RBM14 Antibody

What is RBM14 and why is it significant in cellular research?

RBM14 (RNA-binding motif protein 14) is a multifunctional nuclear protein that plays critical roles in several cellular processes. It contains two RNA recognition motifs (RRMs) at the N-terminus and multiple hexapeptide repeat domains at the C-terminus that interact with various proteins .

Key functions include:

• Transcriptional regulation: Isoform 1 functions as a nuclear receptor coactivator enhancing transcription through coactivators like NCOA6 and CITED1, while isoform 2 acts as a transcriptional repressor

• Centriole biogenesis regulation: Prevents formation of aberrant centriolar protein complexes by interfering with STIL-CENPJ interaction

• Innate immune response regulation: Assembles into the HDP-RNP complex that serves as a platform for IRF3 phosphorylation and immune response activation through the cGAS-STING pathway

• Pre-mRNA alternative splicing modulation

• Spindle integrity maintenance during cell division

RBM14's diverse functions make it a significant target for studying fundamental cellular mechanisms and disease processes.

Proper storage and handling of RBM14 antibodies are crucial for maintaining their functionality and specificity:

Storage conditions:

• Store at -20°C for long-term storage

• For short-term use (up to 2 weeks), refrigeration at 2-8°C is acceptable

• Aliquot antibodies to prevent freeze-thaw cycles, which can degrade antibody quality

Buffer composition varies by manufacturer but typically includes:

• PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• PBS with 0.09% (W/V) sodium azide

Important handling practices:

• Thaw aliquots completely before use and mix gently

• Avoid repeated freeze-thaw cycles by creating small working aliquots

• Some preparations (20μl sizes) may contain 0.1% BSA as a stabilizer

• Follow manufacturer recommendations for specific antibodies as storage buffers may vary

How can RBM14 antibodies be used to investigate spindle morphology and tubulin acetylation?

Recent research has revealed RBM14's critical role in regulating spindle morphology through tubulin acetylation, particularly during meiosis. A comprehensive methodology for investigating this function includes:

• Experimental approach:

  • RNA interference (RNAi) using RBM14-specific morpholino (RBM14-MO) in oocytes

  • Co-immunoprecipitation (Co-IP) to detect physical interactions between RBM14 and α-tubulin

  • Western blot and immunofluorescence to assess α-tubulin acetylation levels

• Key findings:

  • RBM14 knockdown results in spindle defects and chromosome abnormalities during oocyte maturation

  • RBM14-depleted oocytes showed symmetric division compared to controls

  • RBM14 depletion causes significant α-tubulin hyperacetylation (1.33 ± 0.06 vs. 1.0, P < 0.05 by Western blot; 2.14 ± 0.25 vs. 1.0, P < 0.05 by immunofluorescence)

  • Co-IP experiments confirm physical interaction between RBM14 and α-tubulin in mammalian cells

• Methodology details:

  • For Co-IP: Use RBM14 or α-tubulin antibodies with whole cell lysates (e.g., NIH/3T3 cells)

  • For western blot: Standard SDS-PAGE (7.5%) followed by immunoblotting

  • For immunofluorescence: Compare acetylated α-tubulin levels between control and RBM14-depleted samples

This approach enables researchers to elucidate RBM14's role in modulating microtubule stability through regulation of α-tubulin acetylation .

What is the optimal protocol for using RBM14 antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) is a powerful technique for investigating protein-protein interactions involving RBM14. Based on published research, here is an optimized protocol:

• Cell preparation:

  • Use appropriate cell lines where RBM14 is expressed (e.g., NIH/3T3, Jurkat, HeLa, or HEK-293 cells)

  • Harvest cells at 80-90% confluence

• Lysis procedure:

  • Wash cells with cold PBS

  • Lyse cells in IP lysis buffer containing protease inhibitors

  • Incubate on ice for 30 minutes with occasional vortexing

  • Centrifuge at 14,000g for 15 minutes at 4°C to remove cell debris

• Immunoprecipitation:

  • Pre-clear lysate with Protein A/G beads

  • Incubate cleared lysate with RBM14 antibody (recommended dilution dependent on specific antibody)

  • Add Protein A/G beads and incubate overnight at 4°C with gentle rotation

  • Wash beads 4-5 times with wash buffer

  • Elute bound proteins by boiling in SDS sample buffer

• Detection:

  • Separate proteins by SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane

  • Probe with antibodies against potential interacting proteins

  • For reverse Co-IP, immunoprecipitate with antibodies against suspected binding partners and probe with RBM14 antibody

This protocol has been validated for detecting interactions between RBM14 and α-tubulin and can be adapted to investigate other potential RBM14 binding partners .

How do different RBM14 isoforms affect experimental outcomes when using RBM14 antibodies?

RBM14 exists in multiple isoforms with distinct functions, which can significantly impact experimental outcomes and interpretation when using RBM14 antibodies:

• Functional differences between isoforms:

  • Isoform 1: Functions as a nuclear receptor coactivator, enhancing transcription through coactivators like NCOA6 and CITED1

  • Isoform 2: Acts as a transcriptional repressor, modulating transcriptional activities of coactivators including isoform 1, NCOA6, and CITED1

• Antibody selection considerations:

  • Target region specificity: N-terminal antibodies (like ab228692) may detect both isoforms as RRMs are located at the N-terminus

  • C-terminal antibodies (like OAAB07799) may have differential recognition of isoforms if C-terminal sequences vary

  • Validation of antibody specificity against each isoform is crucial for accurate data interpretation

• Expected molecular weights:

  • Calculated molecular weight: 70 kDa

  • Observed molecular weight range: 70-75 kDa

  • Migration differences on SDS-PAGE may reflect post-translational modifications or isoform variations

• Experimental recommendations:

  • Use isoform-specific antibodies when studying distinct functions

  • Include positive controls expressing known isoforms

  • Consider complementary techniques (e.g., RT-PCR with isoform-specific primers) to confirm which isoforms are present in your experimental system

  • Validate findings with multiple antibodies targeting different regions when possible

Understanding which isoforms are detected by your selected antibody is essential for correctly interpreting experimental results, especially when studying transcriptional regulation or protein-protein interactions .

What are the optimal dilutions and conditions for RBM14 antibody use in Western blotting?

Western blotting is one of the most common applications for RBM14 antibodies. Based on validated protocols, here are the optimal conditions:

• Sample preparation:

  • Positive controls: Jurkat cells, HeLa cells, HEK-293 cells

  • Proteins typically extracted using RIPA buffer with protease inhibitors

  • Standard SDS-PAGE (7.5%) recommended for optimal separation

• Detection considerations:

  • Expected molecular weight: 70-75 kDa

  • Incubation times: Typically overnight at 4°C for primary antibody

  • Secondary antibody: Anti-rabbit IgG at manufacturer-recommended dilutions

  • Both chemiluminescent and fluorescent detection methods are compatible

• Optimization tips:

  • Titrate antibody concentration for each experimental system

  • Include positive and negative controls

  • For challenging samples, consider extended blocking times or alternative blocking reagents

  • Sample-dependent optimization may be necessary for optimal results

These conditions have been validated in multiple studies investigating RBM14 expression and function in various cellular contexts .

How can RBM14 antibodies be used effectively in immunofluorescence studies of subcellular localization?

RBM14 exhibits specific subcellular localization patterns that provide insights into its function. Effective immunofluorescence protocols for studying RBM14 localization include:

• Cell/tissue preparation:

  • Fixation: 4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilization: 0.1-0.5% Triton X-100 (5-10 minutes)

  • Blocking: 5% BSA or normal serum (1 hour at room temperature)

• Expected subcellular localization patterns:

  • Primary location: Nucleus, nucleolus

  • Specific subnuclear structures: Punctate structures often adjacent to splicing speckles (paraspeckles)

  • During cell division: Co-localization with α-tubulin in spindle structures

• Co-staining recommendations:

  • α-tubulin: To study RBM14's role in spindle formation and integrity

  • Nuclear markers: To confirm nuclear/nucleolar localization

  • Treatment with spindle-perturbing agents reveals RBM14 co-localization with microtubules

• Special considerations:

  • RBM14 distribution varies at different stages of meiosis in oocytes

  • In MI- and MII-stage oocytes, RBM14 shows overlapped localization patterns with α-tubulin

  • Age-related effects: RBM14 expression is down-regulated in oocytes from old mice

• Control recommendations:

  • Include RBM14-depleted cells (using siRNA or morpholino) as negative controls

  • Secondary antibody-only controls to assess background

This approach has been successfully used to demonstrate RBM14's dynamic localization during cell division and its association with microtubule structures .

What technical considerations should be addressed when using RBM14 antibodies to study its role in innate immunity?

RBM14 plays a significant role in DNA virus-mediated innate immune responses through the cGAS-STING pathway. When investigating this function, several technical considerations should be addressed:

• Experimental design factors:

  • Cell systems: Choose cell types relevant to viral infection and innate immunity (e.g., macrophages, dendritic cells)

  • Viral stimulation: Use DNA virus infection or synthetic DNA (e.g., poly(dA:dT)) to activate the cGAS-STING pathway

  • Timing: Consider temporal dynamics of the immune response and RBM14 recruitment

• Key methodological approaches:

  • Co-immunoprecipitation to detect RBM14 interaction with components of the HDP-RNP complex

  • Immunofluorescence to visualize RBM14 redistribution during viral infection

  • RBM14 knockdown/knockout to assess functional impact on IRF3 phosphorylation and downstream signaling

• Critical controls:

  • RNA virus infection (as negative control, since RBM14's role is specific to DNA virus response)

  • RBM14-depleted cells to confirm antibody specificity

  • Inhibitors of the cGAS-STING pathway to confirm specificity of observed effects

• Readouts to measure:

  • IRF3 phosphorylation levels by Western blot

  • Type I interferon production by ELISA or reporter assays

  • RBM14 localization changes during immune activation

  • Formation of the HDP-RNP complex

• Potential pitfalls and solutions:

  • Cell type-specific effects: Test multiple relevant cell types

  • Temporal dynamics: Perform time-course experiments

  • Antibody cross-reactivity: Use multiple antibodies targeting different epitopes

  • Background signal: Include appropriate negative controls

This approach leverages RBM14's established role in assembling into the HDP-RNP complex that serves as a platform for IRF3 phosphorylation and subsequent innate immune response activation .

How can researchers troubleshoot common issues when using RBM14 antibodies across different applications?

When working with RBM14 antibodies, researchers may encounter various technical challenges. Here are solutions to common issues:

• Western blot troubleshooting:

  Issue	Possible Causes	Solutions
  No signal	Insufficient protein, degraded antibody	Increase protein loading (70-75 kDa expected), use fresh antibody aliquot
  Multiple bands	Cross-reactivity, protein degradation	Use antibody targeting different epitope, add protease inhibitors
  Unexpected MW	Post-translational modifications, isoforms	Compare with positive control cells (Jurkat, HeLa, HEK-293)
  High background	Insufficient blocking, excess antibody	Optimize blocking, titrate antibody (start with 1:500-1:2000)

• Immunofluorescence troubleshooting:

  Issue	Possible Causes	Solutions
  Weak nuclear signal	Fixation issues, epitope masking	Optimize fixation time, try antigen retrieval
  Cytoplasmic signal only	Fixation artifacts, cell cycle stage	Compare with different fixation methods, synchronize cells
  No co-localization with expected partners	Cell cycle stage, experimental conditions	Use spindle-perturbing agents to verify microtubule association
  Non-specific staining	Antibody concentration, blocking	Titrate antibody, increase blocking time/concentration

• Immunoprecipitation troubleshooting:

  Issue	Possible Causes	Solutions
  Failed to pull down RBM14	Insufficient antibody, weak binding	Increase antibody amount, optimize binding conditions
  No co-IP of interacting proteins	Interaction disrupted by lysis buffer	Try milder lysis conditions, crosslinking
  Non-specific bands	Antibody cross-reactivity	Include IgG control, use more stringent washing
  Inconsistent results	Experimental variation	Standardize cell growth conditions and lysis procedures

• General recommendations:

  • Validate antibody in your specific experimental system before conducting complex experiments

  • Include appropriate positive controls (e.g., cell lines known to express RBM14)

  • Consider using multiple antibodies targeting different epitopes to confirm findings

  • Perform complementary experiments (e.g., RNA interference) to validate antibody-based results

These troubleshooting approaches are based on established protocols and research findings using RBM14 antibodies across various applications .

How can RBM14 antibodies be used to investigate its role in cancer biology and therapeutic resistance?

RBM14 has emerged as a significant factor in cancer biology, particularly in therapeutic resistance. Research methodologies using RBM14 antibodies in cancer studies include:

• Radio-resistance in glioblastoma:

  • Differential expression analysis: Compare RBM14 levels between radio-resistant and radio-sensitive tumors using Western blot and IHC

  • Functional studies: Assess the impact of RBM14 knockdown/overexpression on radiation sensitivity

  • Mechanistic investigation: Examine RBM14's role in DNA repair pathways and cell differentiation processes in glioblastoma models

• Methodological approach:

  • Patient-derived samples: Use RBM14 antibodies for IHC to correlate expression with clinical outcomes

  • Cell line models: Western blot and immunofluorescence to monitor RBM14 expression and localization before and after radiation treatment

  • Survival assays: Correlate RBM14 expression with cell survival following radiation therapy

  • DNA repair assays: Co-localization studies with DNA damage markers

• Technical considerations:

  • Use multiple antibodies targeting different RBM14 epitopes to confirm findings

  • Include appropriate controls (normal brain tissue, radio-sensitive glioblastoma lines)

  • Consider the impact of tumor heterogeneity on RBM14 expression patterns

  • Analyze both protein expression levels and subcellular localization

This approach has revealed that RBM14 promotes radio-resistance in glioblastoma by regulating DNA repair and cell differentiation processes, suggesting its potential as a therapeutic target or biomarker .

What are the best practices for using RBM14 antibodies in aging and reproductive biology research?

RBM14 plays a significant role in reproductive biology, particularly in oocyte maturation. Recent research has identified age-related changes in RBM14 expression that may contribute to reproductive aging:

• Key research findings:

  • RBM14 expression is down-regulated in oocytes from old mice compared to young mice

  • RBM14 depletion results in spindle defects and chromosome abnormalities during oocyte maturation

  • RBM14 regulates α-tubulin acetylation, affecting spindle morphology

• Methodological approach for age-related studies:

  • Sample collection: Compare oocytes from young vs. aged animal models

  • Western blot: Quantify age-related differences in RBM14 expression levels

  • Immunofluorescence: Assess changes in RBM14 localization patterns with age

  • Functional studies: RBM14 knockdown with morpholinos to mimic age-related decline

• Technical considerations:

  • Age-appropriate controls: Include age-matched controls for all experiments

  • Careful staging of oocytes: RBM14 distribution varies at different meiotic stages

  • Co-staining recommendations: Include α-tubulin and DNA markers to assess spindle morphology and chromosome alignment

  • Quantitative assessment: Measure α-tubulin acetylation levels as a functional readout of RBM14 activity

• Potential applications:

  • Biomarker development: RBM14 as a potential indicator of oocyte quality

  • Therapeutic target: Modulating RBM14 function to address age-related decline in oocyte quality

  • Diagnostic applications: Assess RBM14 in cases of oocyte maturation failure

These approaches leverage RBM14's established role in spindle integrity and chromosome alignment during oocyte maturation, providing insights into reproductive aging mechanisms .

What emerging research directions are likely to require RBM14 antibodies in the near future?

Based on current research trends and recent findings, several emerging research directions will likely require RBM14 antibodies:

• Paraspeckle biology and liquid-liquid phase separation:

  • RBM14's localization in paraspeckles suggests involvement in membraneless organelle formation

  • Investigations into its role in RNA processing within these structures

  • Studies of protein-protein and protein-RNA interactions that drive phase separation

• Innate immunity and viral response mechanisms:

  • Deeper exploration of RBM14's role in the cGAS-STING pathway

  • Investigation of potential antiviral therapeutics targeting RBM14-dependent mechanisms

  • Studies of RBM14's interaction with other innate immunity pathways

• Reproductive biology and aging:

  • Development of RBM14 as a biomarker for oocyte quality

  • Exploration of interventions to modulate RBM14 function in aging oocytes

  • Investigations into RBM14's role in male gametogenesis and fertility

• Cancer biology and therapeutic resistance:

  • RBM14's involvement in DNA repair mechanisms and therapeutic resistance

  • Development of combination therapies targeting RBM14-dependent pathways

  • Exploration of RBM14 as a prognostic or predictive biomarker in various cancers

• Neurodegenerative diseases:

  • Investigation of RBM14's potential role in RNA metabolism disorders

  • Studies of its function in stress granule formation and neuronal health

  • Exploration of its interaction with other RNA-binding proteins implicated in neurodegeneration