rbm18 Antibody

What is RBM18 and why are antibodies against it important for molecular biology research?

RBM18 (RNA binding motif protein 18) is a probable RNA-binding protein with a canonical length of 190 amino acid residues and a molecular mass of 21.6 kDa in humans . The protein contains specific RNA recognition motifs that enable it to interact with RNA molecules, potentially regulating post-transcriptional processes.

RBM18 antibodies are crucial research tools for:

• Detecting expression patterns across different tissues and cell types

• Investigating protein-RNA interactions

• Studying subcellular localization

• Examining potential roles in RNA processing pathways

Current research indicates that RBM18 interacts with genes RAD23B, TMEM27, EPHA3, and NDUFAB1 , suggesting potential roles in DNA repair, membrane protein regulation, receptor signaling, and mitochondrial function.

What applications are RBM18 antibodies validated for in research settings?

Based on comprehensive validation studies, RBM18 antibodies have been confirmed effective for multiple applications:

The versatility of these applications allows researchers to investigate RBM18 at both protein expression levels and spatial distribution within cells and tissues .

What experimental considerations should be made regarding species reactivity when selecting RBM18 antibodies?

RBM18 is evolutionarily conserved across multiple species. When selecting an antibody, consider the following reactivity profiles available in commercial antibodies:

When designing cross-species experiments, sequence alignment analysis between the immunogen and target species should be performed to predict potential cross-reactivity .

How can RBM18 antibodies be optimized for dual immunofluorescence studies with other RNA binding proteins?

For dual labeling experiments investigating potential co-localization or interactions between RBM18 and other RNA binding proteins:

• Host species selection: Choose primary antibodies raised in different host species (e.g., rabbit anti-RBM18 and mouse anti-partner protein) to avoid cross-reactivity with secondary antibodies .

• Epitope optimization: Select antibodies targeting different regions of RBM18 when studying protein complexes:

  • Middle region-targeting antibodies are suitable for detecting full-length RBM18

  • N-terminal or C-terminal antibodies may be better for detecting RBM18 in protein complexes where the middle domain might be occluded

• Sequential staining protocol:

  • Apply the first primary antibody (anti-RBM18) at optimal dilution (typically 1-4 μg/ml)

  • Detect with appropriate fluorophore-conjugated secondary antibody

  • Block remaining binding sites with excess unconjugated secondary antibody

  • Apply the second primary antibody against partner protein

  • Detect with differently labeled secondary antibody

  • Include appropriate controls for each antibody separately

• Spectral compatibility: Select fluorophores with minimal spectral overlap to reduce bleed-through artifacts in confocal microscopy .

What methodological approaches can be used to investigate potential changes in RBM18 expression during cellular stress responses?

To study stress-induced changes in RBM18 expression:

• Time-course western blot analysis:

  • Subject cells to stress conditions (oxidative stress, heat shock, ER stress)

  • Harvest cells at multiple time points (0, 2, 4, 8, 24 hours)

  • Perform western blot using validated anti-RBM18 antibodies (1:100-200 dilution)

  • Quantify relative to housekeeping proteins

  • Include phosphorylation-specific antibodies if available to detect post-translational modifications

• Subcellular fractionation with immunoblotting:

  • Separate nuclear, cytoplasmic, and membrane fractions

  • Probe each fraction with RBM18 antibodies

  • Monitor potential stress-induced translocation between compartments

• Immunofluorescence microscopy for localization changes:

  • Fix cells at different stress time points

  • Stain with anti-RBM18 antibodies (1-4 μg/ml)

  • Co-stain with markers for stress granules and P-bodies

  • Analyze co-localization coefficients quantitatively

• Controls for specificity validation:

  • RBM18 knockdown or knockout cells as negative controls

  • Recombinant RBM18 protein as a positive control

  • Pre-absorption of antibody with immunizing peptide

How can researchers effectively use RBM18 antibodies to investigate post-translational modifications?

Current understanding of RBM18 post-translational modifications is limited, but methodological approaches include:

• 2D gel electrophoresis followed by western blotting:

  • Separate proteins by isoelectric point and molecular weight

  • Transfer to membrane and probe with anti-RBM18 antibodies

  • Identify potential modified forms by shifts in pI or molecular weight

• Phosphorylation-specific analysis:

  • Treat samples with/without phosphatase

  • Compare migration patterns on western blots

  • Use phosphorylation-dependent protein mobility shift assays

• Co-immunoprecipitation with modification-specific antibodies:

  • Immunoprecipitate with anti-RBM18 antibodies

  • Probe with antibodies against common modifications (phospho-serine/threonine/tyrosine, ubiquitin, SUMO)

  • Alternatively, immunoprecipitate with modification-specific antibodies and probe for RBM18

• Mass spectrometry analysis:

  • Immunoprecipitate RBM18 using validated antibodies

  • Perform LC-MS/MS analysis to identify modifications

  • Validate findings with site-specific mutagenesis

What protocol optimizations are recommended for using RBM18 antibodies in western blotting?

For optimal western blot results with RBM18 antibodies:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for total protein extraction

  • Include phosphatase inhibitors if studying phosphorylation states

  • Sonicate briefly to shear DNA and reduce sample viscosity

• Gel selection and running conditions:

  • 12-15% SDS-PAGE gels are recommended for optimal resolution of the 21.6 kDa RBM18 protein

  • Run at 100-120V to prevent overheating and protein degradation

• Transfer optimization:

  • Use PVDF membrane for enhanced protein binding

  • Transfer at 100V for 1 hour or 30V overnight at 4°C

  • Verify transfer efficiency with reversible staining (Ponceau S)

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST (1 hour, room temperature)

  • Incubate with primary RBM18 antibody at 1:100-200 dilution overnight at 4°C

  • Wash thoroughly (3-5 times, 5 minutes each) with TBST

  • Incubate with HRP-conjugated secondary antibody (1:2000-1:5000) for 1 hour at room temperature

• Detection and controls:

  • Use ECL substrate appropriate for expected expression level

  • Include positive control (tissue/cell line with known RBM18 expression)

  • Include negative control (RBM18-negative cell line if available)

  • Use recombinant RBM18 protein as reference standard

What are the critical considerations for immunohistochemical detection of RBM18 in tissue samples?

For successful IHC detection of RBM18:

• Tissue preparation and fixation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues are suitable

  • Section thickness of approximately 4 μm is recommended

  • Place sections on positively charged slides to prevent detachment

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) is recommended

  • Use citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Pressure cooker or microwave heating for 10-20 minutes

• Blocking and antibody application:

  • Block endogenous peroxidase with 3% H₂O₂ for 5-10 minutes

  • Apply protein block (normal serum matching secondary antibody species)

  • Incubate with RBM18 primary antibody at 1:50-1:200 dilution

  • Optimal incubation: 1 hour at room temperature or overnight at 4°C

• Detection systems:

  • Polymer-based detection systems offer enhanced sensitivity

  • DAB (3,3'-diaminobenzidine) is the recommended chromogen

  • Counterstain with hematoxylin for nuclear visualization

• Controls and validation:

  • Include positive control tissues

  • Use isotype control antibody at the same concentration

  • Consider dual staining with established RNA-binding protein markers

  • Expected localization: Primarily nuclear, with potential cytoplasmic components

What strategies can be employed to validate antibody specificity in RBM18 research?

Thorough validation is essential for confident interpretation of results:

• Genetic approaches:

  • Compare staining in wild-type versus RBM18 knockout/knockdown models

  • Rescue experiments with RBM18 re-expression

  • Use siRNA/shRNA-mediated depletion with gradient concentrations

• Biochemical validation:

  • Pre-absorption with recombinant RBM18 protein or immunizing peptide

  • Peptide competition assays with increasing concentrations of blocking peptide

  • Western blot verification showing a single band at the expected molecular weight (21.6 kDa)

• Orthogonal methods:

  • Compare protein expression with mRNA levels (qRT-PCR)

  • Use multiple antibodies targeting different epitopes of RBM18

  • Protein array testing against target protein plus non-specific proteins

• Cross-platform validation:

  • Confirm localization patterns across multiple techniques (IF, IHC, subcellular fractionation)

  • Mass spectrometry identification following immunoprecipitation

  • Correlation with tagged RBM18 expression constructs

What approaches can resolve weak or absent signals when using RBM18 antibodies?

When facing detection challenges:

• Signal enhancement strategies:

  • Increase antibody concentration (test range: 1:50 to 1:200)

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

  • Use signal amplification systems (TSA, polymer-based detection)

  • Optimize antigen retrieval conditions (test both citrate and EDTA buffers)

• Sample-related considerations:

  • Test fresh samples to rule out epitope degradation

  • Ensure proper fixation (over-fixation can mask epitopes)

  • Check expression levels in different cell types/tissues

  • Consider enrichment by subcellular fractionation

• Antibody selection:

  • Test antibodies targeting different epitopes of RBM18

  • Consider polyclonal antibodies for increased epitope recognition

  • Verify antibody lot consistency and storage conditions

• Technical optimizations:

  • Reduce washing stringency (lower salt concentration, shorter washes)

  • Test alternative blocking reagents (BSA, casein, commercial blockers)

  • For western blots, transfer to PVDF instead of nitrocellulose membrane

  • For IHC/IF, test different mounting media to reduce photobleaching

How can researchers differentiate between RBM18 and other RNA-binding motif proteins in their experiments?

Ensuring specificity when studying similar RBM family proteins:

• Epitope selection and antibody validation:

  • Choose antibodies targeting unique regions rather than conserved RNA-binding motifs

  • Verify specificity using recombinant proteins of multiple RBM family members

  • Perform peptide competition assays with both target and related protein sequences

• Cross-reactivity testing:

  • Express tagged versions of multiple RBM proteins and test antibody specificity

  • Use protein arrays containing RBM18 and related proteins (like RBM8A)

  • Compare migration patterns on western blots (RBM18: 21.6 kDa vs. other RBM proteins)

• Experimental controls:

  • Include knockout/knockdown controls for RBM18 specifically

  • Use parallel detection with antibodies against other RBM family members

  • Compare with fluorescently tagged RBM18 in overexpression systems

• Advanced methods for improved specificity:

  • Design sequential immunoprecipitation to deplete cross-reactive proteins

  • Use targeted mass spectrometry with immunoprecipitation to confirm identity

  • Consider DyAb sequence-based antibody design approaches for enhanced specificity

What experimental design considerations are important when using RBM18 antibodies in co-immunoprecipitation studies?

For successful co-IP experiments investigating RBM18 interactions:

• Lysis buffer optimization:

  • Test multiple buffers with varying detergent strengths:

    • NP-40 buffer (0.5% NP-40) for preserving most interactions

    • RIPA buffer for stronger protein extraction but may disrupt some interactions

    • Include RNase inhibitors to preserve RNA-dependent interactions

    • Add protease and phosphatase inhibitors freshly

• Antibody selection and binding conditions:

  • Choose antibodies validated for immunoprecipitation

  • Determine optimal antibody-to-lysate ratio (typically 2-5 μg antibody per 500 μg protein)

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

  • Consider dynamic interaction conditions (vary salt concentration, temperature)

• Controls and validation:

  • Include "no antibody" and isotype control immunoprecipitations

  • Perform reverse co-IP with antibodies against suspected interacting partners

  • Include RNase treatment to distinguish RNA-dependent from direct protein interactions

  • Validate interactions with alternative methods (proximity ligation assay, FRET)

• Detection strategies:

  • Western blot for known/suspected interactors

  • Mass spectrometry for unbiased interaction discovery

  • RNA sequencing of co-precipitated RNAs to identify bound transcripts

How might RBM18 antibodies contribute to understanding RNA processing mechanisms in disease contexts?

While specific disease associations of RBM18 are still being investigated, several methodological approaches can be employed:

• Comparative expression analysis:

  • Use RBM18 antibodies to compare protein levels in normal versus disease tissues

  • Perform quantitative immunohistochemistry across tissue microarrays

  • Correlate expression with clinical outcomes in patient samples

• RNA-protein interaction studies:

  • Combine RBM18 immunoprecipitation with RNA sequencing (RIP-seq)

  • Identify differential RNA binding in disease states

  • Map interaction changes during disease progression

• Integration with RNA splicing analysis:

  • Correlate RBM18 expression with alternative splicing events

  • Investigate co-localization with spliceosome components using dual immunofluorescence

  • Determine if disease-associated splice variants correlate with RBM18 expression changes

• Therapeutic target assessment:

  • Evaluate RBM18 expression in response to various treatments

  • Investigate potential as a biomarker for disease progression

  • Explore correlations with immune checkpoint inhibitors similar to findings with other RBM proteins

What emerging antibody technologies might enhance RBM18 research in the near future?

Advanced antibody technologies are transforming RNA-binding protein research:

• DyAb sequence-based antibody design:

  • Computational methods for designing antibodies with customized specificity profiles

  • Potential for generating RBM18-specific antibodies with reduced cross-reactivity to related proteins

  • Machine learning approaches integrating small training datasets to predict binding improvements

• Single-domain antibodies and nanobodies:

  • Smaller binding fragments with enhanced tissue penetration

  • Potential for accessing cryptic epitopes in complex RNA-protein structures

  • Improved access to structured domains of RBM18

• Intracellular antibodies (intrabodies):

  • Expression of functional antibody fragments within living cells

  • Real-time tracking of RBM18 dynamics during RNA processing events

  • Potential for targeted disruption of specific RBM18 interactions

• Proximity labeling with antibody-enzyme fusions:

  • Antibody-BioID or antibody-APEX fusions for proximity labeling

  • Identification of the RBM18 protein interactome in specific subcellular compartments

  • Temporal mapping of dynamic interactions during cellular responses

How can RBM18 antibodies be integrated with other technologies for multi-omics research?

Combining antibody-based detection with complementary technologies:

• Spatial transcriptomics integration:

  • Correlate RBM18 protein localization with RNA expression profiles in tissue sections

  • Combine immunofluorescence with in situ RNA detection methods

  • Create spatial maps of RBM18-RNA interactions in complex tissues

• Single-cell protein and RNA analysis:

  • Use RBM18 antibodies in single-cell western blotting or CyTOF analysis

  • Correlate with single-cell RNA sequencing data

  • Identify cell populations with unique RBM18 expression/function signatures

• Live-cell imaging of RNA processing:

  • Combine fluorescently-labeled antibody fragments with RNA tracking technologies

  • Monitor dynamic changes in RBM18-RNA interactions during cellular processes

  • Correlate with functional readouts of RNA processing events

• Structural biology integration:

  • Use antibodies as crystallization chaperones for structural studies of RBM18

  • Combine with cryo-EM to visualize larger RBM18-containing complexes

  • Map functional domains through epitope-specific antibody binding