WAT) expansion during postnatal growth

  • Hyperinsulinemia and hepatic steatosis despite normal body weight

  • Pronounced local inflammation in WAT and loss of serum adipokines

• Metabolic consequences:

  • Ube2i a-KO mice develop metabolic inflexibility and cold intolerance

  • Inflammation and caspase activation of cell death occur in Ube2i a-KO adipocytes

  • Ube2i deletion affects expression of thermogenic genes (Ucp1, Prdm16, Cidea, and Cited1)

• Cellular mechanisms:

  • UBE2I appears essential for adipocyte survival

  • Knockout adipocytes show fewer lipid droplets and higher cleaved Caspase-8 levels

  • These findings are consistent with observations from human subcutaneous adipocytes

These discoveries highlight UBE2I as a critical regulator of adipocyte function and whole-body metabolism, with potential implications for metabolic diseases .

What is the latest evidence regarding UBE2I's role in cancer progression and therapy resistance?

Recent studies have expanded our understanding of UBE2I in cancer biology:

• Cholangiocarcinoma (CCA):

  • UBE2I knockdown inhibits tumorigenesis in CCA models

  • UBE2I targeting enhances chemosensitivity via modulating p27kip1 nuclear localization

  • Abnormal cytoplasmic p27kip1 localization is related to chemotherapy resistance in CCA and other cancers

• Mechanistic insights:

  • UBE2I-dependent SUMOylation regulates the protein levels of tumor suppressors and oncogenes

  • UBE2I dysregulation contributes to cancer pathogenesis through altered SUMOylation patterns

  • Post-translational modifications of UBE2I itself, such as auto-SUMOylation or phosphorylation, can alter its activity and target recognition

• Therapeutic implications:

  • UBE2I represents a potential therapeutic target in cancer treatment strategies

  • Inhibition of UBE2I may sensitize resistant cancer cells to conventional chemotherapies

  • Combined approaches targeting UBE2I and its downstream effectors might provide more effective anti-cancer strategies