UBR7 Antibody

What is UBR7 and why is it significant in research?

UBR7 (previously known as C14orf130) is a 425-amino-acid putative E3 ubiquitin-protein ligase that functions as a component of the N-end rule pathway. This pathway is a ubiquitin-dependent system where E3 ligases called N-recognins (including UBR1, UBR2, and UBR7) lead to target protein ubiquitination and subsequent degradation. UBR7 contains both PHD-type and UBR-type zinc finger domains, which are critical for its function .

Recent research has established UBR7 as a critical regulator of nucleotide metabolism through interaction with phosphoribosyl pyrophosphate synthetase (PRPS) enzyme complexes. Its significance has been highlighted in oncology research, particularly in NOTCH1-driven T-cell acute lymphoblastic leukemia (T-ALL), where UBR7 is overexpressed and supports cancer cell proliferation by promoting nucleotide biosynthesis .

How do UBR7 antibodies assist in characterizing protein interactions in cellular contexts?

UBR7 antibodies serve as essential tools for investigating UBR7's interactions with protein complexes such as PRPS enzyme complexes. Methodologically, researchers can use coimmunoprecipitation (Co-IP) approaches with UBR7 antibodies to pull down protein complexes containing UBR7 and its binding partners.

For example, researchers have used UBR7 antibodies in Co-IP experiments to demonstrate that endogenous UBR7 strongly associates with endogenous PRPS1 and PRPS2 . These interactions have revealed UBR7's role in regulating nucleotide metabolism. The specificity of the antibody ensures reliable detection of these protein-protein interactions while minimizing background signals that could confound research findings.

What are the validated applications for UBR7 antibody in research settings?

Current UBR7 antibodies have been validated for several critical research applications:

For Western blotting applications, UBR7 antibody typically detects a band at 48-60 kDa, corresponding to the calculated molecular weight of 48 kDa . When using the antibody for novel applications or in different cellular contexts, researchers should conduct preliminary titration experiments to determine optimal antibody concentration and validate specificity through appropriate controls.

How should researchers optimize Western blot protocols when using UBR7 antibody?

For optimal Western blot results with UBR7 antibody, researchers should consider the following methodological approach:

• Sample preparation: Prepare whole-cell lysates in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors.

• Protein quantification: Determine total protein content using bicinchoninic acid (BCA) reagent to ensure equal loading across samples.

• Gel selection: Use 10-12% SDS-PAGE gels for optimal resolution of UBR7 (48-60 kDa).

• Transfer conditions: Transfer proteins to nitrocellulose or PVDF membranes at appropriate voltage/time to ensure complete transfer of proteins in UBR7's molecular weight range.

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute UBR7 antibody between 1:2000 and 1:10000 in blocking buffer. Incubate overnight at 4°C with gentle rocking .

• Visualization: For enhanced sensitivity when detecting endogenous UBR7, consider using HRP-labeled UBR7 antibody as described in published protocols, which can be prepared using antibody labeling kits .

What are the key considerations when designing coimmunoprecipitation experiments with UBR7 antibody?

When designing coimmunoprecipitation experiments to investigate UBR7 protein interactions:

• Buffer selection: For standard Co-IP, use lysis buffer containing 20 mM HEPES (pH 7.9), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, supplemented with protease and phosphatase inhibitors .

• For interactions involving ubiquitinated proteins: Include deubiquitinase inhibitors such as N-ethylmaleimide (5 mM) in the lysis buffer.

• Antibody amount: Use approximately 0.6 μg of anti-UBR7 antibody per sample, with normal rabbit IgG as a negative control .

• Bead selection: Protein A+G magnetic beads are recommended for rabbit polyclonal UBR7 antibodies.

• Washing conditions: Perform at least five washes with lysis buffer to reduce non-specific binding.

• Protein elution: Elute immunoprecipitated proteins in 2× Laemmli sample buffer before SDS-PAGE analysis.

• Detection optimization: For detecting endogenous interactions, consider using HRP-conjugated UBR7 antibody to minimize background from IgG heavy chains that may overlap with proteins of interest .

How can UBR7 antibody be utilized to investigate NOTCH1-driven leukemia pathways?

UBR7 antibody serves as a crucial tool for investigating NOTCH1-driven leukemia pathways through multiple methodological approaches:

• Expression profiling: Western blotting with UBR7 antibody can establish correlation between UBR7 expression and NOTCH1 activation status across T-ALL cell lines. Research has demonstrated that UBR7 is highly abundant in NOTCH1 intracellular domain (NICD1)-positive cell lines compared to NICD1-negative ones .

• Pathway inhibition studies: UBR7 antibody can be used to monitor protein level changes following γ-secretase inhibitor (GSI) treatment. For example, inhibition of NOTCH1 cleavage by the GSI DAPT in NICD1-driven T-ALL cell lines (DND41 and ALL-SIL) results in down-regulation of UBR7, which can be detected via Western blotting .

• Correlation analysis: Quantitative Western blot analysis with UBR7 antibody has revealed that UBR7 protein levels are highly correlated with both PRPS1 and PRPS2 levels in T-ALL cell lines, with correlation coefficients of 0.84 and 0.68, respectively .

How do you interpret UBR7 expression patterns in relation to PRPS enzyme complex regulation?

When analyzing UBR7 expression patterns in relation to PRPS enzyme complex regulation:

• Expression correlation analysis: Research has shown that UBR7 protein levels positively correlate with PRPS1 and PRPS2 levels in T-ALL cell lines, suggesting coordinated regulation .

• Knockdown effects: UBR7 knockdown studies demonstrate that depletion of UBR7 leads to decreased levels of both PRPS1 and PRPS2 proteins, without corresponding reduction in their transcript levels. This indicates post-transcriptional regulation of PRPS enzymes by UBR7 .

• Functional pathway analysis: Nucleotide metabolism assays following UBR7 depletion show reduced de novo pyrimidine synthesis in T-ALL cell lines, consistent with UBR7's role in maintaining PRPS enzyme complex functionality .

• Patient sample correlation: Analysis of patient data from the pediatric cancer genome project portal shows significant up-regulation of UBR7 and PRPS2 in NOTCH1-mutated T-ALL compared to NOTCH1 wild-type T-ALL, providing clinical relevance to experimental findings .

What are the strategies for investigating UBR7's role in protein ubiquitination and degradation pathways?

To investigate UBR7's role in protein ubiquitination and degradation:

• Denaturing immunoprecipitation: Use a specialized protocol to detect ubiquitinated proteins by first lysing cells in buffer containing 1% SDS, heating the lysates at 95°C for 10 minutes, and then diluting to 0.1% SDS before immunoprecipitation .

• Ubiquitination assays: When conducting ubiquitination studies involving UBR7, include 5 mM N-ethylmaleimide in lysis buffers to inhibit deubiquitinase activity and preserve ubiquitination modifications .

• Domain-specific analysis: Utilize UBR7 truncation mutants to determine which domains are required for specific interactions and functions. For example, studies have shown that N-terminal deletion mutants (117-425 and 217-425) retain the ability to interact with PRPS1 and PRPS2, while C-terminal truncation eliminates this interaction .

• Target validation: For suspected UBR7 ubiquitination targets like PRPSAP1, conduct proteasome inhibition experiments (e.g., with MG132) to accumulate ubiquitinated forms of target proteins for detection.

What controls are essential when validating UBR7 antibody specificity in experimental contexts?

When validating UBR7 antibody specificity:

• Negative controls: Include UBR7 knockdown samples in Western blotting experiments to confirm reduction or disappearance of the specific band.

• Positive controls: Include cell lines known to express UBR7 at detectable levels, such as HeLa or HepG2 cells, which have been validated with existing UBR7 antibodies .

• Immunoprecipitation controls: Always include an isotype-matched IgG control (e.g., normal rabbit IgG for rabbit polyclonal UBR7 antibodies) to distinguish specific from non-specific pull-downs .

• Multiple detection methods: Confirm findings using alternative antibodies targeting different epitopes of UBR7, or tagged UBR7 constructs in recombinant expression systems.

• Signal specificity: For Co-IP experiments involving UBR7, verify that the interaction is maintained across different lysis conditions but abolished by appropriate controls (e.g., domain mutants known to disrupt the interaction).

How do researchers differentiate between UBR7's chromatin-associated functions and its role in nucleotide metabolism?

Differentiating between UBR7's chromatin-associated functions and its role in nucleotide metabolism requires specific methodological approaches:

• Subcellular fractionation: Separate chromatin-bound proteins from soluble nuclear and cytoplasmic fractions before analyzing UBR7 interactions. Evidence suggests that UBR7-PRPS interaction occurs in the soluble non-chromatin fraction .

• Domain-function correlation: Utilize UBR7 PHD finger mutants deficient in chromatin association to assess their ability to interact with PRPS. Research indicates that these mutants actually have enhanced ability to interact with PRPS, suggesting separation of these functions .

• Functional assays: Employ nucleotide metabolism assays (such as isotope-labeled precursor incorporation) to distinguish metabolic effects from transcriptional effects. Studies show that UBR7 knockdown reduces de novo nucleotide synthesis without affecting precursor uptake .

• Rescue experiments: Perform functional rescue experiments with UBR7 mutants that specifically lack either chromatin binding or PRPS interaction capabilities to determine which function is essential for specific cellular phenotypes.

What analytical approaches should be used when studying UBR7's impact on nucleotide biosynthesis pathways?

For analyzing UBR7's impact on nucleotide biosynthesis:

• Metabolic tracing: Use isotope-labeled precursors such as 14C-glycine and 3H-hypoxanthine (for purine synthesis) or 14C-aspartate and 3H-uridine (for pyrimidine synthesis) to trace nucleotide biosynthesis pathways. This approach can distinguish between de novo and salvage pathways .

• Pathway-specific analysis: Separately analyze purine and pyrimidine synthesis pathways, as UBR7 may differentially impact these processes. For instance, research has shown that UBR7 knockdown reduces de novo pyrimidine synthesis in certain cell lines while affecting both de novo and salvage pathways in others .

• Uptake controls: Include precursor uptake measurements as controls to ensure that observed changes in nucleotide synthesis are not due to altered cellular uptake of precursors .

• Protein-level correlation: Quantify the correlation between UBR7 levels and PRPS enzyme levels across multiple samples. Strong correlation coefficients (e.g., 0.84 with PRPS1 and 0.68 with PRPS2 in T-ALL cell lines) support functional relationships .

How can UBR7 antibody be used to investigate links between NOTCH1 signaling and metabolic regulation?

To investigate links between NOTCH1 signaling and metabolic regulation using UBR7 antibody:

• Temporal analysis: Monitor UBR7 protein levels using Western blotting at different time points following NOTCH1 modulation (activation or inhibition) to establish the kinetics of regulatory relationships.

• Pathway inhibition studies: Use γ-secretase inhibitors like DAPT or DBZ to block NOTCH1 cleavage, and then monitor changes in UBR7, PRPS1, and PRPS2 protein levels via Western blotting. This approach has revealed that NOTCH1 inhibition causes down-regulation of all three proteins in T-ALL cell lines .

• Patient stratification: Apply UBR7 antibody in immunoblotting of patient samples to correlate UBR7 expression with NOTCH1 mutation status and clinical outcomes. Analysis of pediatric cancer genome project data shows significant up-regulation of UBR7 in NOTCH1-mutated T-ALL compared to wild-type .

• Metabolic impact assessment: Combine UBR7 detection with metabolic assays to establish connections between NOTCH1 activity, UBR7 expression, and nucleotide metabolism in cancer cells.

What are the methodological considerations when investigating UBR7 domain-specific functions?

When investigating UBR7 domain-specific functions: