DBR1 Antibody

What is DBR1 and why is it important in RNA processing?

DBR1 (lariat debranching enzyme) is an enzyme that hydrolyzes the 2'-5' phosphodiester linkage at branch points of excised lariat intron RNA, converting them into linear molecules that can be subsequently degraded . This process is critical for ribonucleotide turnover in cells and represents a rate-limiting step in lariat RNA processing . DBR1 functions primarily in the nucleus where it facilitates the recycling of intronic material following the splicing process . Beyond its role in RNA processing, DBR1 may also participate in retrovirus replication via an RNA lariat intermediate in cDNA synthesis and has been implicated in antiviral cell-intrinsic defense functions in the brainstem .

What applications are DBR1 antibodies suitable for?

DBR1 antibodies have been validated for multiple research applications including Western blotting (WB), immunohistochemistry (IHC), immunocytochemistry (ICC), and immunofluorescence (IF) . These antibodies can detect DBR1 in various human tissues including brain, placenta, and stomach, as well as in cell lines such as HepG2, HeLa, and A431 . Typically, rabbit polyclonal DBR1 antibodies show reactivity with human samples, and some have been confirmed to cross-react with mouse and rat species, though this may vary by product . When selecting a DBR1 antibody for your research, it's important to verify the specific applications and species reactivity that have been validated for your chosen antibody.

How should DBR1 antibodies be stored and handled?

DBR1 antibodies are typically provided in a stabilized solution containing PBS with additives such as sodium azide and glycerol to maintain integrity during storage . For optimal preservation of antibody activity, store the antibody at -20°C as supplied and avoid repeated freeze-thaw cycles that could degrade the protein . Many manufacturers recommend storage for up to 12 months from the date of receipt at -20°C, or 6 months at 2-8°C after reconstitution . When working with the antibody, maintain sterile conditions and aliquot the stock solution to minimize freeze-thaw cycles if frequent use is anticipated. Some formulations include 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain antibody stability during storage .

What are the optimal dilutions for different applications of DBR1 antibodies?

The optimal working dilutions for DBR1 antibodies vary depending on the specific application and the manufacturer's recommendations . For Western blotting (WB), recommended dilutions typically range from 1:500 to 1:10,000, with some products showing optimal results at 1:500-1:5000 . For immunohistochemistry (IHC), a starting dilution of 1:20 to 1:200 is often suggested, with some studies using 1:100 for paraffin-embedded tissues . For immunofluorescence (IF) applications, dilutions ranging from 1:10 to 1:100 are commonly recommended, with specific examples of 1:25 for HeLa cells . For immunocytochemistry (ICC), dilutions around 4 μg/ml have been validated with specific cell lines such as A431 . Always perform titration experiments to determine the optimal antibody concentration for your specific experimental system and detection method.

How should I validate the specificity of a DBR1 antibody for my experimental system?

To validate DBR1 antibody specificity, implement a multi-faceted approach that confirms target recognition . Begin with positive and negative controls—use tissues/cells known to express DBR1 (such as human brain tissue or HepG2 cells) as positive controls, and consider using DBR1 knockout cells, if available, as negative controls . The observed molecular weight by Western blot should be approximately 62 kDa, which corresponds to the predicted size of the DBR1 protein . For immunohistochemistry, compare staining patterns with published literature and confirm cellular localization is predominantly nuclear, consistent with DBR1's function . Consider performing peptide competition assays, where pre-incubation of the antibody with its specific immunogen peptide should abolish signal. For advanced validation, RNA interference (siRNA) targeting DBR1 should reduce both protein levels by Western blot and staining intensity in microscopy applications.

What protocols are recommended for immunofluorescent detection of DBR1?

For optimal immunofluorescent detection of DBR1, begin with appropriate fixation and permeabilization steps as DBR1 is primarily a nuclear protein involved in RNA processing . Use paraformaldehyde (PFA) fixation (typically 4%) followed by permeabilization with Triton X-100 (0.1-0.5%), which has been validated for DBR1 detection in cells such as A431 and HeLa . For immunofluorescence staining, use the DBR1 antibody at dilutions ranging from 1:10 to 1:100, with some protocols specifically recommending 1:25 for HeLa cells or 4 μg/ml for A431 cells . Incubate with the primary antibody overnight at 4°C for best results, followed by appropriate fluorophore-conjugated secondary antibodies (such as Rhodamine-labeled goat anti-rabbit IgG) . Include DAPI or another nuclear counterstain to visualize nuclei, which helps confirm the predominantly nuclear localization of DBR1 . For advanced co-localization studies, consider dual staining with markers of nuclear speckles, as DBR1 has been shown to co-localize with AQR in these structures .

How does DBR1 interact with the spliceosome and what methods can detect these interactions?

DBR1 interacts with components of the spliceosome, particularly during the process of intron turnover after splicing . Research has shown that DBR1 is recruited to branchpoints by AQR (Aquarius), an RNA helicase and component of the intron-binding complex . This interaction can be detected through co-immunoprecipitation followed by mass spectrometry, which has successfully identified AQR and other spliceosomal proteins as DBR1-interacting partners . Western blot analysis of both DBR1 and AQR immunoprecipitations can confirm these interactions . To study the temporal aspects of DBR1's association with spliceosomes, researchers have used ADAR fusions to timestamp lariats, demonstrating that in the absence of DBR1, spliceosomal components remain associated with the lariat for a longer period of time . Enhanced crosslinking and immunoprecipitation (eCLIP) has been employed to map binding sites of AQR and other RNA-binding proteins that may recruit DBR1 to specific introns . Researchers investigating these interactions should include appropriate controls and consider the use of RNase treatment to distinguish RNA-dependent from direct protein-protein interactions.

What is the role of DBR1 in disease models and how can antibodies help investigate this?

DBR1 has been implicated in various disease models, particularly in neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) . Research has shown that inhibition of RNA lariat debranching enzyme can suppress TDP-43 toxicity in ALS disease models, suggesting a potential therapeutic target . DBR1 antibodies can be invaluable tools for investigating these disease connections through several approaches: immunohistochemistry of patient tissues to examine DBR1 expression and localization patterns in diseased versus healthy tissue; Western blotting to quantify DBR1 protein levels in disease models; and immunoprecipitation followed by RNA sequencing to identify disease-specific RNA targets of DBR1 . Additionally, DBR1 has potential roles in retroviral replication and antiviral defense, making it relevant to infectious disease research . Researchers can use DBR1 antibodies in combination with viral infection models to track changes in DBR1 localization, expression, or post-translational modifications in response to infection. The creation of DBR1 knockout cell lines has enabled more detailed investigation of its role in disease mechanisms, revealing that DBR1 depletion leads to exon skipping and spliceosome recycling defects that may contribute to disease pathology .

How can DBR1 antibodies be used to study branchpoint selection and lariat turnover?

DBR1 antibodies serve as crucial tools for investigating branchpoint selection and lariat turnover mechanisms in splicing research . Through RNA immunoprecipitation (RIP) using DBR1 antibodies, researchers can isolate and sequence lariat RNAs that are actively being processed by DBR1, providing insights into substrate preferences . Recent studies have shown that DBR1 preferentially debranches substrates containing canonical U2 binding motifs and exhibits specificity for particular 5' splice site sequences . To study the spatial and temporal dynamics of lariat processing, immunofluorescence with DBR1 antibodies can be combined with RNA FISH (fluorescent in situ hybridization) to visualize co-localization of DBR1 with specific intron lariats . For quantitative analysis of lariat accumulation in response to DBR1 manipulation, researchers can perform RT-qPCR with primers spanning the branch point, following immunoprecipitation with the DBR1 antibody . Advanced approaches include ChIP-seq (chromatin immunoprecipitation sequencing) with DBR1 antibodies to identify genomic regions where co-transcriptional splicing and debranching occur, and proximity ligation assays to detect interactions between DBR1 and other splicing factors at specific genomic loci .

What are common issues with DBR1 antibody experiments and how can they be resolved?

Researchers using DBR1 antibodies may encounter several common issues that can affect experimental outcomes . One frequent problem is high background in immunostaining applications, which can be addressed by optimizing blocking conditions (try different blocking agents such as BSA, normal serum, or commercial blockers), increasing washing steps, and titrating the antibody to find the optimal concentration . Non-specific bands in Western blot may occur; to resolve this, optimize protein extraction methods to ensure complete denaturation, consider using gradient gels for better separation, and verify the expected molecular weight (approximately 62 kDa for DBR1) . Weak or absent signal can result from insufficient antigen retrieval in fixed tissues or cells, particularly for nuclear proteins like DBR1; try different antigen retrieval methods such as heat-induced epitope retrieval using citrate or EDTA buffers . Batch-to-batch variability is another concern; maintain detailed records of antibody lot numbers and perform validation tests with each new lot . For inconsistent results across different experimental systems, remember that DBR1 expression levels vary between tissues and cell types; adjust antibody dilutions accordingly and consider using loading controls specific for nuclear proteins when performing Western blots .

How can I optimize DBR1 detection in different subcellular compartments?

DBR1 is predominantly a nuclear protein involved in RNA processing, but optimizing its detection in different subcellular compartments requires specific considerations . For nuclear detection, ensure complete nuclear permeabilization using adequate concentrations of detergents such as Triton X-100 (0.1-0.5%) during immunofluorescence procedures . Consider using nuclear extraction protocols that effectively separate nuclear and cytoplasmic fractions for Western blotting; this approach has been validated using Xenopus tropicalis oocyte nuclear and cytoplasmic lysates . For co-localization studies, DBR1 has been shown to concentrate in nuclear speckles along with other splicing factors; use established markers of nuclear speckles such as SC35 for co-immunofluorescence . When examining potential cytoplasmic roles of DBR1, particularly in relation to retroviral replication, optimize fixation protocols to preserve both nuclear and cytoplasmic structures and signals . For high-resolution subcellular localization, consider super-resolution microscopy techniques or electron microscopy with gold-labeled secondary antibodies . Remember that different fixation methods may affect epitope accessibility; compare paraformaldehyde fixation with methanol fixation, which can sometimes better preserve nuclear antigens .

What quantitative methods can be used to measure DBR1 levels in experimental systems?

Several quantitative methods can be employed to accurately measure DBR1 levels in experimental systems . Western blotting with DBR1 antibodies provides a semi-quantitative assessment of protein levels; for accurate quantification, use appropriate loading controls (preferably nuclear proteins with similar abundance), generate standard curves with recombinant DBR1 protein, and employ image analysis software for densitometry . Enzyme-linked immunosorbent assay (ELISA) offers more precise quantification; commercial DBR1 ELISA kits may be available, or researchers can develop their own sandwich ELISA using two different DBR1 antibodies recognizing distinct epitopes . Flow cytometry with intracellular staining protocols can quantify DBR1 levels at the single-cell level, allowing for analysis of cell-to-cell variability and correlation with other cellular parameters . For mRNA quantification as a proxy for protein levels, RT-qPCR targeting DBR1 transcripts can be performed, though post-transcriptional regulation may lead to discrepancies between mRNA and protein levels . Advanced mass spectrometry-based proteomics offers absolute quantification of DBR1 protein; selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) with isotope-labeled peptide standards can provide highly accurate measurements .

How does AQR recruit DBR1 to branchpoints and what are the implications for splicing research?

The recruitment of DBR1 to branchpoints by AQR represents a significant mechanistic insight into RNA processing pathways . AQR (Aquarius), an RNA helicase and intron-binding protein, has been shown to physically interact with DBR1 through co-immunoprecipitation mass spectrometry and confirmed by western blot analysis of both DBR1 and AQR immunoprecipitations . This interaction appears to be functionally significant, as introns identified as AQR-bound exhibited a three-fold higher level of lariat increase upon DBR1 depletion compared to introns not bound by AQR . The specificity of this relationship is notable, as none of the other DBR1 binding partners with available eCLIP data showed a similar response in lariat levels . Further supporting this recruitment model, knockdown of AQR through siRNA led to an increase in lariat reads despite normal DBR1 levels, mimicking the effect of DBR1 depletion . Additionally, AQR depletion resulted in a relative increase in lariats with 'A' branchpoints, which are preferentially debranched by DBR1 . The GC-rich motif enriched in AQR binding regions has been mapped to human introns and predicts increased sensitivity to DBR1 . This recruitment mechanism has significant implications for splicing research, suggesting a coordinated pathway for lariat processing and potential regulation of splicing efficiency through controlled debranching activity. Researchers investigating splicing mechanisms should consider the AQR-DBR1 axis as a potential regulatory step in the splicing cycle.

What are the implications of DBR1 research for understanding neurodegenerative diseases?

DBR1 research has revealed significant implications for understanding neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS) . Studies have shown that inhibition of RNA lariat debranching enzyme can suppress TDP-43 toxicity in ALS disease models, suggesting a potential therapeutic approach . TDP-43 is a major disease protein in ALS, and its toxicity mechanisms may be influenced by RNA processing pathways involving DBR1 . The discovery that DBR1 depletion leads to increased exon skipping suggests a connection between lariat turnover and splicing fidelity, which could be relevant to neurodegenerative conditions where aberrant splicing contributes to pathology . Furthermore, DBR1's potential antiviral cell-intrinsic defense function in the brainstem indicates broader neurological implications beyond RNA processing . Research using DBR1 knockout cell lines has demonstrated a 20-fold increase in lariats and defects in spliceosome recycling, potentially affecting the expression of genes critical for neuronal function . The interaction between DBR1 and AQR provides a mechanistic link that could be exploited for therapeutic development, as targeting this recruitment pathway might modulate lariat processing in disease contexts . Future research directions should explore the specific splicing events affected by DBR1 dysfunction in neuronal cells, the potential role of DBR1 in other neurodegenerative conditions beyond ALS, and the development of small molecules that could modulate DBR1 activity as potential therapeutic agents.

Data Tables and Experimental Reference Guide

Effects of DBR1 Depletion on RNA Processing