rbm24 Antibody, HRP conjugated

What is the molecular function of RBM24 in normal tissue homeostasis?

RBM24 functions primarily as an RNA-binding protein that regulates mRNA splicing and stability. In normal tissues, RBM24 maintains cellular homeostasis by binding to GT-rich regions in the 3'-UTR of target mRNAs, such as PTEN . This binding activity prolongs mRNA half-life and enhances protein expression of critical tumor suppressors . Experimental evidence from RBM24 knockout mice reveals that loss of this protein results in spontaneous colorectal adenomas, indicating its essential role in preventing aberrant cell proliferation under normal physiological conditions .

How does RBM24 expression vary across different tissue types?

Immunohistochemical studies using RBM24 antibodies have demonstrated variable expression patterns across tissues. Significant expression has been documented in mouse heart tissue, human skeletal muscle tissue, and mouse skeletal muscle tissue . In contrast, RBM24 expression is markedly lower in colorectal tumors compared to adjacent normal tissues . This differential expression pattern suggests tissue-specific regulatory mechanisms that control RBM24 levels and may explain its varied functions across different organ systems.

What structural domains of RBM24 are targeted by commercially available antibodies?

The RBM24 antibody described in the search results (ABIN1929622) targets amino acids 4-32 from the N-terminal region of human RBM24 . This region is particularly important as it likely contains critical functional domains for RNA recognition. Polyclonal antibodies generated against this region demonstrate reactivity with human and mouse samples . The specificity of these antibodies has been validated through multiple applications including Western blotting, immunohistochemistry, and immunofluorescence techniques.

What are the optimal experimental conditions for Western blotting with RBM24 HRP-conjugated antibodies?

For optimal Western blotting results with RBM24 HRP-conjugated antibodies, researchers should use dilutions between 1:500-1:1000 . The expected molecular weight of RBM24 is 18-25 kDa, with a calculated molecular weight of 20 kDa from its 191 amino acid sequence . Sample preparation should include proper denaturation and reduction of proteins. Positive controls should incorporate mouse heart tissue or HeLa cells, where RBM24 expression has been confirmed . For membrane blocking, 5% non-fat dry milk in TBST is generally effective, followed by overnight primary antibody incubation at 4°C for maximum sensitivity.

How should antigen retrieval be performed for RBM24 immunohistochemistry?

For immunohistochemistry applications using RBM24 antibodies, the recommended antigen retrieval method involves TE buffer at pH 9.0 . Alternatively, citrate buffer at pH 6.0 can be used if the preferred method yields suboptimal results . The antibody should be diluted between 1:20-1:200 for IHC applications . Following antigen retrieval, sections should be blocked with appropriate serum (typically 5-10% normal goat serum) for 1 hour at room temperature before primary antibody application. For HRP-conjugated antibodies, direct visualization can be achieved using DAB substrate without requiring secondary antibody incubation, which streamlines the protocol and potentially reduces non-specific background.

What controls are essential when using RBM24 antibodies in RNA immunoprecipitation (RIP) assays?

RIP assays using RBM24 antibodies require rigorous controls to ensure valid results. Essential controls include:

This experimental design allowed researchers to convincingly demonstrate that RBM24 directly binds to the GT-rich region at positions 8101-8251 in the 3′-UTR of PTEN mRNA , establishing a critical mechanism for its tumor suppressive function.

How can researchers address weak or inconsistent signal when using RBM24 HRP-conjugated antibodies?

When encountering weak signals with RBM24 HRP-conjugated antibodies, researchers should first optimize antibody concentration. While recommended dilutions are 1:500-1:1000 for WB, titration experiments starting at 1:250 may be necessary for some samples . Extending primary antibody incubation time to overnight at 4°C can enhance signal strength. Additionally, ensure adequate antigen is present in samples; RBM24 expression varies significantly between tissue types, with stronger expression in heart and skeletal muscle compared to other tissues . For enhanced chemiluminescence detection, using fresh ECL substrate and longer exposure times (up to 10 minutes) may reveal faint signals. If signal remains weak, consider sample enrichment through immunoprecipitation before Western blotting.

What strategies can resolve non-specific binding in RBM24 immunohistochemistry?

Non-specific binding in RBM24 immunohistochemistry can be addressed through several methodological modifications:

• Increase blocking stringency by using 5% BSA with 5% normal serum from the species of the secondary antibody

• Add 0.1-0.3% Triton X-100 during antibody incubation to reduce hydrophobic interactions

• Implement additional washing steps (5-6 washes of 5 minutes each) with 0.1% Tween-20 in PBS

• Validate antibody specificity by pre-adsorbing with immunizing peptide from RBM24 amino acids 4-32

• Utilize antigen retrieval optimization with gradient testing of pH conditions from 6.0-9.0

Researchers should additionally compare results with RBM24 knockout controls when available to conclusively identify specific staining patterns.

How can researchers distinguish between different isoforms of RBM24 in experimental applications?

Although the calculated molecular weight of RBM24 is 20 kDa (191 amino acids), the observed molecular weight in Western blotting ranges between 18-25 kDa , suggesting potential post-translational modifications or isoform variations. To distinguish between different isoforms:

• Employ high-resolution SDS-PAGE (12-15% acrylamide) with extended running times

• Utilize 2D electrophoresis to separate isoforms by both molecular weight and isoelectric point

• Perform immunoprecipitation followed by mass spectrometry to identify specific isoforms

• Use phospho-specific antibodies to detect phosphorylated forms if available

• Compare expression patterns between tissues known to express different isoforms (e.g., heart versus skeletal muscle)

Sequence alignment analysis of potential isoforms can identify unique epitopes that might be targeted by specific antibodies in future research.

How can RBM24 antibodies be used to investigate its role in mRNA stability regulation?

RBM24 antibodies can be employed in several advanced techniques to investigate its role in regulating mRNA stability:

• RNA Immunoprecipitation (RIP): As demonstrated in the research results, RIP assays using RBM24 antibodies successfully identified direct binding between RBM24 and the GT-rich region (positions 8101-8251) in PTEN mRNA's 3'-UTR . This technique can be applied to identify novel mRNA targets beyond PTEN.

• Crosslinking Immunoprecipitation (CLIP): By combining UV crosslinking with immunoprecipitation using RBM24 antibodies, researchers can map binding sites at single-nucleotide resolution, providing deeper insights into the sequence specificity of RBM24-RNA interactions.

• Pulse-Chase mRNA Decay Assays: Following RBM24 overexpression or knockdown, researchers can measure target mRNA half-life changes using actinomycin D treatment followed by qPCR analysis at different time points. This approach revealed that RBM24 knockdown decreased PTEN mRNA half-life, while overexpression extended it .

• Polysome Profiling: HRP-conjugated RBM24 antibodies can be used in polysome fractionation experiments to determine whether RBM24 influences not only mRNA stability but also translational efficiency of target transcripts.

What experimental approaches can assess the impact of RBM24 on cancer cell phenotypes?

Multiple experimental approaches can evaluate RBM24's influence on cancer cell phenotypes, as demonstrated in the research findings:

These approaches collectively demonstrated that RBM24 functions as a tumor suppressor by inhibiting cell proliferation, migration, and invasion in colorectal cancer .

How can researchers investigate the relationship between RBM24 and the PI3K/Akt signaling pathway?

To investigate interactions between RBM24 and PI3K/Akt signaling, researchers can employ several advanced approaches:

• Western Blot Analysis: Using HRP-conjugated RBM24 antibodies alongside antibodies against pathway components (PTEN, phosphorylated Akt, total Akt) to monitor expression changes. Research showed RBM24 overexpression upregulated PTEN and reduced Akt phosphorylation without affecting total Akt expression .

• Pharmacological Inhibition Studies: Research demonstrated that PTEN inhibitor SF1670 reversed the effects of RBM24 overexpression, while Akt inhibitor MK-2206 or PI3K inhibitor PI3K-IN-6 reversed effects of RBM24 knockdown , confirming the pathway's involvement.

• Rescue Experiments: Experimental designs can include PTEN overexpression in RBM24-knockdown cells to determine if PTEN restoration can rescue the phenotype, further validating the RBM24-PTEN-PI3K/Akt axis.

• Downstream Effector Analysis: Measuring expression of matrix metalloproteinases (MMP-2, MMP-9) which were downregulated with RBM24 overexpression , providing mechanistic insights into how RBM24 influences cancer cell invasion.

• In Vivo Pathway Validation: Analyzing tumor samples from Rbm24-knockout mice for PI3K/Akt pathway activation markers correlating with adenoma development .

How can RBM24 antibodies be applied in patient stratification for personalized cancer treatment?

RBM24 antibodies can be utilized in patient stratification through several clinical applications:

• Immunohistochemical Analysis: RBM24 expression in CRC tissues was lower than in adjacent normal samples and positively correlated with PTEN expression while negatively correlating with Ki-67 levels . This pattern suggests RBM24 IHC scoring could help identify patients with higher PI3K/Akt pathway activation.

• Prognostic Assessment: Research indicated that CRC patients with high RBM24 expression had favorable outcomes , suggesting RBM24 immunostaining could serve as a prognostic marker in patient biopsies.

• Predictive Biomarker Development: Studies showed RBM24 overexpression increased sensitivity to 5-FU and cisplatin in CRC cells , indicating potential value as a predictive biomarker for chemotherapy response. Patients with low RBM24 expression might benefit from combination therapies targeting the PI3K/Akt pathway.

• Therapy Selection Guidance: Given RBM24's role in regulating PTEN and the PI3K/Akt pathway, patients with low RBM24 expression might be candidates for PI3K/Akt inhibitor therapies already in clinical development.

What are the methodological considerations for developing RBM24-targeted cancer therapeutics?

Development of RBM24-targeted therapeutics should consider several methodological approaches:

• Gene Therapy Approaches: Since RBM24 acts as a tumor suppressor, viral vector-mediated restoration of RBM24 expression in tumors could potentially suppress cancer progression through PTEN stabilization .

• Small Molecule Screening: High-throughput screening for compounds that enhance RBM24 expression or mimic its RNA-binding functions could identify candidate therapeutics.

• RNA Stabilization Strategies: Alternative approaches could target the RBM24-binding region in PTEN mRNA (positions 8101-8251 in the 3′-UTR) with oligonucleotide therapies designed to protect this region from degradation even in the absence of RBM24.

• Combination Therapy Design: Based on findings that RBM24 overexpression increased sensitivity to 5-FU and cisplatin , researchers should investigate optimal dosing schedules and drug combinations for enhanced efficacy.

• Mouse Model Testing: Therapeutic candidates should be evaluated in Rbm24-knockout mice and Apc^min/+^ mice, which showed spontaneous colorectal adenomas with downregulated RBM24 expression compared to adjacent normal tissues .

The development pathway should include assessment of safety, specificity, and efficacy profiles before clinical translation.

How can RBM24-mediated RNA regulation inform broader cancer therapeutic strategies?

The mechanistic insights from RBM24 research provide several frameworks for broader therapeutic development:

• RNA Stability Modulation: Research showing RBM24 binding to specific GT-rich regions to enhance mRNA stability presents a paradigm for targeting other tumor suppressors regulated at the post-transcriptional level. This could inform development of RNA-stabilizing therapeutic approaches beyond RBM24/PTEN.

• Alternative Signaling Pathway Integration: Studies demonstrated that RBM24's tumor-suppressive effects operate through the PTEN/PI3K/Akt axis , suggesting potential synergies between RBM24-targeted therapies and existing PI3K/Akt inhibitors in clinical development.

• Multi-Cancer Applications: Beyond colorectal cancer, research showed RBM24 suppresses lymph node metastasis and epithelial-mesenchymal transition in hypopharyngeal squamous cell carcinoma by regulating Twist1 , indicating potential therapeutic applications across multiple cancer types.

• Biomarker-Guided Therapy: The correlation between RBM24 expression and patient outcomes provides a model for integrating RNA-binding protein expression profiles into precision oncology approaches, potentially informing treatment decisions across broader cancer populations.