RBM3 Antibody

What applications are RBM3 antibodies validated for?

RBM3 antibodies have been validated for multiple applications, with specific performance characteristics depending on the antibody clone and manufacturer. Based on comprehensive validation data, RBM3 antibodies can be reliably used in:

It is recommended to optimize antibody concentrations for each specific experimental system to obtain optimal results . Sample-dependent variations may require titration to achieve ideal signal-to-noise ratios.

What are the recommended buffers and conditions for RBM3 antibody storage?

To maintain antibody activity, most RBM3 antibodies should be stored at -20°C in PBS containing 0.02% sodium azide and 50% glycerol at pH 7.3. Under these conditions, the antibodies remain stable for approximately one year after shipment. Aliquoting is generally unnecessary for -20°C storage. Some preparations may contain 0.1% BSA in smaller sizes (20μl) . Always refer to the specific storage recommendations provided by the manufacturer of your particular antibody.

What are the recommended fixation and antigen retrieval methods for RBM3 immunohistochemistry?

For optimal RBM3 detection in tissue sections:

• Paraformaldehyde fixation has been successfully employed in cell lines

• For FFPE tissue sections, antigen retrieval with TE buffer pH 9.0 is recommended

• Alternatively, citrate buffer pH 6.0 may be used for antigen retrieval

These parameters have been validated across multiple tissue types including human testis, lung, brain, kidney, and spleen tissues .

What is the cellular distribution pattern of RBM3 protein?

RBM3 shows predominantly nuclear localization in most cell types when detected by immunohistochemistry or immunofluorescence. Nuclear expression is particularly associated with favorable prognosis in several cancer types . While cytoplasmic expression can also be observed, it is the nuclear fraction that is typically quantified and correlated with clinical outcomes .

When performing immunofluorescence visualization, RBM3 (green) can be effectively counterstained with cytoskeletal markers such as alpha-tubulin (red) to clearly delineate nuclear versus cytoplasmic localization .

How does RBM3 expression vary across normal and cancerous tissues?

RBM3 shows differential expression patterns:

• Upregulated in multiple cancer types compared to corresponding normal tissues

• In normal tissues, expression has been documented in multiple organs with varying intensity

• Different cancer types show distinct patterns of RBM3 expression

In breast cancer, high nuclear RBM3 expression correlates with:

• Low grade tumors (P<0.001)

• Smaller tumor size (P<0.001)

• Estrogen receptor positivity (P<0.001)

• Ki-67 negativity (P<0.001)

In lung cancer, adenocarcinomas show higher RBM3 expression levels than squamous cell carcinomas (P<0.0001) .

What is the molecular weight and structure of RBM3 protein?

RBM3 is characterized by:

• Calculated molecular weight: 17 kDa

• Observed molecular weight in Western blots: 17-20 kDa

• Structure: N-terminal RNA recognition motif (RRM) consisting of 84 residues that adopts a βαββαβ topology

• C-terminal region: Rich in RGG and YGG motifs and is intrinsically disordered

The solution NMR structure shows that the RRM domain forms the RNA-binding interface through its beta-sheet and two loops. The key interactions between RNA and the RRM domain include hydrogen bonding, pi-pi, and pi-cation interactions .

How does temperature affect RBM3 protein function and detection?

As a cold-shock protein, RBM3 exhibits interesting temperature-dependent properties:

• Oligomerization of RBM3 is favored by decreased temperature, which may be linked to its function as a cold-shock protein

• Temperature-dependent NMR studies reveal that the oligomerization of the RRM domain occurs via nonspecific interactions

• RBM3 enhances global protein synthesis at both physiological and mild hypothermic temperatures

• When studying temperature effects on RBM3, experimental design should account for potential oligomerization artifacts

These temperature-dependent properties may be particularly relevant for researchers studying neuroprotection or hypothermia-related cellular processes, as high levels of RBM3 expression correlate with improved outcomes in hypothermia-treated stroke and trauma patients .

What methods are most effective for validating RBM3 antibody specificity?

Comprehensive validation of RBM3 antibodies should include multiple complementary approaches:

• siRNA gene silencing to confirm signal reduction

• Western blotting to confirm specific band detection at 17-20 kDa

• Immunohistochemistry on known positive/negative tissue controls

• Cell line panels with differential expression patterns

• Epitope mapping using:

  • Bacterial display (identified five distinct epitopes for polyclonal antibodies)

  • Peptide suspension bead array assays

  • Testing against recombinant RBM3 protein

In published studies, correlation between antibodies from different sources provides additional validation. For example, in colorectal cancer cohorts, two different antibodies (one polyclonal and one monoclonal) showed high correlation (R=0.81, p<0.001) .

How is RBM3 expression associated with patient outcomes in different cancer types?

RBM3 has emerged as a significant prognostic marker across multiple cancer types:

How do RBM3 mRNA and protein expression levels correlate in cancer studies?

An interesting discrepancy exists between mRNA and protein expression levels of RBM3 in cancer studies:

This discrepancy highlights the importance of protein-level analyses, particularly examining subcellular localization, when investigating RBM3 as a biomarker.

What is known about RBM3's role in cisplatin sensitivity and resistance?

RBM3 has been implicated in modulating response to cisplatin treatment:

• RBM3 mRNA and protein expression levels were significantly higher in the cisplatin-sensitive A2780 cell line compared to the cisplatin-resistant A2780-Cp70 derivative

• siRNA-mediated silencing of RBM3 in A2780 cells resulted in decreased sensitivity to cisplatin as demonstrated by:

  • Increased cell viability following treatment

  • Reduced proportion of cells arrested in G2/M-phase

• These findings suggest RBM3 may serve as both a prognostic marker and a potential predictor of treatment response

This mechanism may explain why high RBM3 expression correlates with better clinical outcomes in several cancer types, potentially through enhanced sensitivity to platinum-based chemotherapy regimens.

How does RBM3's RNA-binding function relate to its biological effects?

The RNA-binding properties of RBM3 are central to its biological functions:

• The N-terminal RRM domain forms the primary RNA-binding interface via its beta-sheet and two loops

• Key interactions between RNA and the RRM domain include hydrogen bonding, pi-pi, and pi-cation interactions

• RBM3 can reduce the relative abundance of microRNAs when overexpressed

• It enhances phosphorylation of translation initiation factors and promotes active polysome formation

• The protein participates in synaptic plasticity, which is essential for learning and memory

Understanding the specific RNA targets and binding mechanisms of RBM3 is an active area of research that may explain its diverse roles in cellular processes and disease states.

How should researchers address discrepancies between RBM3 antibodies from different sources?

When encountering inconsistent results with different RBM3 antibodies:

• Compare epitope specificity - different antibodies may target distinct regions of RBM3:

  • In one study, a polyclonal antibody recognized five distinct epitopes, while four monoclonal antibodies bound to just three of these epitopes

  • Some antibodies may differentially detect post-translational modifications

• Validate each antibody using multiple approaches:

  • Western blotting to confirm the correct molecular weight (17-20 kDa)

  • siRNA knockdown to demonstrate specificity

  • Use positive and negative control tissues/cell lines

• Consider performing correlation analyses between antibodies:

  • In colorectal cancer studies, different antibodies showed high correlation (R=0.81, p<0.001)

  • This approach helps validate findings across different detection reagents

• Document and report the specific clone and catalog number in publications to enhance reproducibility

What are the optimal scoring methods for RBM3 immunohistochemistry in prognostic studies?

For consistent and reproducible evaluation of RBM3 expression in tissue samples:

• Use a combined scoring approach that considers both:

  • Nuclear fraction (percentage of positive cells)

  • Staining intensity (typically on a 0-3 scale)

  • This approach has been validated in multiple studies

• Consider different scoring thresholds for different histological subtypes:

  • In lung cancer studies, different cutoffs were used for adenocarcinoma versus squamous cell carcinoma due to different baseline expression levels

• Employ digital image analysis when possible to reduce subjectivity

• Validate scoring methods through:

  • Independent observer assessment

  • Correlation with clinical outcomes

  • Comparison with established biomarkers

• In multivariate analyses, control for relevant clinical variables such as stage, grade, treatment, and patient characteristics

How might RBM3's temperature-dependent properties be exploited in therapeutic applications?

The cold-shock properties of RBM3 present intriguing therapeutic possibilities:

• Neuroprotection strategies:

  • RBM3's neuroprotective abilities correlate with improved outcomes in hypothermia-treated stroke and trauma patients

  • Research into RBM3 induction or mimetics could potentially provide neuroprotection without requiring physical cooling

• Cancer therapy approaches:

  • RBM3's association with cisplatin sensitivity suggests potential for combination therapies

  • Understanding the molecular basis of RBM3's temperature-dependent oligomerization could lead to novel targeted approaches

• Therapeutic temperature modulation:

  • Local or systemic temperature modulation might alter RBM3 expression and function in targeted tissues

  • This could potentially enhance chemosensitivity in tumors or provide protection in normal tissues

• Synaptic plasticity and cognitive enhancement:

  • RBM3's role in synaptic plasticity, essential for learning and memory , suggests potential applications in neurodegenerative diseases

Future research should examine how these temperature-dependent properties might be selectively modulated in different tissue contexts.

What explains the tissue-specific prognostic implications of RBM3 expression?

The divergent prognostic implications of RBM3 across different tissue types present an intriguing research question:

• In lung cancer, high RBM3 expression is associated with better outcomes in adenocarcinoma but shows a trend toward worse outcomes in squamous cell carcinoma

• Potential explanations requiring further investigation include:

  • Tissue-specific RNA targets and binding partners

  • Different post-translational modifications in different cellular contexts

  • Varying subcellular localization patterns

  • Interaction with tissue-specific transcription factors or signaling pathways

• Future research directions should include:

  • Comprehensive RNA-binding studies in different tissue contexts

  • Identification of tissue-specific interaction partners

  • Analysis of post-translational modifications across tissue types

  • Integration with tissue-specific gene expression programs

Understanding these tissue-specific effects will be crucial for developing targeted therapeutic approaches based on RBM3 modulation.