UBR5 Antibody

What is UBR5 and why is it important to study?

UBR5 is a large (~309.4 kDa) HECT-type E3 ubiquitin ligase that is highly conserved in metazoans, with murine and human proteins sharing 98% sequence identity . It plays multifunctional roles in:

• Antiviral immunity: UBR5 positively regulates RIG-I-like receptor (RLR) transcription by mediating K63-linked ubiquitination of TRIM28, an epigenetic repressor of RLRs

• Transcriptional regulation: UBR5 cooperates with transcription factors like TFIIS to regulate RNA polymerase II activity

• Cancer progression: UBR5 is overexpressed in breast and ovarian cancers, and regulates proliferation and radiosensitivity in laryngeal carcinoma cells

• DNA damage response: UBR5 participates in DNA damage signaling by controlling activities of Chk2, TopBP1, and RNF168

• Immune cell function: UBR5 regulates RORγt stability and IL-17 production by Th17 cells

Its broad functional implications make UBR5 a significant target for both basic research and therapeutic development.

What applications are UBR5 antibodies typically used for?

UBR5 antibodies are employed in multiple research applications including:

• Western blotting (WB): For detecting UBR5 protein expression levels (typically at ~300kDa)

• Immunoprecipitation (IP): For studying UBR5 protein-protein interactions

• Immunohistochemistry (IHC): For visualizing tissue distribution and subcellular localization

• Immunofluorescence (IF): For examining cellular localization patterns of UBR5

• Chromatin immunoprecipitation (ChIP): For studying UBR5 association with chromatin regions, particularly relevant for its role in transcriptional regulation

• ELISA: For quantitative detection of UBR5 protein levels

Different applications may require specific antibody validation strategies and optimization of experimental conditions.

What are the key considerations for selecting an appropriate UBR5 antibody?

When selecting a UBR5 antibody, researchers should consider:

Epitope recognition:

• N-terminal antibodies (detecting regions like AA 1-50, 550-760) vs. C-terminal antibodies (detecting C-terminus)

• Domain-specific antibodies targeting functional domains like HECT, UBR, or MLLE domains for studying specific functions

Species reactivity:

• Human-specific UBR5 antibodies

• Mouse/rat-specific UBR5 antibodies

• Multi-species reactive antibodies (human/mouse/rat)

Antibody format:

• Host species (rabbit, goat, sheep) impacts secondary antibody selection and potential cross-reactivity

• Polyclonal vs. monoclonal (most commercial UBR5 antibodies are polyclonal)

• Conjugation status (unconjugated vs. fluorescent/enzyme-conjugated)

Validation data:

• Verification in knockout models

• Western blot bands at expected molecular weight (~300 kDa)

• Cross-reactivity with other species

Application compatibility:

• Antibodies validated for specific applications (WB, IP, IHC, IF, etc.)

What are the standardized methods for validating UBR5 antibodies?

Proper antibody validation is critical for reliable UBR5 research. Standard validation methods include:

Genetic validation:

• Testing in UBR5 knockout cell lines created by CRISPR-Cas9 (e.g., using gRNAs targeting different UBR5 regions as described in research)

• Using UBR5 siRNA knockdown to confirm antibody specificity

Western blot validation:

• Confirming single band at expected molecular weight (~300 kDa)

• Testing across multiple cell lines (e.g., Jurkat human T cells, HT-2 mouse T cells)

• Comparing reactivity in different tissues (e.g., rat ovary tissue)

Immunoprecipitation validation:

• Coupled with mass spectrometry to confirm target identity

• Reciprocal co-IP for interaction partners (e.g., with TRIM28, CDK9)

Immunohistochemistry/Immunofluorescence validation:

• Comparison with mRNA expression patterns

• Blocking peptide competition assays

• Comparison of staining patterns across multiple antibodies targeting different epitopes

How can UBR5 antibodies be optimized for studying its role in antiviral immunity?

Investigating UBR5's role in antiviral immunity requires specialized experimental approaches:

Cell models and stimulation conditions:

• Apply poly(I:C) treatment to mimic viral RNA and monitor UBR5 expression/localization changes

• Use RNA virus infection models (e.g., EMCV, SARS-CoV-2) for functional studies

• Compare responses in wild-type vs. UBR5 knockout cells generated using CRISPR-Cas9

Co-localization studies:

• Use dual immunostaining with UBR5 antibodies and markers for:

  • RLRs (RIG-I, MDA5) to study sensor interactions

  • TRIM28 to investigate epigenetic regulation

  • Viral proteins to detect potential direct interactions

ChIP-seq experimental design:

• Use UBR5 antibodies for ChIP followed by sequencing to map genomic binding sites

• Focus on promoter regions of RLR genes (e.g., γFBG gene)

• Compare binding patterns before and after viral stimulation

• Perform parallel ChIP for TRIM28 and histone modifications (H3K9me3) to correlate with UBR5 binding

Ubiquitination analysis:

• Immunoprecipitate TRIM28 and probe for K63-linked ubiquitination in the presence/absence of UBR5

• Use UBR5 antibodies for IP followed by ubiquitin detection to identify other substrates

• Consider denaturing conditions (6M urea) to disrupt non-covalent interactions

Experimental controls:

• Include MDA5−/− and MAVS−/− cells as positive controls for antiviral pathway disruption

• Compare DNA virus (e.g., HSV-1) vs. RNA virus responses to establish specificity

What specialized techniques are needed to study UBR5's ubiquitination activity using antibodies?

Studying UBR5's E3 ligase activity requires specialized ubiquitination assays:

In vitro ubiquitination assays:

• Use immunopurified UBR5 (e.g., via TAP purification)

• Combine with E1, E2 enzymes, ubiquitin, ATP, and substrate protein

• Detect ubiquitinated products via western blot using anti-ubiquitin or substrate-specific antibodies

• Consider different ubiquitin linkage-specific antibodies (K48 vs. K63)

In vivo ubiquitination analysis:

• Transfect cells with His-tagged ubiquitin and putative substrate

• Perform nickel-NTA pulldown under denaturing conditions (6M urea)

• Immunoblot for substrate protein to detect ubiquitination

• Alternative approach: IP the substrate and immunoblot for ubiquitin

TUBE (Tandem Ubiquitin Binding Entity) assays:

• Use TUBEs to enrich for ubiquitinated proteins from cell lysates

• Immunoblot for UBR5 substrates (e.g., TRIM28, CDK9, MYC)

• Compare wild-type and catalytically inactive UBR5 mutants

Domain-specific analysis:

• Compare full-length UBR5 with HECT domain deletion mutants to assess E3 ligase dependency

• Use antibodies specific to different UBR5 domains to investigate domain-specific interactions

Technical considerations:

• Include proteasome inhibitors (MG132) to prevent degradation of ubiquitinated substrates

• Use deubiquitinating enzyme inhibitors (N-ethylmaleimide)

• Consider cycloheximide chase experiments to assess protein stability and half-life

How should researchers approach studying UBR5 in cancer models?

Investigating UBR5 in cancer contexts requires specific methodological considerations:

Expression analysis in clinical samples:

• Use validated UBR5 antibodies for IHC on patient tissue microarrays

• Correlate UBR5 expression with clinicopathological features (e.g., TNM stage, survival)

• Compare tumor tissue with adjacent non-tumor tissues as controls

Functional studies in cancer cell lines:

• Generate stable UBR5 knockdown/knockout lines using shRNA or CRISPR-Cas9

• Create UBR5-overexpressing lines to assess oncogenic potential

• Assess effects on:

  • Cell proliferation

  • Cell cycle distribution (flow cytometry)

  • Apoptosis (Bcl-2 expression, Annexin V staining)

  • Migration/invasion

  • Radiosensitivity

Signaling pathway analysis:

• Study UBR5's effects on various cancer-related pathways:

  • p38/MAPK signaling (phosphorylation status)

  • MYC degradation and stability

  • AKT-mTOR pathway

  • DNA damage response

Mechanistic investigations:

• Identify cancer-specific substrates via IP-MS approaches

• Perform domain-specific mutant studies (HECT domain vs. MLLE domain)

• Investigate co-amplification of UBR5 with oncogenes like MYC

In vivo models:

• Use conditional Ubr5 knockout mice to study tissue-specific cancer development

• Xenograft models with UBR5-manipulated cancer cells

• Consider radiation treatments in mouse models to assess UBR5's role in radioresistance

What are the critical controls when investigating UBR5 with antibodies in different experimental systems?

Robust experimental design requires appropriate controls:

Genetic controls:

• UBR5 knockout cells (complete knockout or domain-specific deletions)

• Graded knockdown using different siRNAs/shRNAs with varying efficiency

• Rescue experiments with wild-type UBR5 or domain mutants

Antibody controls:

• IgG isotype controls for IP experiments

• Blocking peptide competition

• Multiple antibodies targeting different epitopes

• Secondary antibody-only controls

Domain-specific controls:

• HECT domain mutants (catalytically inactive) to differentiate ubiquitin ligase-dependent vs. independent functions

• MLLE domain mutants to assess PABC-dependent functions

• UBR domain mutants to evaluate N-recognin activity

Cell type-specific considerations:

• Test across multiple cell lines representing different tissues

• Compare immune cells (e.g., Jurkat) vs. epithelial cells

• Consider species-specific differences (human vs. mouse systems)

Stimulus-specific controls:

• Compare RNA virus vs. DNA virus responses

• Include positive controls for pathway activation (e.g., poly(I:C), IFN-β)

• Time-course experiments to capture dynamic responses

How can researchers use UBR5 antibodies to investigate its interactions with transcriptional machinery?

Studying UBR5's role in transcriptional regulation requires specific approaches:

Chromatin-associated protein analysis:

• Perform cellular fractionation to isolate chromatin-bound UBR5

• Use ChIP with UBR5 antibodies to identify genomic binding sites

• Sequential ChIP (re-ChIP) to identify co-occupancy with transcription factors (e.g., TFIIS)

Transcription factor interaction studies:

• Co-IP UBR5 with RNA polymerase II components

• Investigate CDK9 interaction and ubiquitination patterns

• Examine association with chromatin remodeling complexes

Promoter-specific investigations:

• ChIP-qPCR focusing on specific gene promoters (e.g., RLR genes, γFBG)

• Compare UBR5 binding before and after stimulus (e.g., viral infection)

• Correlate with changes in histone modifications

Transcriptional output analysis:

• Luciferase reporter assays (e.g., ISRE-Luc) to measure UBR5's impact on transcription

• RNA-seq in UBR5 wild-type vs. knockout cells

• Compare effects on different promoters (e.g., IFN-β vs. ISG promoters)

Technical considerations:

• Use cross-linking conditions optimized for detecting transient interactions

• Consider nuclease digestion optimization for ChIP applications

• Perform size fractionation to identify UBR5-containing complexes

Why might Western blotting for UBR5 be challenging and how can these issues be addressed?

Western blotting for UBR5 presents several technical challenges:

High molecular weight detection issues:

• UBR5 is a large protein (~300 kDa), requiring specialized gel separation and transfer protocols

• Use low percentage gels (6-8%) or gradient gels

• Extend transfer time or use specialized transfer systems for high molecular weight proteins

• Consider wet transfer rather than semi-dry transfer

Antibody selection considerations:

• Different antibodies target distinct epitopes, potentially giving variable results

• Compare N-terminal vs. C-terminal targeting antibodies

• Test multiple antibodies to confirm results

• Verify specificity using UBR5 knockout or knockdown samples

Sample preparation optimization:

• Use phosphatase inhibitors to preserve potential phosphorylation sites

• Include protease inhibitors to prevent degradation

• Consider non-denaturing conditions for conformation-specific antibodies

• For ubiquitination studies, include deubiquitinase inhibitors (N-ethylmaleimide)

Troubleshooting weak signals:

• Optimize primary antibody concentration (typically 1:1000 dilution)

• Extend incubation time (overnight at 4°C)

• Use signal enhancement systems

• Consider enrichment of UBR5 by immunoprecipitation before Western blotting

Validation strategies:

• Confirm band identity using siRNA knockdown

• Verify against recombinant protein controls

• Test across multiple cell lines known to express UBR5

What approaches can resolve conflicting results when studying UBR5 function with different antibodies?

Conflicting results can emerge when using different UBR5 antibodies due to several factors:

Epitope-specific differences:

• Different antibodies target distinct domains (HECT domain, UBR domain, MLLE domain)

• Domain-specific antibodies may detect specific conformations or post-translational modifications

• Some epitopes may be masked in protein complexes

Methodological approach:

• Map the epitopes recognized by each antibody

• Test multiple antibodies targeting different regions in parallel

• Verify with genetic approaches (siRNA, CRISPR/Cas9) focusing on different UBR5 regions

• Compare polyclonal vs. monoclonal antibodies

Function-specific considerations:

• Some UBR5 functions are E3 ligase-dependent while others are scaffolding/structural roles

• HECT domain antibodies may be more relevant for studying ubiquitination functions

• MLLE domain antibodies for PAM2-related interactions

Controls to resolve discrepancies:

• Generate domain-specific UBR5 knockouts to validate antibody specificity

• Use reciprocal approaches (e.g., substrate IP vs. UBR5 IP)

• Employ alternative techniques (mass spectrometry, proximity labeling)

• Validate with recombinant UBR5 domain proteins

Data interpretation:

• Consider that discrepancies may reflect biological reality rather than technical issues

• Different UBR5 isoforms or post-translational modifications may exist

• UBR5 may form different protein complexes in different contexts

How should researchers interpret UBR5 antibody data in the context of its multiple cellular functions?

UBR5's diverse roles require careful interpretation of antibody-based data:

Context-dependent function:

• UBR5 functions differently in viral infection vs. cancer vs. DNA damage contexts

• Interpret results within specific biological contexts

• Consider stimulus-specific effects (e.g., radiation, viral infection, growth factors)

Subcellular localization:

• UBR5 may function in different cellular compartments (nucleus, cytoplasm)

• Use fractionation approaches with appropriate compartment markers

• Verify with immunofluorescence microscopy using multiple antibodies

Cell type-specific considerations:

• UBR5 functions may vary between immune cells and epithelial cells

• Compare results across multiple cell types

• Consider tissue-specific interactors

Pathway integration:

• Connect UBR5 findings to established pathways (antiviral, DNA damage, etc.)

• Use pathway inhibitors to dissect specific functions

• Consider compensatory mechanisms in knockout models

Temporal dynamics:

• UBR5 functions may be transient or induced by specific stimuli

• Perform time-course experiments after stimulation

• Consider protein half-life and turnover in different conditions

What are the optimal conditions for immunoprecipitating UBR5 and its interaction partners?

Successful UBR5 immunoprecipitation requires specific optimization:

Lysis buffer optimization:

• For general interactions: Use buffers containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, protease inhibitors

• For chromatin-associated complexes: Include nuclease treatment

• For ubiquitination studies: Add deubiquitinase inhibitors (N-ethylmaleimide)

• For weak interactions: Reduce salt concentration and use milder detergents

Antibody selection and application:

• Amount: Typically 10 μg of antibody per IP from standard lysate volume

• Incubation: Overnight at 4°C with rotation

• Pre-clearing: Use protein A/G beads to reduce background

• Consider cross-linking antibody to beads to avoid IgG contamination

Washing conditions:

• More stringent washes for highly specific interactions

• Gentler washes to preserve weaker interactions

• Gradual salt concentration reduction in sequential washes

• Include detergent in initial washes, buffer only in final washes

Elution strategies:

• Peptide competition for gentle elution

• SDS sample buffer for complete elution

• Native elution for downstream functional assays

Verification approaches:

• Reciprocal IP (IP interaction partner, detect UBR5)

• Input control (5-10% of starting material)

• IgG control to identify non-specific binding

• Size-exclusion chromatography to verify complex formation

What specialized approaches are needed for detecting UBR5 in different tissue types?

Tissue-specific detection of UBR5 requires tailored approaches:

Tissue preparation optimization:

• Fixation: Optimize fixation time for different tissues (typically 24-48h in 10% neutral buffered formalin)

• Antigen retrieval: Test different methods (heat-induced vs. enzymatic) and buffers (citrate vs. EDTA)

• Blocking: Use tissue-specific blocking reagents to reduce background

IHC protocol adjustments:

• Primary antibody concentration: Titrate for each tissue type

• Incubation time: Typically overnight at 4°C, but may require optimization

• Detection systems: Consider signal amplification for tissues with low expression

• Counterstaining: Adjust based on tissue morphology

Controls for tissue analysis:

• Include UBR5-high and UBR5-low expressing tissues as controls

• Use tissue from knockout models when available

• Perform peptide competition controls

• Compare with RNAscope or in situ hybridization for mRNA localization

Multiplex approaches:

• Co-stain with cell type-specific markers

• Combine with markers for UBR5 substrates or interactors

• Use spectral imaging to reduce autofluorescence in certain tissues

• Consider cycling techniques for multiple antigen detection

Image analysis considerations:

• Establish scoring systems for UBR5 expression levels

• Use digital pathology tools for quantification

• Account for heterogeneity within tissue samples

• Consider subcellular localization patterns

UBR5 Antibody Selection Guide Based on Research Applications

Troubleshooting Guide for Common UBR5 Antibody Issues

UBR5 Domain-Specific Functions and Antibody Applications