rbm24 Antibody, FITC conjugated

What is RBM24 and why is it significant in research?

RBM24 functions as a critical splicing regulator protein that plays an essential role in organ development and is frequently dysregulated in human cancers. The RNA-binding proteins like RBM24 have pivotal roles in post-transcriptionally regulating gene expression, exhibiting temporally and spatially regulated expression patterns during early development . Recent evidence has highlighted its role in suppressing colorectal cancer by attenuating the PI3K/Akt signaling pathway through PTEN regulation, making it an increasingly important target for cancer research .

What are the key specifications of commercially available RBM24 antibodies?

Commercial RBM24 antibodies are available in several formats with different specifications. For example, polyclonal RBM24 antibodies (like 18178-1-AP) target the full RNA binding motif protein 24 (calculated molecular weight: 20 kDa, observed molecular weight: 18-25 kDa) and show reactivity with human, mouse, and rat samples . Monoclonal options like RBM24/38 Antibody (G-6) target epitopes 26-86 in humans. These antibodies typically come in unconjugated form, though conjugated versions (including FITC-conjugated) are also available for specialized applications .

How should RBM24 antibody be stored to maintain optimal activity?

RBM24 antibodies should be stored at -20°C where they remain stable for approximately one year after shipment. Most formulations come in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability. Interestingly, aliquoting is typically unnecessary for -20°C storage, simplifying laboratory management. For smaller volumes (around 20μl), preparations may contain 0.1% BSA as a stabilizer . Always avoid repeated freeze-thaw cycles as they can compromise antibody integrity and performance.

What are the recommended dilutions for different applications of RBM24 antibody?

Optimal dilutions vary based on the specific application:

• For Western Blot (WB): 1:500-1:1000

• For Immunohistochemistry (IHC): 1:20-1:200

• For Immunofluorescence (IF-P): 1:50-1:500

These ranges provide starting points, but researchers should titrate the antibody in each testing system to obtain optimal results, as the performance can be sample-dependent. When using FITC-conjugated versions, the fluorophore might affect binding characteristics, potentially requiring adjustments to these dilution ranges.

How should sample preparation differ when using RBM24 antibody for various tissues?

Sample preparation requirements vary significantly depending on the tissue type. For IHC applications with RBM24 antibody on human skeletal muscle tissue, antigen retrieval with TE buffer at pH 9.0 is suggested, though citrate buffer at pH 6.0 can serve as an alternative . When working with mouse heart tissue or HeLa cells for Western blotting, standard protein extraction protocols are typically sufficient, while muscle tissues often require more rigorous lysis conditions due to their dense protein networks . For immunofluorescence procedures with FITC-conjugated antibodies, fixation and permeabilization steps must be optimized to preserve both antigen accessibility and fluorophore activity.

What controls should be included in experiments using RBM24 antibody?

A robust experimental design using RBM24 antibody should include multiple controls:

• Positive controls: Use tissues or cells known to express RBM24, such as mouse heart tissue, mouse skeletal muscle tissue, or HeLa cells .

• Negative controls: Include samples where RBM24 is known to be absent or use CRISPR/Cas9 knockout samples as definitive negative controls .

• Isotype controls: Include a non-specific antibody of the same isotype (e.g., Rabbit IgG for polyclonal or mouse IgG 1 κ for monoclonal antibodies) to assess non-specific binding .

• Loading controls: For Western blots, include housekeeping proteins like GAPDH, β-actin, or H2AX as standardization references .

For FITC-conjugated antibodies specifically, additional controls should include autofluorescence assessment in the FITC channel and blocking of non-specific binding sites.

How can RBM24 antibody be utilized in RNA immunoprecipitation (RIP) protocols?

RBM24 antibody has been successfully employed in RNA immunoprecipitation (RIP) assays to investigate RNA-protein interactions . When designing RIP protocols with RBM24 antibody, researchers should:

• Cross-link protein-RNA complexes using UV or formaldehyde treatment

• Lyse cells under conditions that preserve RNA integrity

• Pre-clear lysates to reduce background

• Incubate with RBM24 antibody at concentrations higher than those used for Western blotting (typically starting at 5 μg per immunoprecipitation)

• Capture antibody-protein-RNA complexes using protein A/G beads

• Reverse cross-links and purify RNA for downstream analysis such as RT-PCR or sequencing

For FITC-conjugated versions, fluorescence can be used to monitor immunoprecipitation efficiency, though the fluorophore might sterically hinder some protein-RNA interactions.

How can RBM24 antibody be used to investigate the PI3K/Akt signaling pathway?

RBM24 has been demonstrated to attenuate the PI3K/Akt signaling pathway by regulating PTEN expression, thereby suppressing colorectal cancer cell proliferation, migration, and invasion . When investigating this pathway:

• Use RBM24 antibody in combination with antibodies against pathway components (PTEN, PI3K, Akt, phosphorylated Akt)

• Perform overexpression or knockdown of RBM24 to observe effects on pathway activation

• Include pathway-specific inhibitors as controls (e.g., PTEN inhibitor SF1670, Akt inhibitor MK-2206, or PI3K inhibitor PI3K-IN-6)

• Measure downstream effects on matrix metalloproteinases (MMP-2, MMP-9) and cell behavior

The research shows that RBM24 overexpression upregulates PTEN and reduces Akt phosphorylation, while RBM24 knockdown produces opposite effects. These changes can be reversed by pathway-specific inhibitors, confirming the regulatory role of RBM24 in this signaling cascade .

What are the considerations when using RBM24 antibody in multiplexed immunofluorescence?

When designing multiplexed immunofluorescence experiments with FITC-conjugated RBM24 antibody:

• Select complementary fluorophores that have minimal spectral overlap with FITC (excitation ~495 nm, emission ~520 nm)

• Consider sequential staining approaches if antibodies are derived from the same host species

• Perform proper controls for each fluorophore channel independently

• Adjust exposure times to compensate for differences in fluorophore intensity

• Account for potential cross-reactivity between antibodies in the multiplex panel

• Use appropriate blocking steps to minimize non-specific binding

• Consider the relative expression levels of target proteins when designing the panel (pair low-expression targets with brighter fluorophores)

This approach allows simultaneous visualization of RBM24 alongside other proteins of interest, such as components of the PI3K/Akt pathway or markers of cell proliferation and apoptosis.

How can researchers address weak or absent signal when using RBM24 antibody?

When troubleshooting weak or absent signal with RBM24 antibody:

• Verify sample expression: Confirm RBM24 expression in your samples; it shows tissue-specific expression primarily in heart and skeletal muscle tissues .

• Optimize antibody concentration: Titrate the antibody using a broader range than the recommended dilutions.

• Enhance antigen retrieval: For IHC applications, compare TE buffer (pH 9.0) versus citrate buffer (pH 6.0) for optimal epitope exposure .

• Extend incubation times: Consider overnight incubation at 4°C instead of shorter incubations at room temperature.

• Check secondary antibody compatibility: Ensure secondary antibody properly recognizes the primary antibody's host species and isotype.

• For FITC-conjugated antibodies specifically, protect from photobleaching by minimizing exposure to light during all handling steps.

• Verify buffer compatibility: Some buffer components may interfere with antibody-antigen binding or fluorophore activity.

What could cause unexpected bands or staining patterns with RBM24 antibody?

Unexpected results with RBM24 antibody may arise from several factors:

• Post-translational modifications: RBM24 may undergo modifications affecting its molecular weight (observed 18-25 kDa versus calculated 20 kDa) .

• Alternative splicing: Different splice variants may produce bands of unexpected sizes.

• Cross-reactivity: RBM24 antibodies may cross-react with the closely related RBM38, particularly antibodies targeting conserved domains (e.g., RBM24/38 Antibody G-6) .

• Sample preparation issues: Incomplete protein denaturation or degradation can create fragment bands.

• Non-specific binding: Insufficient blocking or high antibody concentrations can lead to background staining.

To address these issues, include appropriate controls, optimize blocking conditions, and consider using RBM24 knockout or knockdown samples to confirm signal specificity .

How should researchers interpret contradictory results between different applications using RBM24 antibody?

When facing contradictory results across different applications:

• Consider epitope accessibility: The RBM24 epitope may be differentially accessible in various applications due to protein folding, fixation, or embedding.

• Evaluate detection sensitivity: Applications differ in sensitivity; WB can detect denatured epitopes while IF requires native conformations.

• Assess quantitative differences: Compare relative expression rather than absolute values across techniques.

• Check application compatibility: Not all antibodies perform equally well in all applications despite manufacturer claims.

• Review isoform specificity: Different techniques may preferentially detect certain isoforms.

To resolve contradictions, perform validation experiments using multiple antibodies targeting different epitopes of RBM24, implement CRISPR/Cas9 knockout controls, and compare results with mRNA expression data .

How can RBM24 antibody be used to investigate cell cycle and proliferation mechanisms?

RBM24 has been implicated in cell cycle regulation and proliferation control in cancer models . To investigate these mechanisms:

• Combine RBM24 staining with proliferation markers: Use antibodies against Ki-67, CyclinD1, CDK4, or phosphorylated histone H3 (pHH3) .

• Perform cell cycle analysis: Synchronize cells and analyze RBM24 expression at different cell cycle phases.

• Assess effect on cell cycle regulators: Measure p21 expression, which can be affected by RBM24 activity .

• Examine DNA damage response: Analyze γH2AX, ATM, and ATR phosphorylation in relation to RBM24 expression .

Research shows that RBM24 overexpression can inhibit proliferation through multiple mechanisms including PTEN upregulation and subsequent PI3K/Akt pathway modulation .

What are the best practices for using RBM24 antibody in investigating epithelial-mesenchymal transition (EMT)?

When studying EMT mechanisms involving RBM24:

• Monitor both epithelial and mesenchymal markers: Pair RBM24 antibody with antibodies against E-Cadherin (epithelial) and Vimentin (mesenchymal) .

• Assess matrix metalloproteinase activity: Measure MMP-2 and MMP-9 expression, which are downstream targets affected by RBM24 expression levels .

• Analyze migration and invasion: Correlate RBM24 expression with functional changes in cell behavior.

• Examine pathway connections: Investigate the relationship between RBM24, PTEN, and the PI3K/Akt signaling pathway in the context of EMT .

Studies have shown that RBM24 overexpression leads to decreased MMP-2 and MMP-9 expression, which can be reversed by PTEN inhibition, suggesting a regulatory role for RBM24 in EMT-related processes .

What approaches should be considered when using gene editing tools alongside RBM24 antibody?

When combining gene editing approaches with RBM24 antibody techniques:

• Generate proper controls: Use CRISPR/Cas9 knockout systems (e.g., RBM24 CRISPR/Cas9 KO Plasmid) to create definitive negative controls .

• Verify knockout efficiency: Confirm gene editing success using the RBM24 antibody itself via Western blot or immunofluorescence.

• Consider gene activation alternatives: CRISPR activation plasmids and lentiviral particles can upregulate RBM24 expression for gain-of-function studies .

• Use homology-directed repair: HDR plasmids allow tagging or modifying the endogenous RBM24 protein for advanced studies .

• Implement double nickase approaches: These provide higher specificity for RBM24 targeting with reduced off-target effects .

The combination of gene editing tools with antibody-based detection provides a powerful approach for understanding RBM24 function in normal and pathological contexts.