RBM38 Antibody, HRP conjugated

What is RBM38 and what cellular functions does it perform?

RBM38 (RNA Binding Motif Protein 38) is a cell cycle protein found in both the cytosol and nucleus that exists as two alternatively spliced isoforms: isoform 1 (RNPC1a, 239 amino acids) and isoform 2 (RNPC1b, 121 amino acids) . RBM38 functions as a critical regulator of mRNA stability and splicing through its RNA recognition motif (RRM) domain. Independent of p53 expression, RBM38 isoform 1 induces cell cycle arrest in G1 phase by maintaining transcript stability at the 3'-UTR of p21, a key regulator of cell cycle progression at S phase . Additionally, RBM38 acts as an mRNA splicing factor that regulates the expression of proteins such as FGFR2 . Recent research has also identified RBM38 as playing significant roles in pathways related to muscle cell differentiation and skeletal muscle fiber development .

What are the key technical specifications of RBM38 antibodies researchers should consider?

When selecting an RBM38 antibody for research applications, several technical specifications warrant consideration:

• Host Species: Most RBM38 antibodies are produced in rabbit or mouse hosts, with rabbit polyclonal being the most common .

• Clonality: Both polyclonal and monoclonal options are available, with polyclonals offering broader epitope recognition .

• Target Epitopes: Different antibodies target specific regions of RBM38, including N-terminal, C-terminal, and middle regions, each potentially yielding different experimental outcomes .

• Species Reactivity: Available antibodies show reactivity to human and mouse RBM38, with some cross-reacting with rat, dog, cow, guinea pig, zebrafish, monkey, pig, bat, and Xenopus laevis samples .

• Applications: RBM38 antibodies are validated for various techniques including ELISA, IHC, Western blotting, and immunofluorescence, with application-specific dilution recommendations (e.g., 1:50-1:300 for IHC) .

How does HRP conjugation affect antibody performance and experimental design?

HRP (Horseradish Peroxidase) conjugation provides several methodological advantages in RBM38 research:

• Signal Amplification: HRP enzymatically converts substrates to generate detectable signals, offering significant amplification compared to directly labeled antibodies.

• Detection Methods: HRP-conjugated antibodies are compatible with both chromogenic detection (using substrates like TMB, DAB) and chemiluminescent detection (using luminol-based substrates), offering flexibility in visualization approaches.

• Experimental Design Considerations: When using HRP-conjugated RBM38 antibodies, researchers should:

  • Include appropriate quenching steps to neutralize endogenous peroxidase activity in tissues/cells

  • Optimize substrate exposure time to prevent signal saturation

  • Consider using tyramide signal amplification (TSA) for detection of low-abundance RBM38

  • Ensure proper storage conditions (typically -20°C with glycerol and sodium azide) to maintain conjugate stability

What storage and handling protocols maximize RBM38 antibody stability?

To maintain optimal activity of RBM38 antibodies, particularly HRP-conjugated variants:

• Storage Temperature: Store at -20°C as recommended by manufacturers .

• Buffer Conditions: Typical formulation includes PBS with 0.05% sodium azide and 50% glycerol, pH 7.4 .

• Freeze/Thaw Cycles: Minimize freeze/thaw cycles by aliquoting upon first thaw .

• Safety Considerations: Be aware that sodium azide, a common preservative in antibody preparations, is a poisonous and hazardous substance requiring trained handling .

• Long-term Storage: For HRP-conjugated antibodies specifically, avoid prolonged exposure to light and consider oxygen-free storage environments to prevent oxidative inactivation of the HRP enzyme.

How can RBM38 antibodies be optimized for detecting specific protein-RNA interactions?

Investigating RBM38's interactions with target RNAs requires specialized methodological approaches:

The RNA immunoprecipitation (RIP) technique effectively detects RBM38-RNA complexes. In published studies, researchers successfully immunoprecipitated RBM38-RNA complexes from MCF7 cell extracts using RBM38-specific antibodies, followed by RT-PCR and qRT-PCR analysis of bound transcripts . This approach revealed direct binding between RBM38 and target transcripts such as ZO-1, HuR, and p21 .

For quantitative binding analysis, biolayer interferometry can determine binding kinetics. Research has shown that purified RBM38 protein (6×His-tagged) binds to specific RNA sequences with nanomolar affinity. For example, RBM38 bound to ISE2-WT RNA with high affinity (91.5 ± 5.15 nM) compared to mutant sequences (597.8 ± 5.23 nM) . This method provides precise association and dissociation kinetics data for RBM38-RNA interactions.

For visualization of RBM38-RNA interactions, researchers might employ:

• Immunofluorescence with HRP-conjugated antibodies (using tyramide signal amplification)

• Combined fluorescence in situ hybridization (FISH) with immunocytochemistry

• Proximity ligation assays for detecting RBM38 interactions with other RNA-binding proteins

What methodological approaches reveal RBM38's role in TGF-β-induced epithelial-to-mesenchymal transition?

TGF-β signaling induces epithelial-to-mesenchymal transition (EMT) partly through a regulatory axis involving Snail, RBM38, and ZO-1:

Experimental Framework:

• TGF-β Treatment Protocol: Expose breast cancer cells to TGF-β to induce EMT, then measure RBM38 expression changes using Western blotting with anti-RBM38 antibodies .

• Transcriptional Regulation Analysis: Use chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays to investigate the direct relationship between the transcription repressor Snail and RBM38. Research has shown that Snail directly targets E-box elements in the promoter region of the RBM38 gene .

• RNA-Protein Interaction Studies:

  • Perform RNA immunoprecipitation combined with RNA electrophoretic mobility shift assays to demonstrate direct binding between RBM38 and ZO-1 mRNA

  • Employ dual-luciferase reporter assays to confirm that RBM38 positively regulates ZO-1 transcript via direct binding to AU/U-rich elements in its mRNA 3′-UTR

• Functional Validation: Conduct transwell and Matrigel invasion assays following modulation of RBM38 expression to examine effects on cell migratory and invasive capacity .

This multifaceted approach has revealed that TGF-β induces downregulation of RBM38 in breast cancer, which is directly regulated by the transcription repressor Snail. RBM38, in turn, positively regulates ZO-1 transcript levels, with important implications for cell migration and invasion in cancer progression .

How can RBM38 antibodies be utilized in viral research, particularly regarding B19V pre-mRNA processing?

RBM38 plays a crucial role in the processing of parvovirus B19 (B19V) pre-mRNA during viral replication, which can be investigated using specialized techniques:

Recommended Methodological Approach:

• RNA-Protein Binding Analysis:

  • Synthesize 32P-labeled RNA containing the intronic splicing enhancer 2 (ISE2) region of B19V pre-mRNA

  • Incubate labeled RNA with purified GST-RBM38 protein at increasing concentrations

  • Perform gel shift assays to visualize RBM38-RNA complexes

  • Include competition studies with wild-type and mutant cold probes to confirm binding specificity

• Binding Affinity Determination:

  • Express and purify 6×His-tagged RBM38

  • Synthesize biotinylated RNA sequences (wild-type and mutant)

  • Perform biolayer interferometry to measure association and dissociation kinetics

  • Calculate binding affinities (Kd values) for different RNA sequences

Research using these approaches has demonstrated that RBM38 binds specifically to the ISE2 sequence of B19V pre-mRNA with high affinity (91.5 ± 5.15 nM compared to 597.8 ± 5.23 nM for mutant sequences), facilitating viral pre-mRNA splicing at the D2 site .

What optimization strategies should be employed when using HRP-conjugated RBM38 antibodies in multiplex detection systems?

For multiplex detection incorporating HRP-conjugated RBM38 antibodies, consider these optimization strategies:

Methodological Considerations:

• Sequential Detection Protocol:

  • Begin with the lowest-abundance target (often RBM38) using HRP-conjugated antibody

  • Develop signal using specialized substrates like tyramide-fluorophores that covalently bind to nearby proteins

  • Quench HRP activity completely using hydrogen peroxide or sodium azide

  • Proceed to next target with different HRP-conjugated antibody and alternative fluorophore

• Cross-Reactivity Mitigation:

  • Conduct comprehensive blocking using species-specific secondary antibody controls

  • Pre-adsorb antibodies against tissues/cells from knockout models if available

  • Validate staining patterns with multiple antibodies targeting different RBM38 epitopes

  • Include appropriate isotype controls in experimental design

• Signal Optimization Table for HRP-Conjugated Antibodies:

What are the critical validation steps to ensure specificity of RBM38 antibody detection in complex experimental systems?

Ensuring RBM38 antibody specificity requires rigorous validation procedures:

Comprehensive Validation Approach:

• Genetic Controls:

  • Compare staining between wild-type and RBM38 knockout/knockdown samples

  • Rescue experiments by re-expressing RBM38 in knockout models

  • Use cell lines with known differential expression of RBM38 isoforms

• Biochemical Validation:

  • Perform Western blots to confirm single bands at expected molecular weights (239 aa isoform at ~30 kDa and 121 aa isoform at ~14 kDa)

  • Conduct peptide competition assays using immunogenic peptides

  • Employ immunoprecipitation followed by mass spectrometry to confirm target identity

• Cross-Platform Confirmation:

  • Compare protein detection with mRNA expression data

  • Use orthogonal detection methods (e.g., fluorescent protein tagging)

  • Validate functional outcomes of RBM38 through RNA binding assays

• Spatial Localization:

  • Confirm expected subcellular localization patterns (both cytosolic and nuclear)

  • Perform fractionation studies to verify distribution between compartments

  • Co-localize with known RBM38 interaction partners (e.g., p21 mRNA)