RBM26 Antibody

What is RBM26 and what is its biological function?

RBM26 (RNA-binding motif protein 26) is a ~114 kDa protein that functions as an RNA-binding protein. Recent research using the C. elegans ortholog (rbm-26) has revealed that RBM26/27 plays a critical role in protecting against mitochondrial dysfunction during neurodevelopment . The protein negatively regulates the expression of MALS-1 (MALSU1), a mitoribosomal assembly factor, which helps protect against axonal defects and mitochondrial dysfunction . RBM26 has been found to be widely expressed in multiple tissues, including neurons, hypodermis, muscle, and intestine, with nuclear localization observed in neurons and hypodermis . It is also known as CTCL tumor antigen se70-2 in some literature .

What applications are RBM26 antibodies suitable for?

RBM26 antibodies are primarily validated for Western blot (WB) applications, with some antibodies also suitable for ELISA . For Western blot applications, the recommended dilution ranges from 1:500-1:1000, while for ELISA, a dilution of 1:10000 may be appropriate . The antibodies are typically designed to detect endogenous levels of RBM26 protein . When selecting an RBM26 antibody, researchers should verify the specific applications for which the antibody has been validated, as this may vary between suppliers and specific antibody clones.

What species reactivity can I expect with commercial RBM26 antibodies?

Commercial RBM26 antibodies typically demonstrate reactivity with human, mouse, and rat samples . This cross-reactivity makes these antibodies versatile for comparative studies across these three mammalian species. When working with other species, it is advisable to perform preliminary validation experiments or contact the manufacturer to determine potential cross-reactivity. Sequence homology analysis between your species of interest and the immunogen sequence can provide insights into potential reactivity.

How should RBM26 antibodies be stored and handled for optimal performance?

RBM26 antibodies are typically shipped on wet ice and should be stored at -20°C for long-term preservation . The antibodies are commonly formulated in buffered aqueous solutions, often containing phosphate buffered saline (PBS) with 0.05% sodium azide at approximately pH 7.3 . For optimal performance, avoid repeated freeze-thaw cycles by aliquoting the antibody upon first thaw. When working with the antibody, maintain cold chain practices by keeping it on ice during experimental procedures. Check the manufacturer's specific recommendations for your antibody, as storage and handling conditions may vary slightly between different products.

What are the optimal protocols for using RBM26 antibodies in Western blot applications?

For optimal Western blot results with RBM26 antibodies, follow these methodological considerations:

• Sample preparation: Extract proteins using RIPA or NP-40 lysis buffers containing protease inhibitors. For nuclear proteins like RBM26, consider nuclear extraction protocols.

• Gel electrophoresis: Use 8-10% SDS-PAGE gels to properly resolve the ~114 kDa RBM26 protein .

• Transfer conditions: For large proteins like RBM26, use a wet transfer system with 10% methanol for optimal transfer efficiency.

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature.

• Primary antibody incubation: Dilute RBM26 antibody 1:500-1:1000 in blocking buffer and incubate overnight at 4°C .

• Secondary antibody: Use an appropriate anti-rabbit HRP-conjugated secondary antibody at 1:5000-1:10000 dilution .

• Detection: Visualize using enhanced chemiluminescence (ECL) detection reagents.

• Expected band size: Look for a specific band at approximately 114 kDa .

Always include positive and negative controls, and consider the use of loading controls such as β-actin or GAPDH for normalization purposes.

How can I validate RBM26 antibody specificity for my experimental system?

Validating antibody specificity is crucial for ensuring reliable experimental results. For RBM26 antibodies, consider these validation approaches:

• RNAi or knockout controls: Compare samples with RBM26 knockdown or knockout to wild-type samples. An authentic RBM26 antibody should show reduced or absent signal in the knockdown/knockout samples .

• Overexpression controls: Compare samples overexpressing RBM26 with normal expression samples. The antibody should detect increased signal in overexpression samples.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before immunoblotting. This should abolish specific binding.

• Multiple antibodies approach: Use different antibodies raised against distinct epitopes of RBM26 to confirm detection of the same protein.

• Cross-species validation: If the antibody is expected to be cross-reactive, test it on samples from different species to confirm conservation of the epitope.

• Molecular weight verification: Confirm that the detected band corresponds to the expected molecular weight of ~114 kDa .

The validation data from the manufacturer can provide a starting point, but independent validation in your specific experimental system is strongly recommended.

What are the considerations for using RBM26 antibodies in ELISA applications?

When using RBM26 antibodies for ELISA applications, consider these methodological aspects:

• Antibody dilution: For ELISA applications, a higher dilution (1:10000) is typically recommended compared to Western blot applications .

• Coating conditions: For direct ELISA, optimize coating buffer (carbonate/bicarbonate buffer pH 9.6 is often effective) and coating concentration of capture antibody or antigen.

• Blocking buffer optimization: Test different blocking agents (BSA, non-fat dry milk, normal serum) to minimize background while maintaining specific signal.

• Sample preparation: Consider using different extraction methods to optimize antigen presentation in your samples.

• Standard curve: Generate a standard curve using recombinant RBM26 protein if available for quantitative analysis.

• Cross-reactivity testing: Evaluate potential cross-reactivity with related proteins, particularly other RNA-binding motif proteins.

• Detection system: Choose appropriate detection systems (colorimetric, fluorescent, or chemiluminescent) based on your sensitivity requirements.

• Validation controls: Include positive and negative controls in each assay to ensure reliability.

A titration experiment comparing different antibody dilutions can help identify the optimal concentration that maximizes specific signal while minimizing background.

How can RBM26 antibodies be used to study mitochondrial dysfunction in neurodevelopmental disorders?

RBM26 antibodies can be powerful tools for investigating mitochondrial dysfunction in neurodevelopmental disorders based on recent findings about RBM26's role in mitochondrial protection . Consider these methodological approaches:

• Immunofluorescence co-localization studies: Use RBM26 antibodies in combination with mitochondrial markers to examine potential associations between RBM26 expression patterns and mitochondrial distribution in neuronal cells.

• Biochemical fractionation: Combine subcellular fractionation techniques with Western blot analysis using RBM26 antibodies to determine the distribution of RBM26 in different cellular compartments, particularly in relation to mitochondria.

• Proximity ligation assays: Use RBM26 antibodies with antibodies against mitochondrial proteins to detect potential protein-protein interactions.

• Disease model comparisons: Compare RBM26 expression and localization in normal versus disease model neurons (using patient-derived iPSCs or animal models of autism) using immunocytochemistry or Western blot analysis.

• Functional mitochondrial assays: Correlate RBM26 expression levels (detected by antibodies) with measurements of mitochondrial function such as:

  • Mitochondrial membrane potential

  • Reactive oxygen species (ROS) production

  • Mitochondrial density in axons

  • ATP production

• Comparison of variants: Use RBM26 antibodies to compare expression levels of wild-type RBM26 versus autism-associated variants (such as P79L and L13V in human RBM27) to determine if these mutations affect protein stability .

Recent research has shown that loss of RBM-26 function causes mitochondrial dysfunction characterized by increased ROS production and decreased mitochondrial density in axons, which could be relevant to neurodevelopmental disorders such as autism .

What experimental approaches can be used to investigate RBM26's RNA-binding targets?

To investigate RBM26's RNA-binding targets, researchers can combine RBM26 antibodies with various molecular techniques:

• RNA immunoprecipitation (RIP): Use RBM26 antibodies to immunoprecipitate RBM26 protein along with bound RNA molecules, followed by RT-PCR or RNA sequencing to identify bound transcripts. This approach has successfully identified mals-1 mRNA as a binding partner for RBM-26 in C. elegans .

• Cross-linking immunoprecipitation (CLIP): Combine UV cross-linking with immunoprecipitation using RBM26 antibodies to identify direct RNA-protein interactions at single-nucleotide resolution.

• Electrophoretic mobility shift assay (EMSA): Use purified RBM26 protein (detected with RBM26 antibodies) to assess binding to candidate RNA sequences in vitro.

• RNA pull-down assays: Use biotinylated RNA probes followed by Western blot with RBM26 antibodies to confirm specific interactions between candidate RNAs and RBM26.

• Immunofluorescence co-localization: Combine RBM26 antibodies with RNA FISH (fluorescence in situ hybridization) to visualize co-localization of RBM26 with specific RNA transcripts in cells.

• Functional validation: After identifying candidate targets, use RBM26 knockout/knockdown systems to assess changes in target RNA levels, stability, or splicing patterns.

• Structure-function analysis: Use RBM26 antibodies to detect expression of mutant RBM26 proteins with alterations in RNA-binding domains to correlate RNA-binding activity with functional outcomes.

Research has shown that in C. elegans, RBM-26 negatively regulates the expression of mals-1 mRNA, which encodes a mitoribosomal assembly factor, suggesting a potential regulatory mechanism that could be conserved in humans .

How can RBM26 antibodies be used to study the effects of autism-associated mutations?

RBM26 antibodies can be instrumental in studying the effects of autism-associated mutations through various experimental approaches:

• Expression level analysis: Use Western blot with RBM26 antibodies to compare expression levels of wild-type versus mutant RBM26 proteins. Research in C. elegans has shown that autism-associated missense variants (P80L and L13V, equivalent to P79L and L13V in humans) cause a sharp decrease in RBM-26 protein expression .

• Subcellular localization studies: Use immunofluorescence with RBM26 antibodies to determine if autism-associated mutations alter the nuclear localization pattern of RBM26 observed in neurons and other cell types .

• Protein stability assays: Combine cycloheximide chase experiments with Western blot detection using RBM26 antibodies to compare the stability of wild-type versus mutant RBM26 proteins.

• Functional rescue experiments: Use RBM26 antibodies to confirm expression of wild-type RBM26 in rescue experiments where wild-type RBM26 is introduced into cells expressing mutant RBM26.

• Protein-protein interaction studies: Use co-immunoprecipitation with RBM26 antibodies to identify potential differences in protein interaction partners between wild-type and mutant RBM26.

• Post-translational modification analysis: Use RBM26 antibodies in combination with phospho-specific or other modification-specific antibodies to determine if autism-associated mutations alter post-translational modifications of RBM26.

Studies in C. elegans have shown that autism-associated missense variants in RBM-26 cause phenotypes similar to null mutations, including axon degeneration, axon overlap defects, and mitochondrial dysfunction, suggesting these variants are likely gene-disrupting in humans .

What methods are recommended for studying RBM26 protein localization in neuronal and non-neuronal tissues?

For studying RBM26 protein localization in various tissues, researchers can employ these methodological approaches:

• Immunohistochemistry (IHC): Use RBM26 antibodies on tissue sections to visualize protein expression patterns across different cell types and brain regions. Optimize fixation methods (paraformaldehyde vs. methanol) and antigen retrieval techniques for best results.

• Immunocytochemistry (ICC): Apply RBM26 antibodies to cultured cells, including primary neurons, to examine subcellular localization. Research has shown nuclear localization of RBM26 in neurons and hypodermis .

• Fluorescent protein tagging: Complement antibody-based approaches with fluorescent protein tagging of RBM26 (as done with Scarlet tag in C. elegans studies) to visualize protein localization in live cells .

• Super-resolution microscopy: Use techniques such as STED, STORM, or PALM with RBM26 antibodies to achieve high-resolution imaging of RBM26 localization within subcellular compartments.

• Tissue-specific expression analysis: Apply RBM26 antibodies in Western blot analysis of protein extracts from different tissues to compare expression levels across tissue types. RBM26 has been detected in neurons, hypodermis, muscle, and intestine in C. elegans .

• Developmental time-course studies: Use RBM26 antibodies to track changes in protein expression and localization during development, particularly in the context of neurodevelopmental disorders.

• Co-localization with cellular markers: Combine RBM26 antibodies with markers for specific cellular compartments (nuclear envelope, nucleolus, mitochondria, etc.) to precisely define the subcellular localization pattern.

• Electron microscopy: Use immunogold labeling with RBM26 antibodies for ultrastructural localization studies at the electron microscopy level.

For optimal results, consider using multiple fixation and permeabilization protocols, as these can significantly affect antibody accessibility and staining patterns, particularly for nuclear proteins like RBM26.

What are common issues encountered when using RBM26 antibodies and how can they be resolved?

When working with RBM26 antibodies, researchers may encounter several technical challenges. Here are common issues and their potential solutions:

• Weak or no signal in Western blot:

  • Increase primary antibody concentration (try 1:250 if 1:500 doesn't work)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize protein extraction protocol for nuclear proteins

  • Increase protein loading (50-80 μg per lane)

  • Use enhanced chemiluminescence substrates with higher sensitivity

  • Check transfer efficiency, especially for high molecular weight proteins (~114 kDa)

• High background:

  • Increase blocking time or concentration (5-10% blocking agent)

  • Use more stringent washing conditions (increase TBST concentration or washing duration)

  • Optimize antibody dilution to reduce non-specific binding

  • Try alternative blocking agents (switch between milk and BSA)

  • Pre-adsorb secondary antibody with tissue powder from the species being analyzed

• Multiple bands:

  • Verify if additional bands represent isoforms, degradation products, or post-translational modifications

  • Include positive and negative controls to distinguish specific from non-specific bands

  • Use fresh samples with complete protease inhibitor cocktails

  • Consider using gradient gels for better resolution

• Inconsistent results:

  • Standardize sample preparation protocols

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Maintain consistent incubation times and temperatures

  • Include internal controls in each experiment

• Poor reproducibility between different lots:

  • Request lot-specific validation data from manufacturers

  • Validate each new lot against a previously working lot

  • Consider using monoclonal antibodies for more consistent results

For applications studying autism-associated variants, be aware that these mutations may significantly reduce protein expression levels, requiring more sensitive detection methods or increased sample loading .

How can I optimize immunoprecipitation protocols using RBM26 antibodies?

For successful immunoprecipitation (IP) of RBM26 protein, consider these optimization strategies:

• Lysis buffer selection:

  • For nuclear proteins like RBM26, use nuclear extraction buffers containing 0.1-0.3% NP-40 or Triton X-100

  • Include DNase I to reduce chromatin-mediated precipitation

  • Use protease and phosphatase inhibitors to preserve protein integrity

• Pre-clearing step:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Use species-matched normal IgG for pre-clearing

• Antibody amount optimization:

  • Titrate antibody amounts (typically 2-5 μg per mg of total protein)

  • Consider crosslinking the antibody to beads to prevent heavy chain interference in Western blot analysis

• Incubation conditions:

  • Optimize antibody-antigen binding by testing different incubation times (4 hours to overnight)

  • Perform incubation at 4°C with gentle rotation to preserve protein-protein interactions

• Washing stringency:

  • Balance between stringency (to reduce background) and preservation of specific interactions

  • Test a gradient of salt concentrations in wash buffers (150-300 mM NaCl)

  • Consider including low concentrations of non-ionic detergents (0.1% NP-40)

• Elution methods:

  • Compare different elution methods (SDS buffer, low pH, or peptide competition)

  • For downstream analysis of interaction partners, consider native elution conditions

• Controls:

  • Always include an IgG control from the same species as the RBM26 antibody

  • Consider using samples with RBM26 knockdown as negative controls

• Verification:

  • Confirm successful IP by Western blot with a different RBM26 antibody recognizing a different epitope

  • For co-IP experiments, validate interactions with reciprocal IP when possible

For RNA immunoprecipitation applications, include RNase inhibitors in all buffers and consider UV crosslinking to stabilize RNA-protein interactions before cell lysis.

How can RBM26 antibodies be used in neurodevelopmental disorder research models?

RBM26 antibodies can be valuable tools for investigating neurodevelopmental disorders through these methodological approaches:

• Patient-derived cell models:

  • Use RBM26 antibodies to compare protein expression and localization in neurons derived from patient iPSCs versus controls

  • Correlate RBM26 expression levels with mitochondrial function parameters in autism model systems

• Animal models:

  • Apply RBM26 antibodies in immunohistochemistry of brain sections from autism model animals

  • Use Western blot analysis to track developmental expression patterns of RBM26 in different brain regions

• Molecular phenotyping:

  • Compare RBM26 expression, localization, and RNA-binding activities between wild-type and autism model systems

  • Investigate downstream effects of RBM26 dysfunction on target RNA processing

• Rescue experiments:

  • Use RBM26 antibodies to confirm expression of wild-type RBM26 in rescue experiments testing functional complementation

  • Monitor correction of phenotypes (mitochondrial function, axon development) upon restoration of RBM26 function

• Pharmacological studies:

  • Apply RBM26 antibodies to measure changes in protein expression or localization in response to experimental therapeutics

  • Use as biomarkers to monitor treatment efficacy in model systems

Recent research in C. elegans models has shown that autism-associated RBM26 variants lead to protein expression deficits accompanied by axon degeneration, axon overlap defects, and mitochondrial dysfunction, suggesting similar mechanisms may operate in human neurons . These findings indicate that RBM26 antibodies could be particularly useful for studying axonal integrity and mitochondrial function in neurodevelopmental disorder models.

What is the significance of RBM26's role in mitochondrial protection and how can antibodies help elucidate this function?

The discovery of RBM26's role in mitochondrial protection has significant implications for understanding neurodevelopmental disorders. RBM26 antibodies can help elucidate this function through:

• Temporal expression analysis:

  • Use RBM26 antibodies to track protein expression during critical developmental windows when mitochondrial function is essential for neuronal development

  • Create developmental timelines correlating RBM26 expression with mitochondrial biogenesis markers

• Subcellular distribution studies:

  • Apply RBM26 antibodies in subcellular fractionation experiments to determine if a subset of RBM26 localizes to mitochondria or mitochondria-associated membranes

  • Use super-resolution microscopy to visualize potential associations between RBM26 and mitochondrial structures

• Target validation:

  • Use RBM26 antibodies in combination with antibodies against MALS-1/MALSU1 to study their reciprocal expression patterns

  • Investigate how RBM26 levels correlate with expression of other mitoribosomal assembly factors

• Stress response experiments:

  • Apply RBM26 antibodies to measure changes in protein expression or localization under conditions of mitochondrial stress

  • Determine if RBM26 is part of a cellular stress response pathway protecting mitochondrial function

• Mechanistic dissection:

  • Use RBM26 antibodies in chromatin immunoprecipitation experiments to identify potential transcriptional targets related to mitochondrial function

  • Combine with RNA immunoprecipitation to identify RNAs involved in mitochondrial function that are bound by RBM26

Research in C. elegans has shown that RBM-26 negatively regulates MALS-1 expression to protect against mitochondrial dysfunction and axon degeneration during development . RBM26 antibodies can help determine if this protective mechanism is conserved in mammals and potentially identify novel therapeutic targets for neurodevelopmental disorders associated with mitochondrial dysfunction.