UBE2D2 Antibody, HRP conjugated

What is UBE2D2 and what are its primary functions in cellular systems?

UBE2D2 belongs to the UBE2D family of E2 ubiquitin-conjugating enzymes that play essential roles in the ubiquitin-proteasome system. This enzyme collaborates with E3 ubiquitin ligases to catalyze the transfer of ubiquitin to substrate proteins, marking them for degradation or altered trafficking.

Research has demonstrated that UBE2D2 specifically regulates critical membrane receptors including VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) in endothelial cells . Experimental knockdown of UBE2D2 causes approximately 3-fold increase in steady-state VEGFR2 levels, indicating its central role in receptor turnover . UBE2D2 also works with the E3 ligase IDOL to mediate sterol-dependent degradation of LDLR (Low-Density Lipoprotein Receptor) .

What distinguishes HRP-conjugated UBE2D2 antibodies from unconjugated versions?

HRP-conjugated UBE2D2 antibodies have horseradish peroxidase directly linked to the antibody molecule, providing several methodological advantages:

• Direct detection capability without requiring secondary antibody incubation

• Streamlined experimental workflows with fewer washing steps

• Enhanced sensitivity through enzymatic signal amplification

• Compatibility with multiple detection substrates (colorimetric, chemiluminescent)

Unconjugated antibodies, such as the polyclonal UBE2D2 antibody described in the search results, require a secondary detection system and are typically used in conventional two-step immunodetection protocols .

What are the primary laboratory applications for UBE2D2 antibodies?

UBE2D2 antibodies serve multiple critical research applications:

Published studies have successfully used UBE2D2 antibodies to demonstrate specific interactions between UBE2D2 and client proteins such as VEGFR2, with clear discrimination between UBE2D2 and other closely related family members .

What storage conditions maintain optimal HRP-conjugated antibody activity?

For maximum stability and activity retention of HRP-conjugated UBE2D2 antibodies:

• Store at -20°C for up to 1 year from receipt date

• Avoid repeated freeze-thaw cycles that degrade both antibody binding and HRP activity

• Use stabilizing formulations containing glycerol (typically 50%), BSA (0.5%), and sodium azide (0.02%)

• Aliquot working stocks to minimize freeze-thaw events

• Protect from prolonged exposure to light which can compromise HRP enzyme activity

How can researchers optimize detection of UBE2D2-mediated protein ubiquitination?

Effective detection of UBE2D2-catalyzed ubiquitination requires careful methodological consideration:

• Cell lysis protocol optimization:

  • Use non-denaturing conditions with mild detergents to preserve protein interactions

  • Include deubiquitinase inhibitors (e.g., N-ethylmaleimide) to prevent ubiquitin removal

  • Maintain low temperature (4°C) throughout processing

• Immunoprecipitation strategy:

  • Target either UBE2D2 or the substrate protein depending on experimental question

  • Include controls for non-specific binding (e.g., non-relevant membrane receptors)

  • Look for higher molecular weight ubiquitinated species as evidence of modification

• Western blot detection parameters:

  • Use gradient gels (4-12%) for better resolution of high molecular weight ubiquitinated species

  • Optimize transfer conditions for large proteins

  • Test membrane blocking conditions to minimize background

Research demonstrates successful detection of "higher molecular weight ubiquitinated VEGFR2 species in both basal and VEGF-A stimulated conditions followed by UBE2D complex isolation" .

How can researchers experimentally distinguish between UBE2D family members?

Despite high sequence homology, UBE2D family members (UBE2D1-4) show distinct functional profiles. Methodological approaches to differentiate them include:

What experimental controls are essential when studying UBE2D2-mediated ubiquitination pathways?

Rigorous experimental design requires multiple control strategies:

• Specificity controls:

  • Include non-targeting siRNA controls for knockdown experiments

  • Use non-substrate membrane proteins (e.g., transferrin receptor) as negative controls

  • Test unrelated E2 enzymes (e.g., UBE2H) to confirm pathway specificity

• Functional validation approaches:

  • Employ dominant-negative UBE2D2 mutants (C85A) that lack catalytic activity

  • Compare wild-type versus catalytically inactive proteins

  • Include UBE2Z controls, which are uniquely primed by UBA6 rather than UBA1

• Ubiquitination verification:

  • Analyze ubiquitin chain topology (K48 vs. K63-linked) using linkage-specific antibodies

  • Compare patterns between stimulated and unstimulated conditions

  • Include deubiquitinase inhibitors to preserve ubiquitinated intermediates

What methodological approach best characterizes UBE2D2's role in receptor regulation?

A comprehensive experimental strategy should include:

• Receptor dynamics analysis:

  • Combine UBE2D2 knockdown with cycloheximide chase experiments to distinguish between effects on synthesis versus degradation

  • Monitor receptor trafficking through plasma membrane biotinylation

  • Quantify receptor half-life with and without UBE2D2 manipulation

• Signal transduction assessment:

  • Evaluate receptor activation status (phosphorylation)

  • Monitor downstream pathway activation (MAPK, PLCγ1, Akt)

  • Compare signaling kinetics and magnitude between control and UBE2D2-depleted conditions

• Mechanistic investigation:

  • Direct protein introduction through proteofection (shown to cause "~50% decrease in VEGFR2 levels after 3 h")

  • Co-immunoprecipitation to identify adapter proteins or E3 ligases in the complex

  • Subcellular localization studies to track receptor trafficking

How can researchers design experiments to resolve contradictory findings regarding UBE2D2 function?

When conflicting results emerge regarding UBE2D2 function, consider these methodological approaches:

• Cell type-specific effects:

  • Test multiple cell lines or primary cells

  • Compare results in different tissue contexts

  • Control for expression levels of other ubiquitination machinery

• Technical variation sources:

  • Standardize antibody validation procedures

  • Implement quantitative analysis methods

  • Control for protein degradation during sample processing

• Physiological relevance:

  • Complement in vitro findings with in vivo models

  • Test function under various stress conditions

  • Consider temporal dynamics of ubiquitination

• Systematic validation:

  • Implement both gain-of-function and loss-of-function approaches

  • Utilize CRISPR/Cas9 genome editing alongside siRNA methods

  • Complement genetic approaches with pharmacological inhibitors when available

What are the critical steps for optimizing Western blot protocols with UBE2D2 antibodies?

Successful Western blot detection of UBE2D2 requires attention to several technical parameters:

Research shows that these parameters allow successful detection of endogenous UBE2D2 and discrimination between closely related family members .

How can researchers troubleshoot non-specific binding with UBE2D2 antibodies?

When encountering non-specific binding or high background:

• Validation approaches:

  • Confirm antibody specificity using UBE2D2 knockdown controls

  • Test against recombinant UBE2D proteins to assess cross-reactivity

  • Evaluate recognition of endogenous versus overexpressed protein

• Protocol optimization:

  • Increase blocking stringency (time, concentration, detergent)

  • Adjust antibody concentration (typically lower)

  • Modify washing conditions (duration, buffer composition)

  • Test alternative membrane types (PVDF vs. nitrocellulose)

• Sample preparation considerations:

  • Ensure complete protein denaturation for Western blotting

  • Include phosphatase inhibitors to prevent post-lysis modifications

  • Control lysis conditions to minimize protein degradation

What experimental design best demonstrates UBE2D2's direct effect on substrate proteins?

To establish direct causality between UBE2D2 and substrate regulation:

• In vitro ubiquitination assays:

  • Reconstitute ubiquitination using purified components

  • Compare wild-type versus catalytically inactive UBE2D2

  • Analyze ubiquitin chain formation and topology

• Cellular approaches:

  • Implement proteofection of recombinant UBE2D2 (shown to cause "~50% decrease in VEGFR2 levels after 3 h")

  • Use rapid induction systems for controlled UBE2D2 expression

  • Perform time-course analyses to establish temporal relationships

• Proximity-based methods:

  • Employ BioID or APEX2 proximity labeling

  • Utilize FRET-based interaction assays

  • Implement co-immunoprecipitation under various conditions

The research demonstrates that "cytoplasmic delivery of recombinant UBE2D1 or UBE2D2 caused ~50% decrease in VEGFR2 levels after 3 h," providing strong evidence for direct effects .

How can researchers investigate cooperative functions between UBE2D2 and other E2 enzymes?

Recent findings suggest potential functional overlap between UBE2D family members:

• Sequential activity models:

  • Test combinatorial knockdown of multiple UBE2D enzymes

  • Analyze temporal recruitment patterns to substrate proteins

  • Investigate potential priming/extension mechanisms

• E3 ligase interaction profiles:

  • Compare binding affinities between different UBE2D enzymes and E3 partners

  • Identify unique versus shared E3 interaction networks

  • Map binding domains through mutagenesis approaches

• Substrate specificity determination:

  • Conduct proteomic analyses after selective UBE2D family member depletion

  • Implement ubiquitinome profiling techniques

  • Develop computational models to predict enzyme-substrate relationships

Research showing differential effects of UBE2D1/2 versus UBE2D3/4 on VEGFR2 levels highlights the complexity of these relationships .

What methodological approaches can assess UBE2D2's role in non-degradative ubiquitination?

Beyond protein degradation, ubiquitination regulates numerous cellular processes:

• Ubiquitin linkage-specific analysis:

  • Use linkage-specific antibodies to distinguish K48 (degradative) from K63 and other linkages

  • Employ mass spectrometry to identify ubiquitination sites and chain topology

  • Analyze modification patterns with and without proteasome inhibition

• Trafficking pathway investigation:

  • Track receptor internalization rates after UBE2D2 manipulation

  • Evaluate colocalization with endosomal markers

  • Assess recycling versus lysosomal targeting ratios

• Signaling modulation assessment:

  • Analyze phosphorylation cascades downstream of ubiquitinated receptors

  • Compare activation kinetics between wild-type and ubiquitination-deficient mutants

  • Investigate protein-protein interaction networks altered by ubiquitination

Research demonstrates UBE2D2's impact on VEGFR2 signal transduction and membrane trafficking, suggesting roles beyond simple degradation .