rbm4.1 Antibody

What is RBM4 and what cellular functions does it regulate?

RBM4 (RNA-binding motif protein 4) is a multifunctional RNA-binding factor involved in several critical cellular processes including:

• Alternative splicing of pre-mRNA

• Translational regulation

• Muscle cell differentiation

• Adaptation of protein synthesis in response to cellular and environmental cues

• Exon selection and skipping, particularly in muscle cell-specific contexts

• Modulation of circadian clock-related mRNA translation

• Suppression of cap-dependent translation under stress conditions

• Involvement in miRNA-guided RNA cleavage and translation suppression

Through these diverse interactions, RBM4 influences the expression of genes involved in cellular development and differentiation, allowing cells to adapt protein synthesis to environmental conditions . Research has also demonstrated that RBM4 can function as a tumor suppressor by controlling cancer-specific alternative splicing events .

Which applications are RBM4.1 antibodies validated for?

RBM4.1 antibodies have been validated for multiple experimental applications with varying recommended dilutions:

Optimum dilutions may be sample-dependent, so titration is recommended in each specific experimental system to achieve optimal results .

What antigen retrieval methods are recommended for RBM4.1 antibody in IHC applications?

For immunohistochemical applications with RBM4.1 antibody, two primary antigen retrieval methods have been validated:

• Preferred method: TE buffer pH 9.0 has shown optimal results for exposing RBM4 epitopes in formalin-fixed paraffin-embedded (FFPE) tissues .

• Alternative method: Citrate buffer pH 6.0 can also be used when TE buffer is unavailable, though potentially with somewhat reduced sensitivity .

The choice between these methods may depend on tissue type and fixation conditions. For human brain tissues, which show strong RBM4 expression, both methods typically yield acceptable results, while tissues with lower expression levels may benefit from the more efficient TE buffer method. Complete antigen retrieval protocol should include appropriate heating (typically microwave technology), incubation times, and washing steps with PBS before primary antibody application .

What is the molecular weight of RBM4 protein and what band pattern should I expect on Western blots?

When performing Western blot analysis using RBM4.1 antibody, you should observe the following:

• The predicted molecular weight for RBM4 is approximately 40 kDa

• Observed molecular weight on SDS-PAGE gels also typically runs at 40 kDa

• Western blot analysis shows consistent banding patterns across various human cell lines and tissues:

  • RT4 (human urinary bladder cancer cell line)

  • U-251 MG (human brain glioma cell line)

  • Human liver lysate

  • Human tonsil lysate

  • Human plasma (after IgG/HSA depletion)

For accurate molecular weight determination, include appropriate molecular weight markers and positive control samples like HeLa cells or brain tissue lysates, which have been consistently validated to express detectable levels of RBM4 .

What are the optimal sample preparation protocols for different applications of RBM4.1 antibody?

Sample preparation varies significantly depending on the intended application:

For Western Blotting:

• Tissue lysates: Homogenize tissues in RIPA buffer with protease inhibitors

• Cell lysates: Lyse cells directly in sample buffer containing SDS and DTT

• Recommended loading: 20-30 μg total protein per lane

• Include phosphatase inhibitors when studying RBM4 phosphorylation status

• Denaturation: Heat samples at 95°C for 5 minutes before loading

For Immunohistochemistry:

• Formalin fixation for 24-48 hours is compatible with epitope detection

• Paraffin embedding following standard histological protocols

• Section thickness: 4-6 μm sections provide optimal results

• Antigen retrieval: Use TE buffer pH 9.0 with microwave heating

• Blocking: 3% H₂O₂ for 15 minutes to block endogenous peroxidase activity

• Overnight primary antibody incubation at 4°C for maximum specificity

For Immunoprecipitation:

• Cell/tissue lysis in non-denaturing buffers is essential to maintain protein interactions

• Pre-clearing lysates with Protein A/G beads reduces non-specific binding

• Incubate 0.5-4.0 μg antibody with 1-3 mg of total protein lysate

• Capture with appropriate secondary reagents (Protein A/G beads)

The choice of buffer system significantly impacts antibody performance, particularly for applications studying RBM4's RNA-binding properties or protein interactions.

How can I validate the specificity of RBM4.1 antibody results in my experimental system?

Thorough validation of RBM4.1 antibody specificity is critical to ensure experimental rigor. Several complementary approaches are recommended:

• Positive and negative control samples:

  • Known positive controls: Human brain, heart tissues, HeLa cells

  • Negative controls: Replace primary antibody with PBS or non-immune IgG

• Knockdown/knockout validation:

  • RNAi-mediated knockdown of RBM4 should result in reduced signal

  • CRISPR/Cas9-generated knockout cells provide definitive negative controls

  • Multiple published studies have validated antibody specificity using KD/KO approaches

• Peptide competition assays:

  • Pre-incubation of antibody with immunizing peptide should abolish specific signal

  • Commercial immunogen peptides are available as blocking controls

• Cross-validation with multiple antibodies:

  • Use antibodies targeting different epitopes of RBM4

  • Compare results between polyclonal and monoclonal antibodies when available

• Molecular weight verification:

  • Observed band should match the predicted 40 kDa size of RBM4

  • Additional bands may represent isoforms, post-translational modifications, or degradation products

• Recombinant protein controls:

  • Overexpression of tagged RBM4 provides positive control and size reference

  • Detection with both anti-tag and anti-RBM4 antibodies confirms specificity

These validation approaches should be documented and included in publications to enhance experimental reproducibility.

What are the known cross-reactivity issues with RBM4.1 antibodies?

Understanding potential cross-reactivity is essential for accurate interpretation of experimental results:

• RBM4 isoforms cross-reactivity:

  • RBM4.1 antibodies may detect both RBM4A and RBM4B isoforms depending on the epitope

  • Some antibodies are specifically raised against unique regions to distinguish these isoforms

  • Check manufacturer specifications for isoform specificity details

• Species cross-reactivity:

  • Many RBM4.1 antibodies show reactivity with human, mouse, and rat samples

  • Sequence conservation allows cross-species application in most cases

  • Western blot validation in specific species is recommended before proceeding to more complex applications

• Related RBM family proteins:

  • The RNA-binding motif (RBM) family contains numerous proteins with similar domains

  • Potential for cross-reactivity with RBM8A, RBM14, or other family members

  • Antibodies raised against unique C-terminal regions typically show enhanced specificity

• Exclusion validation:

  • Protein array testing with 364 human recombinant protein fragments helps determine cross-reactivity profiles

  • Published antibodies have undergone stringent selection to minimize off-target binding

When interpreting results, particularly in new experimental systems, consider including appropriate controls to rule out potential cross-reactivity issues.

How can RBM4.1 antibody be used to study cancer-related alternative splicing events?

RBM4 has been identified as a key regulator of cancer-related alternative splicing with tumor-suppressive functions. To investigate these mechanisms:

• Specific splicing events analysis:

  • RBM4 controls alternative splicing of apoptotic genes like Bcl-x, shifting the balance between anti-apoptotic and pro-apoptotic isoforms

  • Use RBM4.1 antibody in RIP assays to identify direct binding to target pre-mRNAs

  • Confirm direct binding to the Bcl-x pre-mRNA through RIP followed by qPCR

  • Examine RBM4 binding to sequence motifs like CGGCGG and GTAACG in target mRNAs

• Functional studies in cancer models:

  • Examine RBM4 expression levels across various cancer types using IHC

  • Correlate expression with cellular phenotypes (proliferation, migration, apoptosis)

  • RBM4 has been shown to suppress proliferation and migration of cancer cells

  • In vivo studies have demonstrated that RBM4 overexpression suppresses tumor growth in mouse xenograft models

• Signaling pathway integration:

  • Investigate RBM4's impact on MAPK signaling pathways (ERK1/2, JNK, p38)

  • Use Western blotting to assess phosphorylation states of these kinases in response to RBM4 modulation

  • Study subcellular localization changes in response to stress conditions using IF

• Splicing reporter assays:

  • Employ minigene constructs containing cancer-relevant alternative exons

  • Analyze how RBM4 binding sites influence splicing outcomes

  • Mutational analysis of binding sites confirms sequence-specific interactions

These approaches provide mechanistic insights into how RBM4 functions as a tumor suppressor through control of cancer-specific alternative splicing.

What protein-protein interactions can be identified using RBM4.1 antibodies in co-immunoprecipitation experiments?

RBM4.1 antibodies can be powerful tools for identifying protein interaction networks that regulate splicing and translation:

• Splicing regulatory complexes:

  • Co-IP followed by mass spectrometry can identify RBM4-associated splicing factors

  • Known interactions include antagonistic relationship with PTBP1 (PTB)

  • Investigate interactions with other SR proteins and hnRNPs in tissue-specific contexts

• Translation machinery interactions:

  • RBM4 interacts with translation factors like EIF4A1 during cellular stress

  • These interactions stimulate IRES-dependent translation initiation

  • Co-IP protocols should use non-denaturing buffers to preserve these interactions

• miRNA-related complexes:

  • RBM4 associates with AGO2-containing miRNPs

  • Identify interactions with RISC components using RBM4.1 antibody pulldowns

  • RBM4 promotes association of miRNPs with target mRNAs

• Experimental considerations:

  • Use appropriate lysis conditions (typically NP-40 or CHAPS-based buffers)

  • RNase treatment distinguishes RNA-dependent from direct protein interactions

  • Crosslinking approaches (formaldehyde or DSP) can stabilize transient interactions

  • For IP, use 0.5-4.0 μg antibody per 1-3 mg protein lysate

  • Negative controls should include isotype-matched IgG

By identifying RBM4's protein interaction networks in different cellular contexts, researchers can gain insights into its diverse regulatory functions.

How does phosphorylation affect RBM4 function and how can this be studied?

RBM4 undergoes phosphorylation that regulates its activity, subcellular localization, and protein interactions:

• Detection of phosphorylated RBM4:

  • Western blotting may reveal mobility shifts for phosphorylated forms

  • Phospho-specific antibodies (when available) can directly detect specific modifications

  • Phosphatase treatment of samples confirms phosphorylation-dependent mobility shifts

  • Include phosphatase inhibitors in lysis buffers to preserve in vivo phosphorylation status

• Functional consequences of phosphorylation:

  • Phosphorylation can alter RBM4's subcellular distribution between nucleus and cytoplasm

  • This redistribution affects its participation in splicing versus translation regulation

  • Stress conditions induce phosphorylation and cytoplasmic accumulation

  • During muscle differentiation, phosphorylation modulates RBM4's splicing regulatory activity

• Experimental approaches:

  • Immunofluorescence with RBM4.1 antibody (0.25-2 μg/mL) to track subcellular localization changes

  • Phosphomimetic and phospho-dead mutants to study specific sites

  • Kinase inhibitor treatments to identify regulatory pathways

  • Fractionation studies followed by Western blotting to quantify distribution changes

• Signaling pathway integration:

  • MAPK pathways (ERK1/2, JNK, p38) may regulate RBM4 phosphorylation

  • Western blotting with phospho-specific antibodies against these kinases

  • Correlation between kinase activation and RBM4 phosphorylation status

Understanding RBM4 phosphorylation provides insights into how cells regulate RNA metabolism in response to environmental cues and developmental signals.

What are the most common technical issues with RBM4.1 antibody and how can they be resolved?

Researchers may encounter several technical challenges when working with RBM4.1 antibodies. Here are solutions to common problems:

• Low signal intensity in Western blots:

  • Increase antibody concentration (try 1:500 instead of 1:2000)

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

  • Use more sensitive detection systems (enhanced chemiluminescence)

  • Increase protein loading (40-50 μg per lane)

  • Ensure transfer efficiency for 40 kDa proteins

  • Fresh antibody dilutions may improve performance

• High background in immunohistochemistry:

  • More stringent blocking (5% BSA or 10% normal serum)

  • Reduce primary antibody concentration (try 1:200 instead of 1:50)

  • Increase washing steps (5x5 minutes with PBS-T)

  • Use more specific detection systems

  • Proper antigen retrieval optimization (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Pre-absorption with immunizing peptide to confirm specificity

• Inconsistent results between experiments:

  • Standardize lysate preparation methods

  • Use positive control samples (brain tissue, HeLa cells)

  • Prepare larger antibody aliquots to reduce freeze-thaw cycles

  • Standardize incubation times and temperatures

  • Use automated systems where possible for consistent processing

• Multiple bands in Western blots:

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

  • Use freshly prepared samples with protease inhibitors

  • Compare patterns with published literature

  • Perform peptide competition assays to determine which bands are specific

• Failed immunoprecipitation:

  • Optimize antibody amount (0.5-4.0 μg per 1-3 mg protein)

  • Try different lysis buffers (RIPA vs. NP-40 based)

  • Pre-clear lysates to reduce non-specific binding

  • Cross-link antibody to beads for cleaner results

  • Verify if the epitope is accessible in native conditions

Careful optimization of these parameters will enhance reproducibility and reliability of results.

How should RBM4.1 antibody be stored and handled to maintain optimal performance?

Proper storage and handling of RBM4.1 antibody is critical for maintaining its activity and specificity:

• Long-term storage conditions:

  • Store at -20°C as recommended by manufacturers

  • Antibodies in glycerol buffer (typical formulation is 50% glycerol in PBS with 0.02% sodium azide) are stable for at least one year at -20°C

  • Smaller-volume antibodies (20 μL) may contain BSA (0.1%) as an additional stabilizer

• Working solution preparation:

  • Thaw antibody completely before use

  • Mix gently by inversion rather than vortexing to prevent protein denaturation

  • Brief centrifugation to collect contents at the bottom of the tube

  • Prepare fresh dilutions for each experiment in appropriate buffer

  • Return stock antibody to -20°C immediately after use

• Aliquoting recommendations:

  • For larger volume antibodies, create small working aliquots

  • Aliquoting is generally unnecessary for antibodies already in 50% glycerol at -20°C

  • Minimize freeze-thaw cycles (ideally <5 cycles)

  • Label aliquots with all relevant information (antibody, lot #, date)

• Transport conditions:

  • Transport on wet ice for short periods

  • For longer transport, use dry ice or cold packs

  • Avoid temperature fluctuations during shipping

• Quality control checks:

  • Periodically verify antibody performance using established positive controls

  • Monitor for precipitates or color changes that might indicate degradation

  • Document lot-to-lot variations when receiving new antibody batches

Following these storage and handling guidelines will help ensure consistent experimental results and extend the useful life of RBM4.1 antibodies.

What quantification methods are most appropriate for RBM4 expression analysis in different experimental systems?

Accurate quantification of RBM4 expression requires selecting appropriate methods based on the experimental context:

• Western blot quantification:

  • Densitometric analysis of immunoblots using established software (ImageJ, etc.)

  • Normalization to loading controls (β-actin, GAPDH) is essential

  • Linear dynamic range determination using serial dilutions of positive controls

  • Comparison between multiple independent blots requires inclusion of common reference samples

  • Report relative expression rather than absolute values unless using purified standards

• Immunohistochemistry scoring systems:

  • Combined scoring system incorporating staining intensity and percentage of positive cells

  • Intensity scoring: 0 (no staining), 1 (light), 2 (moderate), 3 (deep staining)

  • Multiply intensity score by percentage of positive cells (0-100%) to obtain total score

  • Blind assessment by two experienced pathologists enhances objectivity

  • Digital image analysis provides more objective quantification

• Immunofluorescence quantification:

  • Mean fluorescence intensity measurement within defined cellular compartments

  • Nuclear/cytoplasmic ratio calculation to assess subcellular distribution

  • Z-stack acquisition for volume-based quantification

  • Standardized exposure settings across experimental conditions

  • Co-localization analysis with markers of specific cellular compartments

• qPCR correlation:

  • Complementary mRNA expression analysis with protein levels

  • Discrepancies may reveal post-transcriptional regulation mechanisms

  • Design primers to distinguish between RBM4 isoforms

• Mass spectrometry approaches:

  • Absolute quantification using isotope-labeled standards

  • Relative quantification across experimental conditions

  • PTM analysis for phosphorylation and other modifications

The choice of quantification method should be justified based on experimental objectives and the specific questions being addressed about RBM4 biology.

How can RBM4.1 antibodies be used to investigate its role in cancer progression?

RBM4 has demonstrated tumor suppressor functions, making it an important target for cancer research:

• Expression analysis across cancer types:

  • Use immunohistochemistry (1:50-1:500 dilution) to examine RBM4 levels in human tumor samples

  • Compare expression between tumor and adjacent normal tissues

  • Correlation with clinical parameters and patient outcomes

  • RBM4 has been studied in various cancers including gastric cancer, brain gliomas, and lung cancer

• Functional validation studies:

  • Overexpression of RBM4 in cancer cell lines suppresses proliferation and migration

  • Western blotting (1:500-1:2000) to confirm expression changes in modified cell lines

  • Xenograft tumor models show reduced tumor growth with RBM4 overexpression

  • H&E staining and IHC of tumor tissues to correlate RBM4 expression with histopathological features

• Molecular mechanism investigations:

  • RBM4 controls cancer-related alternative splicing events

  • Study Bcl-x splicing to understand apoptosis regulation

  • RIP assays demonstrate direct binding to pre-mRNAs of cancer-relevant genes

  • Monitor MAPK signaling pathway components (ERK1/2, JNK, p38) to understand downstream effects

• Therapeutic implications:

  • Potential biomarker for cancer prognosis

  • Target for cancer therapy through modulation of splicing

  • Correlation studies between RBM4 expression and therapeutic responses

The significance of RBM4 in cancer biology makes RBM4.1 antibodies valuable tools for both basic cancer research and translational applications.

What are the recommended protocols for studying RBM4's role in muscle differentiation?

RBM4 functions as a muscle cell differentiation-promoting factor through regulation of alternative splicing:

• Expression dynamics during differentiation:

  • Track RBM4 expression changes during myoblast differentiation

  • Western blotting (1:500-1:2000) at sequential timepoints

  • Immunofluorescence (0.25-2 μg/mL) to monitor subcellular localization changes

  • Correlation with muscle-specific markers (MyoD, myogenin, MHC)

• Alternative splicing regulation:

  • RBM4 activates exon skipping of the PTB pre-mRNA during muscle cell differentiation

  • Analysis of alpha tropomyosin splicing patterns as a model target

  • RT-PCR to detect isoform switches in target genes

  • RBM4 antagonizes PTBP1 to modulate muscle-specific exon selection

• Functional studies in muscle models:

  • C2C12 myoblast differentiation model

  • Primary muscle cell cultures

  • Knockdown/overexpression followed by differentiation assessment

  • Morphological analysis and fusion index calculation

  • Co-staining with RBM4.1 antibody and muscle markers

• Mechanistic investigations:

  • RBM4 binding to intronic pyrimidine-rich sequences in target pre-mRNAs

  • Analysis of RBM4 association with miRNAs during muscle differentiation

  • Phosphorylation status changes during differentiation

  • RNA-seq to identify global splicing changes dependent on RBM4

These approaches provide comprehensive insights into how RBM4 contributes to the complex regulatory networks governing muscle differentiation.

How can the role of RBM4 in stress response pathways be investigated using specific antibodies?

RBM4 plays important roles in cellular stress responses, particularly in translational regulation:

• Stress-induced subcellular redistribution:

  • Immunofluorescence (0.25-2 μg/mL) to track RBM4 localization under various stressors

  • Common stressors: arsenite, thapsigargin, heat shock, hypoxia

  • Co-staining with stress granule markers (G3BP, TIA-1)

  • Time-course analysis of redistribution kinetics

  • Fractionation studies followed by Western blotting for quantification

• Translation regulation under stress:

  • RBM4 exerts suppressive activity on cap-dependent translation under stress conditions

  • RBM4 stimulates IRES-dependent translation via EIF4A1 recruitment

  • Polysome profiling combined with RBM4 detection in fractions

  • Reporter assays with cap-dependent and IRES-dependent constructs

  • RIP assays to identify direct binding to CU-rich elements in mRNA 3'UTRs

• Phosphorylation dynamics:

  • Stress-induced phosphorylation alters RBM4 function

  • Western blotting to detect mobility shifts

  • Phosphatase treatment controls

  • Analysis of upstream kinase pathways (MAPK, AKT)

  • Correlation between stress intensity and phosphorylation levels

• Target mRNA identification:

  • RBM4 binds to specific mRNAs under stress conditions

  • CLIP-seq to identify binding sites genome-wide

  • RIP followed by RT-qPCR for candidate mRNAs

  • Correlation with translational efficiency changes

  • Focus on mRNAs with IRES elements or CU-rich motifs

Understanding RBM4's role in stress responses provides insights into cellular adaptation mechanisms and may reveal therapeutic targets for stress-related pathologies.

How should researchers address conflicting results when studying RBM4 in different experimental systems?

When facing contradictory findings across different experimental systems, a systematic approach is necessary:

• Technical versus biological variation assessment:

  • Evaluate antibody lot-to-lot variation using consistent positive controls

  • Compare different detection methods (WB vs. IHC vs. IF) for consistency

  • Assess if differences reflect actual biological variation between systems

  • Document detailed experimental conditions that might explain discrepancies

• Systematic validation approaches:

  • Use multiple antibodies targeting different epitopes of RBM4

  • Employ complementary detection methods (protein vs. mRNA vs. function)

  • Include appropriate positive and negative controls in all experiments

  • Cross-validate findings using orthogonal techniques

• Context-dependent regulation consideration:

  • RBM4 functions can vary dramatically between:

    • Cell types (e.g., muscle cells vs. cancer cells)

    • Developmental stages

    • Stress conditions

    • Disease states

  • These biological differences may explain apparently contradictory results

• Common sources of discrepancy:

  • Post-translational modifications affecting epitope recognition

  • Alternative splicing of RBM4 itself creating different isoforms

  • Subcellular localization differences affecting detection

  • Protein-protein or protein-RNA interactions masking antibody binding sites

• Reporting recommendations:

  • Transparently document all experimental parameters

  • Include detailed antibody information (catalog number, lot, dilution)

  • Report both positive and negative findings

  • Acknowledge limitations and potential sources of variation

By systematically addressing these factors, researchers can better understand whether conflicting results represent technical artifacts or biologically meaningful context-dependent differences in RBM4 function.

What are the critical controls required for publication-quality RBM4 research?

For robust and publishable research on RBM4, the following controls are essential:

• Antibody validation controls:

  • Positive control samples (brain tissue, HeLa cells)

  • Negative controls (primary antibody omission, isotype-matched IgG)

  • Knockdown/knockout validation demonstrating specificity

  • Peptide competition assays to confirm binding specificity

  • Secondary antibody-only controls to assess non-specific binding

• Expression analysis controls:

  • Loading controls for Western blot (β-actin, GAPDH)

  • Reference gene selection for qPCR normalization

  • Internal control samples across multiple experiments for inter-experimental normalization

  • Biological replicates (minimum n=3) from independent experiments

  • Technical replicates to assess methodological variation

• Functional study controls:

  • Vector-only controls for overexpression studies

  • Non-targeting siRNA/shRNA controls for knockdown studies

  • Rescue experiments to confirm specificity of observed phenotypes

  • Dose-response relationships to establish biological relevance

  • Time-course analyses to distinguish primary from secondary effects

• Cancer model specific controls:

  • Paired normal-tumor tissue comparisons

  • Multiple cell lines representing different cancer subtypes

  • In vivo controls (vector control injection sites in the same animals)

  • Correlation with established cancer markers

• Reproducibility measures:

  • Independent biological replicates with clearly reported n values

  • Statistical analysis appropriate to the experimental design

  • Effect size reporting beyond p-values

  • Blinded assessment for subjective measurements like IHC scoring

These controls should be comprehensively documented in materials and methods sections and included in supplementary data where appropriate.

How can researchers distinguish between RBM4 isoforms and closely related family members?

Distinguishing between highly similar proteins requires careful experimental design:

• Isoform-specific detection strategies:

  • RBM4 has multiple isoforms (e.g., RBM4A/RBM4B)

  • Select antibodies raised against unique regions not shared between isoforms

  • Design isoform-specific primers for RT-PCR analysis

  • Use recombinant isoform proteins as positive controls

  • Mass spectrometry can identify isoform-specific peptides

• Related family member discrimination:

  • The RBM family contains numerous related proteins

  • Verify antibody cross-reactivity profiles against related family members

  • Protein array testing with 364 human recombinant protein fragments helps determine specificity

  • Focus on C-terminal domains which typically show greater divergence

  • Epitope mapping to confirm target specificity

• Functional discrimination approaches:

  • Selective knockdown of specific isoforms using targeted siRNAs

  • Rescue experiments with isoform-specific constructs

  • Analysis of differential subcellular localization patterns

  • Examination of tissue-specific expression profiles

  • Investigation of isoform-specific binding partners

• Technical considerations:

  • Higher percentage gels (12-15%) for better separation of similar molecular weight proteins

  • Extended gel running times to maximize resolution

  • 2D gel electrophoresis to separate based on both pI and molecular weight

  • Careful selection of blocking reagents to minimize non-specific binding

  • Use of monoclonal antibodies when isoform specificity is critical

Proper discrimination between RBM4 isoforms and family members is essential for accurate interpretation of experimental results and for understanding the distinct biological functions of these closely related proteins.

How can RBM4.1 antibodies be utilized in studying circadian rhythm regulation?

RBM4 has emerging roles in circadian clock regulation, presenting exciting research opportunities:

• Translational regulation of clock genes:

  • RBM4 is required for translational activation of PER1 mRNA in response to circadian cues

  • Direct binding to the 3'-UTR of PER1 mRNA has been demonstrated

  • Immunoprecipitation combined with RT-qPCR to identify clock-related target mRNAs

  • Circadian time-course analysis of RBM4-mRNA interactions

  • Polysome profiling to assess translational efficiency of clock genes

• Circadian phosphorylation dynamics:

  • Time-of-day-dependent post-translational modifications of RBM4

  • Western blotting across circadian time points

  • Phosphatase treatment to confirm modifications

  • Correlation with activity of circadian-regulated kinases

  • Mass spectrometry to identify specific modified residues

• Subcellular localization rhythms:

  • Immunofluorescence tracking of RBM4 localization across the circadian cycle

  • Co-localization with clock proteins (PER, CRY, CLOCK, BMAL1)

  • Nuclear/cytoplasmic shuttling related to circadian phase

  • Tissue-specific patterns in clock-relevant tissues (SCN, liver, muscle)

• Functional manipulation studies:

  • Effects of RBM4 knockdown/overexpression on circadian period/amplitude

  • Real-time luminescence monitoring of clock gene reporters

  • Phase-shifting responses to zeitgebers

  • Tissue-specific perturbations in circadian model systems

This research direction links RNA processing mechanisms to circadian biology, potentially revealing novel regulatory nodes in the circadian clock system.

What recent methodological advances enhance the application of RBM4.1 antibodies in research?

Emerging technologies are expanding the utility of RBM4.1 antibodies in research:

• Proximity labeling approaches:

  • BioID or TurboID fusion with RBM4 to identify proximal proteins

  • APEX2 systems for temporal control of labeling

  • Protein interaction networks under different cellular conditions

  • Comparison between nuclear and cytoplasmic interaction partners

  • Combination with RBM4.1 antibodies for validation of identified partners

• Single-cell applications:

  • Single-cell Western blotting for heterogeneity analysis

  • Mass cytometry (CyTOF) with metal-conjugated antibodies

  • Imaging mass cytometry for spatial information in tissues

  • Single-cell immunofluorescence combined with RNA-FISH for protein-RNA correlations

  • Microfluidic platforms for high-throughput single-cell analysis

• Live-cell imaging innovations:

  • Antibody-based fluorescent biosensors for RBM4 activity

  • nanobody development for live-cell applications

  • FRET-based approaches to monitor protein-protein interactions

  • Optogenetic control of RBM4 activity combined with antibody detection

  • Super-resolution microscopy for detailed localization studies

• High-throughput screening applications:

  • Automated immunofluorescence in drug screening platforms

  • CRISPR screens with RBM4 activity readouts

  • Patient-derived organoid screening with RBM4.1 antibody-based assays

  • Tissue microarray analysis across large sample collections

These methodological advances enable more sophisticated investigations into RBM4 biology with higher resolution, throughput, and physiological relevance.

How can integrative multi-omics approaches incorporate RBM4.1 antibody-based data?

Modern research increasingly combines multiple data types for comprehensive understanding:

• Integration of antibody-based data with genomics:

  • ChIP-seq using RBM4.1 antibodies to identify potential DNA interactions

  • Correlation between genetic variants and RBM4 binding/function

  • Integration with GWAS data for disease associations

  • Analysis of expression quantitative trait loci (eQTLs) affecting RBM4 levels

• Combined proteomics and antibody approaches:

  • IP-mass spectrometry to identify RBM4 interactomes

  • Phosphoproteomics to map RBM4 modification sites

  • Correlation between global proteome changes and RBM4 activity

  • Validation of proteomic findings with targeted antibody-based approaches

• RNA-related multi-omics:

  • CLIP-seq or RIP-seq to identify RBM4 RNA targets

  • Integration with RNA-seq for splicing outcome correlations

  • Validation of key targets with RBM4.1 antibody-based methods

  • Ribosome profiling to assess translational impacts

  • miRNA profiling to connect with RBM4's role in miRNA-guided processes

• Computational integration frameworks:

  • Network analysis incorporating RBM4 interaction data

  • Machine learning approaches to predict RBM4 functions

  • Systems biology modeling of splicing and translation regulation

  • Cross-platform data normalization strategies

  • Visualization tools for complex multi-omic datasets

• Physiological context integration:

  • Tissue-specific analyses across multiple platforms

  • Developmental trajectories integrating multiple data types

  • Disease-specific multi-omic profiles

  • Response to perturbations across multiple levels

These integrative approaches provide a holistic view of RBM4 biology beyond what any single technique can reveal, contributing to a deeper understanding of its complex roles in cellular regulation.