UPF3B Antibody, HRP conjugated

What is UPF3B and what cellular process is it involved in?

UPF3B (Regulator of nonsense transcripts 3B) is a key protein involved in nonsense-mediated decay (NMD), a quality control mechanism that eliminates mRNAs containing premature termination codons (PTCs). UPF3B functions by associating with the nuclear exon junction complex (EJC) and serving as a link between the EJC core and NMD machinery . In this process, UPF3B recruits UPF2 at the cytoplasmic side of the nuclear envelope, leading to the formation of a UPF1-UPF2-UPF3 surveillance complex that activates NMD . UPF3B cooperates with UPF2 to stimulate both ATPase and RNA helicase activities of UPF1, which are essential for NMD function . Recent studies have also shown that UPF3B has direct involvement in the termination reaction in human cell extracts, indicating its multifunctional role in mRNA quality control .

What are the specifications of UPF3B Antibody, HRP conjugated?

The UPF3B Antibody, HRP conjugated is a Rabbit Polyclonal antibody specifically designed for the detection of Human UPF3B . The antibody is directly conjugated to horseradish peroxidase (HRP), which facilitates direct detection without the need for secondary antibodies in applications such as ELISA .

How should UPF3B Antibody, HRP conjugated be stored and handled for optimal performance?

For optimal performance and longevity, the UPF3B Antibody, HRP conjugated should be aliquoted and stored at -20°C . Repeated freeze/thaw cycles should be avoided as they can compromise antibody activity and specificity . The antibody is provided in a liquid form containing 0.01 M PBS (pH 7.4) with 0.03% Proclin-300 and 50% glycerol, which helps maintain stability during storage . When working with the antibody, it's advisable to keep it on ice and minimize exposure to room temperature. HRP conjugation makes the antibody sensitive to reducing agents and light, so these should be avoided during handling. For long-term storage, small aliquots are recommended to prevent multiple freeze-thaw cycles that could degrade the HRP enzyme activity.

What detection methods work best with UPF3B Antibody, HRP conjugated?

The UPF3B Antibody, HRP conjugated is specifically tested and validated for ELISA applications . The direct HRP conjugation provides significant advantages for this method, including:

• Elimination of secondary antibody requirements, which reduces background and cross-reactivity issues

• Streamlined protocols with fewer incubation and washing steps

• Increased sensitivity through direct signal generation

• Reduced experimental variability by eliminating secondary antibody binding efficiency as a variable

For ELISA applications, the recommended approach involves coating plates with the target antigen, blocking non-specific binding sites, then adding the HRP-conjugated UPF3B antibody. After washing, a chromogenic or chemiluminescent substrate can be added for detection. While the manufacturer recommends determining optimal dilutions experimentally, typical starting dilutions for HRP-conjugated antibodies in ELISA range from 1:1000 to 1:5000 .

How can I validate UPF3B antibody specificity in UPF3B knockout models?

Validating antibody specificity is crucial for reliable research outcomes, particularly when studying proteins with paralogs like UPF3B and UPF3A. Based on research findings, the following stepwise approach is recommended:

• Generate UPF3B knockout cell lines using CRISPR-Cas9 technology targeting the start codon of UPF3B, as demonstrated in HCT116 cells .

• Confirm complete knockout through genomic PCR, RT-PCR, and Western blotting. In successful knockouts, Western blotting should show complete absence of UPF3B protein band .

• Perform immunoprecipitation experiments with the UPF3B antibody in wild-type and knockout cells to assess specificity. In knockout cells, there should be no detectable UPF3B pull-down .

• Check for cross-reactivity with UPF3A, especially since UPF3A is upregulated approximately 3.5-fold at the protein level in UPF3B knockout cells . This upregulation provides an excellent control for antibody specificity.

• Include positive controls by transfecting knockout cells with GFP-tagged UPF3B constructs, which should restore antibody detection .

Recent studies have successfully validated UPF3B antibodies using these approaches, demonstrating that properly validated antibodies show no signal in UPF3B knockout cells despite the increased expression of the paralog UPF3A .

What experimental controls should be included when studying NMD with UPF3B antibodies?

When studying nonsense-mediated decay using UPF3B antibodies, comprehensive controls are essential for reliable data interpretation:

• Genetic controls:

  • Wild-type cells as baseline controls

  • UPF3B knockout/knockdown cells to assess UPF3B-dependent effects

  • UPF1 knockdown as a positive control for NMD inhibition, since UPF1 is required for all NMD

  • UPF3A knockout/knockdown to differentiate paralog-specific effects

  • Double knockdown/knockout of UPF3A and UPF3B to assess redundancy

• Expression controls:

  • Monitor expression levels of known NMD target transcripts (e.g., RSRC2, SRSF2, ZFAS1, ILK, NFKBIB, RPS9, SRSF3)

  • Include non-NMD regulated transcripts as negative controls

• Functional controls:

  • PTC-containing reporter constructs (e.g., β-globin with PTC at codon 39) to measure NMD efficiency

  • PTC-free versions of the same reporters as negative controls

• Technical controls for antibody specificity:

  • Isotype controls matching the UPF3B antibody class and species

  • Secondary antibody-only controls when using unconjugated primary antibodies

  • Pre-absorption with recombinant UPF3B to confirm specificity

This comprehensive control strategy allows for accurate assessment of both antibody performance and biological effects in NMD research.

How can UPF3B Antibody be used to investigate EJC-UPF complex formation in NMD?

The UPF3B Antibody can be instrumental in studying the exon junction complex (EJC)-UPF interaction, which is central to NMD function. Advanced experimental approaches include:

• Co-immunoprecipitation (Co-IP) studies:
  UPF3B antibodies can be used to pull down UPF3B-containing complexes followed by immunoblotting for EJC components (EIF4A3, MAGOH, Y14, CASC3) and other UPF proteins (UPF1, UPF2) . Research has shown that UPF3B directly interacts with the EJC and serves as a bridge to other NMD factors .

• Tandem affinity purification:
  Using a dual-tagging strategy (e.g., FLAG-MAGOH and MYC-UPF2) allows isolation of intact EJC-UPF complexes through sequential immunoprecipitation steps . This approach revealed that in wild-type cells, UPF3B is the major paralog incorporated into the EJC-UPF complex, while UPF3A incorporation increases dramatically in UPF3B knockout cells .

• RNA-dependent complex analysis:
  Including RNase treatment controls in immunoprecipitation experiments helps distinguish direct protein-protein interactions from RNA-mediated associations. Studies show that the enhanced UPF1-UPF3A association in UPF3B-deficient cells is independent of RNA .

• Proximity ligation assays:
  Using UPF3B antibodies in conjunction with antibodies against EJC components allows visualization and quantification of complex formation in situ, providing spatial information about where these complexes form within the cell.

What is the relationship between UPF3A and UPF3B in NMD regulation, and how can antibodies help elucidate this?

The relationship between UPF3A and UPF3B in NMD regulation has been a subject of debate, with recent research providing clarification. UPF3B Antibody, in combination with UPF3A-specific antibodies, has helped reveal several key aspects of this relationship:

Research using antibodies against both paralogs has reconciled previously conflicting findings, concluding that in human cells, UPF3A is dispensable for NMD under normal conditions but can promote NMD when UPF3B is depleted .

How does UPF3B knockout affect global mRNA expression and NMD efficiency?

UPF3B knockout has complex effects on global mRNA expression and NMD efficiency that have been characterized through antibody-based detection and transcriptome analysis:

These findings illustrate the partial redundancy in the NMD pathway and highlight the value of antibody-based detection methods in characterizing the molecular consequences of UPF3B deficiency.

What are the common pitfalls when using HRP-conjugated antibodies for UPF3B detection, and how can they be addressed?

When working with HRP-conjugated UPF3B antibodies, researchers commonly encounter several technical challenges that can be systematically addressed:

• High background signal:

  • Cause: Insufficient blocking, excessive antibody concentration, or cross-reactivity

  • Solution: Optimize blocking conditions (5% BSA or milk is generally effective); titrate antibody concentration; include additional washing steps with 0.05-0.1% Tween-20 in PBS

• Weak or no signal detection:

  • Cause: Antibody degradation, insufficient antigen, or suboptimal substrate

  • Solution: Verify HRP activity using a direct enzyme assay; increase sample concentration; optimize substrate incubation time; ensure proper storage at -20°C in aliquots to prevent repeated freeze-thaw cycles

• Non-specific bands in Western blotting:

  • Cause: Cross-reactivity with UPF3A (particularly problematic since both proteins are similar in size)

  • Solution: Include UPF3B knockout controls; use more stringent washing conditions; validate specificity using immunoprecipitation followed by mass spectrometry

• Poor reproducibility:

  • Cause: Variability in experimental conditions or antibody performance

  • Solution: Standardize protocols; include positive controls; use internal loading controls; consider automated systems for consistent washing and development

• Signal quenching in multiplex assays:

  • Cause: HRP substrate products can interfere with fluorescent signals

  • Solution: Carefully sequence detection steps; consider using compatible substrates or sequential detection approaches

Following these troubleshooting strategies can significantly improve the reliability and sensitivity of UPF3B detection using HRP-conjugated antibodies.

How can UPF3B Antibody, HRP conjugated be optimized for detecting low-abundance UPF3B in different cell types?

Detecting low-abundance UPF3B requires methodological optimizations tailored to different cell types and experimental contexts:

• Sample enrichment strategies:

  • Perform nuclear/cytoplasmic fractionation to concentrate UPF3B in relevant cellular compartments

  • Use immunoprecipitation as a pre-enrichment step before detection

  • Employ subcellular fractionation to isolate mRNA processing bodies where NMD factors might be concentrated

• Signal amplification techniques:

  • Utilize enhanced chemiluminescence (ECL) substrates specifically designed for low-abundance proteins

  • Implement tyramide signal amplification (TSA) to enhance HRP signal by depositing multiple reactive tyramide molecules

  • Consider enzyme-linked enhancement protocols that use cascading enzyme reactions to amplify signal

• Cell-type specific considerations:

  • For neuronal cells, which express higher levels of UPF3B, standard protocols may be sufficient

  • For stem cells, which have more variable UPF3B expression, longer incubation times and enhanced blocking may be necessary

  • For blood cells, additional red blood cell lysis steps and specialized lysis buffers are recommended

• Detection method optimization:

  • For ELISA: increase sample volume, extend antibody incubation time (overnight at 4°C), and use high-sensitivity substrates

  • For Western blotting: use PVDF membranes for better protein retention, transfer at lower voltage for longer periods, and extend primary antibody incubation

• Validation in multiple cell lines:

  • Compare detection sensitivity across different cell types including HCT116, HEK293, and HeLa cells, which have been well-characterized for UPF3B expression

  • Include positive controls of cells transfected with UPF3B expression constructs

These optimizations have proven effective in detecting physiological levels of UPF3B across diverse experimental systems.

What strategies can resolve data inconsistencies when comparing UPF3B antibody results from different experimental platforms?

Resolving data inconsistencies between different experimental platforms using UPF3B antibodies requires a systematic approach:

• Standardization of reference materials:

  • Use recombinant UPF3B protein standards across all platforms

  • Implement common positive and negative controls (e.g., UPF3B overexpression and knockout samples)

  • Develop a standard curve for quantitative assays using the same reference material

• Cross-platform validation protocol:

  • When comparing results between ELISA and Western blotting, normalize data to total protein concentration

  • For discrepancies between immunofluorescence and biochemical assays, verify subcellular localization with fractionation studies

  • When comparing mass spectrometry data with antibody-based detection, focus on peptide sequences within the antibody epitope region

• Addressing technology-specific biases:

  • For HRP-based detection systems, account for potential substrate depletion in high-expression samples

  • In multiplexed assays, test for antibody cross-reactivity and signal interference

  • When combining data from different imaging platforms, standardize image acquisition and analysis parameters

• Statistical approaches for data integration:

  • Apply rank-based normalization when combining data from different platforms

  • Use paired experimental designs when possible

  • Implement Bland-Altman plots to identify systematic differences between methods

• Biological validation of unexpected results:

  • Verify discrepant findings using orthogonal techniques (e.g., RNA-seq, qPCR)

  • Consider cell-type specific differences in UPF3B function and expression

  • Assess the impact of UPF3A compensation, which varies between cell types

Researchers have successfully applied these approaches to reconcile initially conflicting data about UPF3A/B functions in NMD, ultimately revealing their context-dependent roles .

How is UPF3B involved in neurological disorders, and what research methodologies can investigate this connection?

UPF3B has been implicated in several neurological disorders, predominantly through its role in regulating the neuronal transcriptome via nonsense-mediated mRNA decay. The UPF3B Antibody, HRP conjugated can facilitate investigations into these connections through multiple research methodologies:

• Clinical correlations:
  UPF3B mutations have been associated with neurodevelopmental disorders including X-linked intellectual disability, autism spectrum disorders, and schizophrenia . The OMIM database entry (300298) for UPF3B confirms these associations . Antibody-based techniques can characterize UPF3B expression patterns in patient-derived cells and tissues.

• Molecular mechanisms in neuronal models:

  • Neuronal differentiation studies: Track UPF3B expression and localization during differentiation of neural progenitors

  • Synapse formation analysis: Examine UPF3B localization at synapses using immunocytochemistry

  • mRNA surveillance in neurons: Identify neuron-specific NMD targets regulated by UPF3B using CLIP-seq combined with UPF3B immunoprecipitation

• Disease model systems:

  • Patient-derived iPSCs: Generate neurons from patient-derived induced pluripotent stem cells carrying UPF3B mutations

  • CRISPR-engineered models: Create isogenic cell lines with specific UPF3B mutations found in neurological disorders

  • Conditional knockout animals: Study brain region-specific effects of UPF3B deletion

• Functional assays:

  • Electrophysiology: Correlate UPF3B expression with neuronal activity in wild-type and mutant models

  • Calcium imaging: Assess neuronal network activity in relation to UPF3B function

  • Behavioral phenotyping: Connect molecular findings to behavioral outcomes in animal models

These methodologies, leveraging UPF3B antibodies for protein detection, have revealed that UPF3B regulation of the neuronal transcriptome is critical for proper brain development and function, with dysregulation contributing to various neurological conditions.

What insights have emerged from studying UPF3B in cancer research, and how can antibody-based approaches advance this field?

Research on UPF3B in cancer contexts has revealed several significant insights, with antibody-based approaches offering powerful tools for further investigation:

• Dysregulation of NMD in cancer:
  Studies using UPF3B antibodies have shown altered expression patterns of UPF3B across different cancer types, suggesting dysregulation of the NMD pathway. The HCT116 colorectal carcinoma cell line, which has a near-diploid genome with only one UPF3B copy, has been particularly valuable for studying UPF3B function in cancer contexts .

• Impact on cancer-related transcripts:

  • UPF3B-dependent NMD regulates PTC-containing transcripts of genes involved in cancer progression, including those related to cell cycle control and apoptosis

  • Cancer cells may exploit UPF3B-mediated NMD to degrade tumor suppressor transcripts containing PTCs

  • Antibody-based RNA immunoprecipitation has helped identify cancer-specific NMD targets

• Therapeutic implications:

  • Inhibition of NMD factors including UPF3B has been explored as a potential cancer therapeutic strategy

  • UPF3B antibodies can be used to monitor treatment efficacy and target engagement in experimental models

  • The balance between UPF3A and UPF3B may influence cancer cell responses to NMD-targeting therapies

• Advanced antibody-based methodologies for cancer research:

  • Tissue microarrays: High-throughput analysis of UPF3B expression across tumor samples and matched normal tissues

  • Circulating tumor cell analysis: Detection of UPF3B in liquid biopsies as a potential biomarker

  • Proximity ligation assays: Studying UPF3B interactions with other cancer-relevant proteins in situ

  • Chromatin immunoprecipitation: Investigating potential roles of UPF3B in transcriptional regulation in cancer cells

These approaches have contributed to understanding how NMD pathway alterations influence cancer development and progression, offering new perspectives on potential therapeutic targets.

How might emerging technologies enhance the application of UPF3B antibodies in RNA biology research?

Emerging technologies are poised to revolutionize how UPF3B antibodies can be applied in RNA biology research, opening new frontiers in understanding NMD mechanisms:

• Spatial transcriptomics integration:
  Combining UPF3B immunofluorescence with spatial transcriptomics technologies would allow researchers to correlate UPF3B protein localization with the distribution of NMD target transcripts within single cells and tissues. This approach could reveal microenvironmental influences on NMD efficiency and identify specialized NMD compartments within cells.

• Single-molecule imaging techniques:

  • Single-molecule FISH combined with immunofluorescence: Track individual UPF3B-mRNA interactions in real-time

  • Live-cell single-molecule tracking: Monitor UPF3B dynamics during NMD using fluorescently tagged antibody fragments

  • Super-resolution microscopy: Resolve UPF3B within macromolecular complexes below the diffraction limit

• Multiplexed protein-RNA detection systems:

  • MERFISH with protein detection: Simultaneously visualize dozens of RNA species alongside UPF3B protein

  • Seq-Scope with antibody staining: Integrate transcriptomic data with UPF3B protein information at subcellular resolution

  • Spatial proteogenomics: Map UPF3B protein distribution in relation to its target transcriptome

• High-throughput functional screening:

  • CRISPR screens with UPF3B antibody readouts: Identify genes that modulate UPF3B function using antibody-based detection

  • Antibody-based biosensors: Develop real-time monitoring systems for NMD activity using FRET-based UPF3B conformational sensors

  • Microfluidic antibody arrays: Screen thousands of conditions for effects on UPF3B expression and activity

• Artificial intelligence applications:
  Machine learning algorithms trained on UPF3B antibody staining patterns could identify subtle phenotypes and predict NMD efficiency across diverse cell types and conditions, accelerating the discovery of context-dependent NMD regulation.

These technological advances promise to transform our understanding of UPF3B function from static snapshots to dynamic, spatially resolved, systems-level insights.

What are the key unresolved questions about UPF3B function that antibody-based research could help address?

Despite significant advances in understanding UPF3B biology, several fundamental questions remain unresolved. Antibody-based research approaches offer promising strategies to address these knowledge gaps:

• Structural dynamics during NMD:
  How does UPF3B change conformation during different stages of the NMD process? Antibodies recognizing distinct epitopes could be used in FRET-based assays or hydrogen-deuterium exchange mass spectrometry to map conformational changes during complex formation with UPF1, UPF2, and the EJC.

• Paralog-specific functions:
  Beyond redundancy, do UPF3A and UPF3B have unique functions in specific cellular contexts? Comparative immunoprecipitation studies using paralog-specific antibodies followed by RNA-seq and proteomics could identify unique binding partners and targets .

• Post-translational regulation:
  How do post-translational modifications regulate UPF3B activity? Phospho-specific and other modification-specific antibodies could map the dynamic regulation of UPF3B in response to cellular signals and stresses.

• Tissue-specific NMD regulation:
  Does UPF3B function differently across tissue types, particularly in the nervous system where it has been implicated in developmental disorders? Immunohistochemistry studies across tissues and developmental stages could reveal tissue-specific expression patterns and interacting partners.

• NMD-independent functions:
  Does UPF3B play roles beyond NMD? In vitro studies have shown that UPF3B can stimulate translation independently of its association with UPF2 and EJC components . Antibody-based proximity labeling techniques could identify novel UPF3B protein interactions in different cellular compartments.

• Therapeutic targeting potential:
  Can modulation of UPF3B activity be therapeutically beneficial in diseases involving aberrant NMD? Antibody-based screening assays could identify compounds that selectively modulate UPF3B activity or specific interactions.

These research directions, enabled by specific and well-characterized UPF3B antibodies, would significantly advance our understanding of mRNA quality control mechanisms and their implications in health and disease.