UBE2A/UBE2B Antibody

What are UBE2A and UBE2B, and why are they important research targets?

UBE2A and UBE2B are paralogue ubiquitin-conjugating enzymes (E2s) that play critical roles in the ubiquitin pathway, particularly in protein degradation and cellular signaling. These proteins have emerged as significant research targets due to their involvement in:

• The ubiquitin N-degron pathway with E3 ligases like UBR4

• DNA repair mechanisms and response to genotoxic stress

• Mechanotransduction and contact inhibition processes

• Integrated stress response pathways

• Cancer progression and prognosis in hepatocellular carcinoma and rectal cancer

Understanding these proteins requires specific and validated antibodies for various experimental applications across multiple research disciplines.

How should researchers select the appropriate UBE2A/UBE2B antibody for their specific experimental design?

Selection of appropriate UBE2A/UBE2B antibodies requires careful consideration of several factors:

• Specificity considerations: Due to high sequence homology between UBE2A and UBE2B, many commercially available antibodies cross-react with both proteins. As noted in research literature, "in case of UBE2A it is even described in the respective antibody data sheet that the UBE2A antibodies recognize UBE2B as well, making it impossible to identify knockout cells" .

• Application compatibility: Verify the antibody has been validated for your specific application (Western blot, IHC, IF, etc.)

• Species reactivity: Confirm reactivity with your experimental model species

• Epitope location: Consider whether the epitope is accessible in your experimental conditions

What validation strategies are critical when using UBE2A/UBE2B antibodies?

Proper validation is essential due to the high homology between UBE2A and UBE2B. Recommended validation approaches include:

• Genetic knockout/knockdown controls:

  • Complete knockout of UBE2A or UBE2B can be challenging. Research teams have reported: "In case of UBE2A, UBE2B and UBE2D1 we tried several times to generate knockout cells and used even different gRNAs but were not able to identify knockout cells" .

  • siRNA knockdown (>90% reduction at mRNA level) provides a viable alternative when knockout is not feasible .

• Parallel detection methods:

  • Combine protein detection with mRNA quantification using qPCR

  • Wang et al. demonstrated UBE2A upregulation in HCC using three complementary methods: qPCR for mRNA, western blotting, and immunohistochemistry .

• Cross-reactivity testing:

  • When possible, test antibody against recombinant UBE2A and UBE2B proteins

  • For polyclonal antibodies, consider pre-absorption with the non-target protein

• Positive and negative tissue controls:

  • Use tissues with known differential expression (e.g., HCC tissues show high UBE2A expression compared to adjacent normal tissue) .

What are the optimized protocols for UBE2A/UBE2B western blotting?

Based on published research methodologies, the following optimized protocol is recommended:

• Tissue/cell preparation:

  • Lyse approximately 25 mg tissue with RIPA buffer containing phosphatase inhibitor cocktail

  • Centrifuge at 10,000 × g for 10 min at 4°C

  • Quantify protein using BCA assay

• Gel electrophoresis and transfer:

  • Separate equal amounts of protein on 10% SDS-PAGE

  • Transfer to nitrocellulose membranes at 100 V for 90 min

• Antibody incubation:

  • Primary antibody: Anti-UBE2A/RAD6 (cat. no. ab31917; 1:1,000; Abcam)

  • Incubation: Overnight at 4°C

  • Secondary antibody: HRP-conjugated goat anti-rabbit IgG

  • Loading control: β-actin (cat. no. ab8227; 1:1,500; Abcam)

• Detection considerations:

  • When studying both UBE2A and UBE2B, researchers should be aware of cross-reactivity issues

  • Distinguishing between UBE2A and other UBE family members may require specific antibodies, as researchers have noted: "we finally found a UBE2D3-reactive antibody, which did not recognize UBE2D1, D2, or D4"

How should UBE2A/UBE2B antibodies be used for immunohistochemistry studies?

The following protocol has been validated in research studies:

• Sample preparation:

  • Deparaffinize and rehydrate sections

  • Perform antigen retrieval using citrate buffer (pH 6.0) at high pressure for 2 min

  • Block endogenous peroxidase activity with 3% H₂O₂ for 15 min at room temperature

• Antibody staining:

  • Primary antibody: Rabbit anti-human UBE2A (cat. no. ab31917, 1:200 dilution; Abcam)

  • Incubation: Overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-mouse/rabbit secondary antibody

  • Incubation: 30 min at room temperature

  • Visualization: DAB staining for 1 min followed by hematoxylin counterstaining

• Scoring and evaluation:

  • UBE2A expression can be scored on a scale of 0–3 based on staining intensity:

    • 0: negative expression

    • 1: weak expression

    • 2: moderate expression

    • 3: strong expression

  • Note subcellular localization patterns: UBE2A has been detected in both nuclei and cytoplasm of HCC cells, but predominantly in the cytoplasm

What approaches are recommended for studying UBE2A/UBE2B interactions with E3 ligases?

For studying interactions between UBE2A/UBE2B and E3 ligases like UBR4, researchers should consider:

• Immunoprecipitation strategies:

  • Tag-based approaches using HA-tagged UBR4 have successfully demonstrated interactions with UBE2A/UBE2B

  • Stable expression in HEK293 cells followed by immunoprecipitation has been effective for studying E3-E2 interactions

• Functional interaction assays:

  • Autoubiquitination assays can assess functional interactions between UBE2A/UBE2B and E3 ligases

  • Livernois et al. demonstrated that "HA–UBR4 underwent robust autoubiquitination activity, but only when partnered with UBE2A, or its paralogue, UBE2B"

• Mutational analysis:

  • Specific residues can be targeted to understand mechanism:

    • Asn80 in UBE2A is part of a conserved His–Pro–Asn motif essential for thioester activation

    • Ser120 in UBE2A facilitates E2 transfer to lysine residues

  • Quantitative gel-based autoubiquitination assays using fluorescently labeled ubiquitin can assess the effects of these mutations

How can UBE2A/UBE2B antibodies be used to investigate cancer progression mechanisms?

UBE2A/UBE2B have been implicated in cancer progression, making their study valuable in oncology research:

• Prognostic marker studies:

  • UBE2A overexpression correlates with poor prognosis in hepatocellular carcinoma (HCC)

  • UBE2B expression correlates with tumor regression grade and survival rate in rectal cancer patients receiving preoperative chemoradiotherapy

• Investigation methodology:

  • For HCC studies, researchers used a combination of qPCR, western blotting, and immunohistochemistry to establish UBE2A as a prognostic marker

  • Statistical analysis of correlation between UBE2A/UBE2B expression and clinicopathological parameters requires software like GraphPad Prism and SPSS

• Therapeutic target validation:

  • UBE2B inhibition with compounds like TZ9 has been shown to elevate genotoxicity of ionizing radiation in radioresistant cell lines

  • Investigation of 53BP1 and Rad51 recruitment patterns can help elucidate the mechanism of UBE2B in DNA repair pathways

What control experiments are essential when studying UBE2A/UBE2B with antibodies?

Given the challenges in antibody specificity and protein function overlap, essential controls include:

• Genetic controls:

  • siRNA knockdown of UBE2A/UBE2B with >90% reduction in mRNA (verified by real-time PCR)

  • If using knockout approaches, careful validation is required as complete knockout can be challenging

• Antibody controls:

  • No primary antibody control

  • Isotype control

  • Preabsorption with recombinant protein when available

• Functional controls:

  • When studying UBE2A/UBE2B involvement in specific pathways (e.g., integrated stress response), co-depletion of both proteins may be necessary to observe phenotypes

  • For mechanotransduction studies, appropriate mechanical stimulation controls should be included when using UBE2A/B antibodies to detect translocation or activation

How do UBE2A and UBE2B differ in their biochemical properties and research applications?

While UBE2A and UBE2B are paralogues with significant homology, research has revealed important distinctions:

• Catalytic activity:

  • UBE2A demonstrates "intrinsically high lysine reactivity" which may be important for substrate specificity

  • The observed rate of UBE2A discharge to lysine is at least sixfold higher than the prototypical E2 UBE2D3

• Structural considerations:

  • UBE2A interactions with E3 ligases involve specific residues like Y82 and S120 that guide the ε-amino-group of K48 in ubiquitin toward the catalytic Cys88

  • These structural features are important for K48-specific chain formation

• Pathway involvement:

  • While both proteins are involved in ubiquitination, they may have differential involvement in specific pathways

  • UBE2B has been specifically implicated in DNA repair pathways related to ionizing radiation response

  • Both proteins appear to function in the integrated stress response when co-depleted

What are common troubleshooting strategies for UBE2A/UBE2B antibody experiments?

Researchers frequently encounter challenges when using UBE2A/UBE2B antibodies:

• Cross-reactivity issues:

  • Problem: UBE2A antibodies often recognize UBE2B and vice versa

  • Solution: Use siRNA knockdown of specific proteins and confirm knockdown efficiency with qPCR

  • Alternative: When studying pathways where both proteins might function redundantly, consider co-depletion approaches

• Low signal strength:

  • Problem: Weak detection in western blots or immunostaining

  • Solution: Optimize antibody concentration, incubation time, and detection methods

  • For Wang et al.'s HCC study, a 1:1,000 dilution for western blot and 1:200 for IHC provided optimal results

• Background issues in immunohistochemistry:

  • Problem: High background staining

  • Solution: Optimize blocking (3% H₂O₂ for 15 min) and washing steps

  • Consider counterstaining time adjustments (DAB staining for 1 min)

How can UBE2A/UBE2B antibodies help investigate mechanotransduction pathways?

UBE2A/B has been identified as a force- and contact inhibition-sensing factor in mechanotransduction :

• Experimental approaches:

  • DHS-proteomics methods revealed UBE2A/B as a potential mechanosensing trans-acting factor

  • Antibodies can be used to detect nucleocytoplasmic shuttling of UBE2A/B in response to mechanical stimuli

• Methodological considerations:

  • When investigating mechanotransduction, researchers should combine antibody-based detection with functional assays

  • Controls for mechanical stimulation are essential

  • Consider both localization changes and post-translational modifications of UBE2A/B

• Research applications:

  • UBE2A/B antibodies can help elucidate how mechanical forces regulate gene expression

  • They can be used to study how mechanotransduction connects to cell proliferation, differentiation, and tumorigenesis

What role do UBE2A/UBE2B play in the integrated stress response, and how can this be studied?

Recent research has identified UBE2A/UBE2B involvement in the integrated stress response (ISR):

• Experimental evidence:

  • "UBE2A and UBE2B co-depletion results in increased activation of the integrated stress response, as determined by αATF4-Western blotting"

• Study methodology:

  • Co-depletion approaches (rather than single protein depletion) may be necessary to observe phenotypes

  • Western blotting for stress response markers like ATF4 can help quantify ISR activation

  • Both genetic approaches (siRNA, CRISPR) and chemical inhibition can be used to study UBE2A/B functions

• Research applications:

  • Understanding UBE2A/B roles in ISR could provide insights into cellular stress management

  • Potential therapeutic applications for stress-related conditions and cancer treatments