UBE2D2 Antibody, Biotin conjugated

What is UBE2D2 and what are its cellular functions?

UBE2D2 (Ubiquitin-conjugating enzyme E2D 2) is a 147-amino acid, 17 kDa protein that functions as an essential E2 ubiquitin-conjugating enzyme in the ubiquitin-proteasome system. It plays a crucial role in facilitating the transfer of ubiquitin to substrate proteins, working in conjunction with E1 (ubiquitin-activating) and E3 (ubiquitin-ligase) enzymes. UBE2D2 is primarily localized to the cytoplasm, where it interacts with various E3 ligases to catalyze substrate ubiquitination .

Recent research has demonstrated that UBE2D2 specifically regulates vascular endothelial growth factor receptor 2 (VEGFR2) levels in endothelial cells. Knockdown of UBE2D2 causes approximately 1.6-fold increase in VEGFR2 levels, while introduction of recombinant UBE2D2 results in approximately 50% decrease in VEGFR2 levels after 3 hours, indicating its direct role in regulating this critical angiogenic receptor . Additionally, UBE2D2 catalyzes IκBα ubiquitination in a phosphorylation-dependent manner, which is essential for NFκB activation and subsequent immune and inflammatory responses .

How does biotin conjugation affect UBE2D2 antibody functionality?

Biotin conjugation of UBE2D2 antibodies creates a versatile reagent that maintains target specificity while enabling enhanced detection sensitivity and experimental flexibility. The small biotin moiety (244 Da) minimally alters antibody binding kinetics when properly conjugated, preserving the antibody's ability to recognize native or denatured UBE2D2 protein. Conjugation typically occurs at lysine residues remote from the antigen-binding region, ensuring that biotin addition does not interfere with epitope recognition.

What are the key advantages of biotin-conjugated UBE2D2 antibodies over unconjugated versions?

Biotin-conjugated UBE2D2 antibodies offer several methodological advantages compared to unconjugated versions:

When studying UBE2D2's interactions with VEGFR2 or other client proteins, biotin-conjugated antibodies can be particularly valuable for co-immunoprecipitation experiments, allowing efficient recovery of protein complexes while minimizing interference from heavy and light antibody chains .

Which experimental techniques are most suitable for biotin-conjugated UBE2D2 antibodies?

Biotin-conjugated UBE2D2 antibodies are exceptionally versatile across multiple experimental platforms. Based on the validation data from unconjugated UBE2D2 antibodies, the following applications are particularly well-suited for biotin-conjugated versions:

• Western Blotting: Recommended dilution ranges from 1:500 to 1:2000, detecting UBE2D2 at 14-17 kDa . Biotin conjugation enhances detection sensitivity through streptavidin-HRP systems.

• Immunohistochemistry: Successfully employed at dilutions of 1:250 to 1:1000 . The biotin-conjugated format enables amplified signal detection using streptavidin-based visualization systems.

• Immunoprecipitation: Particularly valuable for isolating UBE2D2 and its binding partners, such as VEGFR2 . The biotin tag allows efficient complex recovery using streptavidin-coated beads.

• Immunofluorescence: Effective for visualizing UBE2D2's cytoplasmic localization , with biotin conjugation permitting flexible secondary detection options.

• ELISA: Validated as a detection antibody in sandwich ELISA formats .

• Flow Cytometry: Enables quantitative analysis of UBE2D2 expression levels across cell populations.

Cell lines successfully used with UBE2D2 antibodies include HEK-293, MCF-7, NIH/3T3, PC-12, U-87MG, K-562, and Jurkat cells, while tissue samples include human breast cancer tissue, mouse testis, mouse brain, mouse kidney, and rat kidney .

What are the optimal fixation and permeabilization methods for immunofluorescence with UBE2D2 antibodies?

For optimal immunofluorescence detection of UBE2D2, the following protocol is recommended based on its cytoplasmic localization and structural characteristics:

Fixation Options:

• Paraformaldehyde (4%): 15 minutes at room temperature, preserves protein-protein interactions and cellular architecture

• Methanol: 10 minutes at -20°C, may better expose certain epitopes but can disrupt some protein complexes

Permeabilization Options:

• Triton X-100 (0.1-0.2%): 10 minutes for paraformaldehyde-fixed cells

• Saponin (0.1%): Gentler alternative that better preserves membrane structure

Optimization Considerations:

• UBE2D2's interaction with client proteins like VEGFR2 may be sensitive to fixation conditions

• The small size of UBE2D2 (17 kDa) may require more aggressive permeabilization

• Antigen retrieval with TE buffer pH 9.0 is recommended for tissue sections; alternatively, citrate buffer pH 6.0 may be used

When using biotin-conjugated antibodies, include a blocking step for endogenous biotin (particularly important in tissues with high biotin content) using a commercial biotin blocking kit before primary antibody application.

How should researchers optimize western blot protocols for UBE2D2 detection?

For optimal western blot detection of UBE2D2 using biotin-conjugated antibodies, consider the following protocol modifications:

Sample Preparation:

• Use RIPA buffer with protease inhibitors

• Include 20 mM N-ethylmaleimide to preserve ubiquitination status

• Sonicate briefly to shear DNA and reduce sample viscosity

Gel Electrophoresis:

• 12-15% SDS-PAGE gels are optimal for resolving UBE2D2's 17 kDa band

• Load 10-30 μg of total protein per lane

• Include positive control samples (e.g., HEK-293, MCF-7, or NIH/3T3 cell lysates)

Transfer and Detection:

• PVDF membranes are preferred over nitrocellulose for better protein retention

• Perform semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour

• Block with 5% non-fat milk or BSA in TBST

• For biotin-conjugated antibodies, use 1:1000 to 1:5000 dilution of streptavidin-HRP

• Develop using enhanced chemiluminescence

Troubleshooting Tips:

• Expected molecular weight: 14-17 kDa

• Multiple bands may represent different UBE2D family members due to high sequence homology

• Consider endogenous biotinylated proteins that may cause background (use appropriate blocking)

This protocol has been validated across multiple cell types including human (HEK-293, MCF-7, U-87MG, K-562, Jurkat), mouse (NIH/3T3), and rat (PC-12) cell lines .

How can researchers address non-specific binding issues with biotin-conjugated UBE2D2 antibodies?

Non-specific binding is a common challenge with biotin-conjugated antibodies due to endogenous biotin and biotinylated proteins in biological samples. For UBE2D2 detection, implement these strategies:

Pre-experiment Validation:

• Run antibody validation with both positive controls (e.g., HEK-293, MCF-7, NIH/3T3 cells) and negative controls (knockdown/knockout samples)

• Confirm specificity using peptide competition assays with the immunogen sequence (amino acids 1-147 of human UBE2D2)

Blocking Optimization:

• Use avidin/biotin blocking kits before applying the biotin-conjugated antibody

• Implement dual blocking with both 5% BSA and 5% normal serum from the species of the detection reagent

• Add 0.1-0.3% Triton X-100 to blocking solutions to reduce hydrophobic interactions

Sample-Specific Strategies:

• For tissues with high endogenous biotin (liver, kidney, brain): Extend avidin/biotin blocking times

• For proximity to UBE2D homologs (UBE2D1, UBE2D3, UBE2D4): Use higher antibody dilutions (1:1000-1:2000)

• For highly ubiquitinated samples: Pre-clear lysates with protein A/G beads

Cross-Reactivity Management Matrix:

Research has shown that while UBE2D2 and UBE2D1 share high homology, they have distinct effects on VEGFR2 levels, with UBE2D1 knockdown causing a 2-fold increase compared to UBE2D2's 1.6-fold increase .

What explains variability in UBE2D2 molecular weight observed in western blots?

UBE2D2 is typically observed at 14-17 kDa in western blots , but researchers may notice variability in its apparent molecular weight. This variation can be attributed to several factors:

Post-translational Modifications:

• Mono-ubiquitination: Adds approximately 8.5 kDa

• Phosphorylation: Subtle shifts of 1-3 kDa

• SUMOylation: Adds approximately 11 kDa

Technical Factors:

• Gel percentage: Higher percentage gels (15-20%) provide better resolution in the 10-20 kDa range

• Buffer systems: Tris-Glycine vs. Tris-Tricine can affect migration patterns

• Sample preparation: Reducing vs. non-reducing conditions

• Sample heating: Excessive heating can cause aggregation or degradation

Biological Factors:

• Tissue-specific isoforms

• Species differences: Human UBE2D2 (17 kDa) vs. mouse/rat homologs

• Cell-specific post-translational modification patterns

When interpreting UBE2D2 western blots, researchers should note that ubiquitinated VEGFR2 species with higher molecular mass have been observed in both basal and VEGF-A-stimulated conditions following UBE2D2 complex isolation . This indicates that UBE2D2 may itself be part of higher molecular weight complexes depending on cellular conditions.

How should researchers interpret dual or multiple bands when detecting UBE2D2?

Multiple bands when detecting UBE2D2 can provide valuable biological information rather than simply indicating technical issues. Interpretation should consider:

Expected Band Patterns:

• Primary UBE2D2 band: 14-17 kDa representing the unmodified protein

• Higher molecular weight bands: May represent:

  • Ubiquitinated forms (~25 kDa and higher)

  • UBE2D2-substrate intermediates

  • Post-translationally modified UBE2D2

Distinguishing True Signal from Artifacts:

Research has demonstrated that UBE2D2 forms complexes with client proteins like VEGFR2, and these complexes can be isolated and detected as higher molecular weight species. Particularly, ubiquitinated VEGFR2 species with higher molecular mass were evident after UBE2D2 complex isolation in both basal and VEGF-A-stimulated conditions .

How can biotin-conjugated UBE2D2 antibodies be used to study E2-E3 ligase interactions?

Biotin-conjugated UBE2D2 antibodies offer sophisticated approaches for investigating the critical interactions between UBE2D2 and various E3 ligases that determine substrate specificity in the ubiquitination pathway.

Methodological Approaches:

• In vitro E2-E3 Binding Assays:

  • Immobilize biotin-conjugated UBE2D2 antibodies on streptavidin surfaces

  • Immunoprecipitate native UBE2D2 complexes from cell lysates

  • Add recombinant E3 ligases to test direct binding

  • Quantify interaction strength through surface plasmon resonance

• Proximity Ligation Assay (PLA):

  • Use biotin-conjugated UBE2D2 antibody with primary antibody against suspected E3 partner

  • Apply streptavidin-conjugated oligonucleotide and secondary antibody-conjugated oligonucleotide

  • Detect amplified signal only when UBE2D2 and E3 are within 40 nm

  • Quantify discrete interaction spots per cell

• ChIP-Sequential Precipitation (ChIP-SeqP):

  • First immunoprecipitate chromatin-associated E3 ligases

  • Elute and re-precipitate using biotin-conjugated UBE2D2 antibody

  • Sequence DNA to identify genomic regions where UBE2D2-E3 complexes associate

Research Applications:
This approach has revealed that UBE2D2 directly interacts with VEGFR2 in endothelial cells, forming complexes that can be co-precipitated. These interactions occur in both basal and VEGF-A-stimulated conditions, suggesting constitutive association . Similar approaches could identify other client proteins and their associated E3 ligases.

What considerations are important when designing UBE2D2 knockdown experiments?

When designing UBE2D2 knockdown experiments to study its functional role, researchers should implement the following evidence-based strategies:

Knockdown Validation:

• Confirm knockdown efficiency at both mRNA (qRT-PCR) and protein (western blot) levels

• Typical knockdown protocols achieve 70-90% reduction in UBE2D2 levels

• Use validated siRNA sequences targeting unique regions of UBE2D2 to avoid off-target effects

Experimental Design Considerations:

Expected Phenotypes:
Research has shown that UBE2D2 knockdown in endothelial cells causes:

• ~1.6-fold increase in VEGFR2 levels

• Altered downstream signaling pathways

• Changes in endothelial cell responses

Conversely, cytoplasmic delivery of recombinant UBE2D2 caused an ~50% decrease in VEGFR2 levels after 3 hours , providing complementary evidence for UBE2D2's regulatory role.

How can researchers quantitatively assess UBE2D2-mediated ubiquitination activity?

Quantitative assessment of UBE2D2-mediated ubiquitination requires specialized assays that capture both the kinetics and substrate specificity of this process. The following methodologies enable precise measurement:

In Vitro Ubiquitination Assays:

• FRET-based real-time monitoring:

  • Label ubiquitin with donor fluorophore

  • Label substrate with acceptor fluorophore

  • Measure FRET signal increase as ubiquitination proceeds

  • Calculate reaction rates under varying E2/E3/substrate concentrations

• TR-FRET (Time-Resolved FRET) Assays:

  • Use biotin-conjugated UBE2D2 antibody with streptavidin-europium

  • Anti-ubiquitin antibody conjugated to acceptor fluorophore

  • Measure energy transfer as indicator of ubiquitination

  • Long fluorescence lifetime reduces background interference

Cellular Ubiquitination Quantification:

Research on VEGFR2 has demonstrated that UBE2D2-mediated ubiquitination significantly impacts receptor turnover. Knockdown of UBE2D2 increases VEGFR2 levels by approximately 1.6-fold, while introduction of recombinant UBE2D2 decreases levels by approximately 50% after 3 hours . These quantitative measurements provide insight into the kinetics and magnitude of UBE2D2's regulatory effects.

What role does UBE2D2 play in disease pathogenesis and therapeutic development?

UBE2D2's central role in protein ubiquitination positions it as a significant factor in multiple disease mechanisms and a potential therapeutic target. Current research highlights its involvement in:

Cardiovascular Disorders:

• UBE2D2 regulates VEGFR2 levels in endothelial cells, directly impacting angiogenic responses

• Knockdown of UBE2D2 increases VEGFR2 levels by ~1.6-fold, potentially enhancing angiogenic responses

• Modulation of UBE2D2 activity could regulate pathological angiogenesis in cancer or therapeutic angiogenesis in ischemic diseases

Inflammatory Conditions:

• UBE2D2 catalyzes IκBα ubiquitination in a phosphorylation and SCFβ-TRCP-dependent manner

• This process is critical for NFκB activation and inflammatory gene expression

• Targeting UBE2D2 could modulate inflammatory responses in autoimmune diseases

Cancer Biology:

• Dysregulation of ubiquitination pathways contributes to cancer development and progression

• UBE2D2 expression has been detected in multiple cancer cell lines including U-87MG, K-562, and Jurkat cells

• UBE2D2 antibodies enable mechanistic studies of ubiquitination in tumor tissues

Therapeutic Development Approaches:

Biotin-conjugated UBE2D2 antibodies serve as valuable tools in these therapeutic development pathways by enabling precise detection and quantification of UBE2D2 in complex biological samples and high-throughput screening platforms.

What are the key considerations for selecting and validating biotin-conjugated UBE2D2 antibodies?

When selecting and validating biotin-conjugated UBE2D2 antibodies for research, scientists should consider multiple factors to ensure experimental success:

Selection Criteria:

• Epitope specificity: Antibodies targeting the middle region of UBE2D2 (such as those based on amino acids 1-147) demonstrate good specificity

• Cross-reactivity profile: Consider whether species cross-reactivity (human, mouse, rat) is desired

• Validation data: Review available western blot, IHC, and IP validation data from manufacturers

• Biotin conjugation method: Preferentially select antibodies with site-specific conjugation rather than random labeling

Validation Strategy:

• Confirm specificity using:

  • Positive control lysates (HEK-293, MCF-7, NIH/3T3, U-87MG, K-562, Jurkat)

  • UBE2D2 knockdown or knockout samples

  • Peptide competition assays

• Assess performance across multiple applications (WB, IF, IHC, IP) at recommended dilutions

• Evaluate lot-to-lot consistency and storage stability

• Verify absence of endogenous biotin interference using appropriate blocking controls