RBM38 Antibody, Biotin conjugated

What is RBM38 and why is it a significant research target?

RBM38 (also known as RNPC1) is an RNA-binding protein that plays a critical role in post-transcriptional regulation of gene expression. It specifically binds to the 3'-UTR of transcripts like CDKN1A, maintaining their stability and acting as a mediator of the p53/TP53 family in regulating cell cycle arrest . RBM38 is also involved in mRNA splicing, particularly regulating the expression of FGFR2-IIIb, an epithelial cell-specific isoform of FGFR2, and plays a role in myogenic differentiation . Its dysregulation has been linked to various diseases, including cancer, making it a promising target for therapeutic interventions .

What are the key applications for RBM38 antibodies in research?

RBM38 antibodies are validated for multiple research applications, including ELISA, immunohistochemistry (IHC) for both paraffin-embedded and frozen sections, and immunofluorescence (IF) . For ELISA applications, dilutions typically range from 1:2000 to 1:10000, while IHC applications require 1:20 to 1:200 dilutions, and IF applications require 1:50 to 1:200 dilutions . These antibodies are valuable tools for detecting and analyzing RBM38 expression in various cell types, making them essential for studies in molecular biology and cancer research, particularly in investigating post-transcriptional regulation mechanisms .

How do biotin-conjugated RBM38 antibodies differ from non-conjugated versions?

Biotin-conjugated RBM38 antibodies offer several advantages over non-conjugated versions, particularly in detection sensitivity and flexibility. The biotin conjugation allows for signal amplification through the strong affinity between biotin and streptavidin, enabling enhanced detection in applications like ELISA and IHC . This conjugation maintains the antibody's specificity while providing a consistent binding site for secondary detection reagents. Unlike non-conjugated antibodies that require species-specific secondary antibodies, biotin-conjugated versions can be detected with any streptavidin-coupled reporter system, allowing for greater experimental flexibility and multiplexing capabilities .

What are the storage and handling requirements for biotin-conjugated RBM38 antibodies?

Biotin-conjugated RBM38 antibodies require specific storage conditions to maintain their functionality. They should be stored at -20°C or -80°C to prevent degradation . The antibodies are typically supplied in liquid form in a storage buffer containing 50% glycerol, 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative . It's important to avoid repeated freeze-thaw cycles which can compromise antibody performance. When handling these antibodies, researchers should be aware that they contain preservatives like Proclin, which is classified as a poisonous and hazardous substance requiring trained staff for handling . Always follow manufacturer's recommendations for thawing, aliquoting, and working dilution preparation.

How can RBM38 antibodies be utilized to investigate TGF-β-induced epithelial-to-mesenchymal transition in cancer?

RBM38 antibodies are instrumental in studying the role of RBM38 in TGF-β-induced EMT. Research shows that RBM38 expression is significantly reduced in breast cancer cells undergoing EMT, functioning as a mediator in the TGF-β signaling pathway . To investigate this relationship, researchers can use RBM38 antibodies in Western blot applications to monitor changes in RBM38 protein levels in response to TGF-β treatment over time (typically 5 days for phenotypic switching observations) . Additionally, immunofluorescence using biotin-conjugated RBM38 antibodies can visualize the cellular localization changes during EMT. These antibodies can also be employed in co-immunoprecipitation experiments to identify interaction partners that regulate RBM38 during EMT, providing insights into the mechanisms by which TGF-β signaling suppresses RBM38 expression .

What experimental approaches can reveal the relationship between RBM38 and ZO-1 in epithelial integrity maintenance?

To investigate the regulatory relationship between RBM38 and ZO-1, researchers can employ several complementary approaches using RBM38 antibodies. RNA immunoprecipitation (RIP) assays can determine whether RBM38 physically binds to ZO-1 transcripts, as demonstrated in breast cancer cell lines where ZO-1 mRNA was detected in RBM38 immunoprecipitates but not in control IgG . For assessing how RBM38 affects ZO-1 mRNA stability, researchers can perform actinomycin-D chase experiments in cells with RBM38 overexpression or knockdown, using biotin-conjugated RBM38 antibodies to confirm expression levels . Dual immunofluorescence or IHC studies in tissue samples can reveal the correlation between RBM38 and ZO-1 expression patterns, with data showing stronger ZO-1 staining in RBM38 high-expressed breast cancers compared to RBM38 low-expressed samples . Additionally, migration and invasion assays following RBM38 modulation can demonstrate functional consequences of the RBM38-ZO-1 regulatory axis in epithelial integrity maintenance .

What are the considerations for using RBM38 antibodies in multi-parameter flow cytometry studies?

When incorporating biotin-conjugated RBM38 antibodies into multi-parameter flow cytometry, researchers must consider several technical aspects. First, since RBM38 is primarily expressed in the cytoplasm , permeabilization protocols need optimization to ensure antibody access while maintaining cellular integrity. The biotin conjugation requires careful panel design to avoid fluorochrome spectral overlap when using streptavidin-coupled fluorophores. Titration experiments are essential to determine optimal antibody concentration that maximizes signal-to-noise ratio while preventing non-specific binding. For co-staining with other biotin-conjugated antibodies, sequential labeling with different detection systems may be necessary. Additionally, appropriate compensation controls must account for the amplification effect of the biotin-streptavidin interaction. When designing panels investigating EMT markers alongside RBM38, inclusion of markers like ZO-1, Snail, and Slug is recommended based on their established relationship with RBM38 in the TGF-β pathway .

How can RBM38 antibodies be employed in RNA-protein interaction studies?

RBM38 antibodies are critical tools for investigating RNA-protein interactions involving this RNA-binding protein. RNA immunoprecipitation (RIP) assays using specific RBM38 antibodies can identify mRNA targets that physically bind to RBM38, as demonstrated in studies where ZO-1, HuR, and p21 transcripts were detected in RBM38 immunoprecipitates . For mapping precise binding sites, researchers can combine RBM38 antibodies with crosslinking and immunoprecipitation (CLIP) techniques, which provide higher resolution of binding regions. To investigate whether RBM38 binds to specific motifs like AREs (AU-rich elements) in the 3'-UTR of target mRNAs, biotin-labeled RNA probes can be used in conjunction with RBM38 antibodies in RNA electrophoretic mobility shift assays . When conducting these studies, it's essential to include appropriate controls such as IgG for immunoprecipitation specificity and β-actin for detecting non-specific RNA binding .

What optimization steps are required for immunohistochemistry using biotin-conjugated RBM38 antibodies?

Optimizing IHC protocols for biotin-conjugated RBM38 antibodies requires systematic adjustments to multiple parameters. Antigen retrieval methods should be compared (heat-induced vs. enzymatic) to maximize epitope accessibility while preserving tissue morphology. For paraffin-embedded sections, testing different dilutions ranging from 1:20 to 1:200 is recommended to determine optimal antibody concentration . When using biotin-conjugated antibodies, it's critical to block endogenous biotin in tissues (particularly liver, kidney, and brain) using biotin blocking kits before antibody application to reduce background . Detection systems should be evaluated for sensitivity, with streptavidin-HRP or streptavidin-fluorophore conjugates typically providing excellent results. Counterstaining protocols should be optimized to clearly distinguish RBM38 cytoplasmic staining from nuclear markers when performing co-localization studies . Validation steps should include both positive controls (tissues known to express RBM38) and negative controls (isotype antibodies and tissues with RBM38 knockdown) to confirm staining specificity .

What controls are essential when using RBM38 antibodies in RNA immunoprecipitation experiments?

For RNA immunoprecipitation (RIP) experiments using RBM38 antibodies, comprehensive controls are essential to ensure valid results. Input controls (typically 10% of cell extracts) should be analyzed alongside immunoprecipitated samples to normalize for RNA abundance variations . Isotype-matched IgG immunoprecipitation serves as a negative control to assess non-specific binding . Known RBM38 targets like HuR and p21 transcripts should be included as positive controls, as their interaction with RBM38 has been previously established . Housekeeping genes such as β-actin are important negative controls that should not be enriched in RBM38 immunoprecipitates . When investigating new potential RBM38 targets, validation through independent methods is recommended, such as using bio-UTP-labeled probes and RNA EMSAs to confirm direct binding . Additionally, RBM38 knockdown or overexpression cell lines should be used to demonstrate specificity of the RNA-protein interactions identified through RIP experiments.

What are the considerations for using biotin-conjugated RBM38 antibodies in multiplex immunofluorescence studies?

Multiplex immunofluorescence using biotin-conjugated RBM38 antibodies requires careful planning to achieve optimal results. Panel design should consider the spectral properties of fluorophores coupled to streptavidin to minimize overlap and ensure clear distinction between markers. Sequential staining protocols may be necessary when including multiple biotin-conjugated antibodies in the same panel, with complete blocking steps between each antibody application. When investigating RBM38 in relation to EMT markers, inclusion of ZO-1, Snail, and Slug is recommended based on their established relationship with RBM38 . Signal amplification systems like tyramide signal amplification can enhance detection sensitivity while allowing for antibody stripping and re-probing of the same tissue section. For quantitative analysis, standardized image acquisition settings and automated analysis algorithms should be established to ensure consistent measurement of marker expression and co-localization. Appropriate controls including single-stained samples for spectral unmixing and secondary-only controls are essential for accurate interpretation of multiplex results.

How should antibody validation be performed to ensure specificity for RBM38?

Comprehensive validation of RBM38 antibodies is crucial to ensure experimental reliability. Western blot analysis should demonstrate a single band at the expected molecular weight (approximately 30 kDa for RBM38) . Antibody performance should be compared in RBM38 overexpression and knockdown models to confirm specificity . Peptide competition assays, where the antibody is pre-incubated with the immunizing peptide, should eliminate specific binding if the antibody is truly specific. Cross-reactivity testing against related RNA-binding proteins is important, particularly for polyclonal antibodies that might recognize conserved epitopes. For biotin-conjugated antibodies, additional controls should verify that the conjugation process hasn't altered binding specificity. Reproducibility across different lots should be confirmed, especially for polyclonal antibodies that may show batch-to-batch variation. Finally, orthogonal method validation, where RBM38 detection by antibody-based methods is compared with mRNA expression data, provides additional confidence in antibody specificity.

How should researchers interpret changes in RBM38 expression during TGF-β treatment experiments?

When interpreting RBM38 expression changes during TGF-β treatment, researchers should consider both temporal dynamics and context-dependent factors. The significant decrease in RBM38 expression observed following TGF-β treatment in breast cancer cell lines indicates its potential role as a downstream mediator of TGF-β signaling . This reduction typically coincides with activation of Smad3, Snail, and Slug, alongside reduction of ZO-1, collectively marking EMT progression . The timing of RBM38 downregulation should be analyzed within the context of the EMT timeline, as early changes may indicate direct regulation by TGF-β signaling. TGF-β receptor inhibitors like SB431542 block the downregulation of RBM38, confirming pathway specificity . When interpreting these results, researchers should consider cell-type variations in response magnitude and timing. For quantitative analysis, normalization to appropriate housekeeping genes is essential, and expression changes should be correlated with functional outcomes such as cell migration and invasion capabilities to establish biological significance .

What are common technical challenges when using biotin-conjugated RBM38 antibodies and how can they be addressed?

Several technical challenges may arise when working with biotin-conjugated RBM38 antibodies. High background staining in IHC or IF applications is commonly caused by endogenous biotin in tissues, which can be addressed by implementing biotin blocking steps before antibody application . Weak or absent signal may result from insufficient permeabilization, as RBM38 is predominantly cytoplasmic ; optimizing permeabilization protocols with different detergents or concentrations can improve accessibility. Signal variability between experiments often stems from inconsistent handling of the antibody; creating single-use aliquots and avoiding repeated freeze-thaw cycles helps maintain consistent performance . Non-specific binding can be minimized by optimizing blocking buffers and antibody dilutions through titration experiments. For multi-color applications, streptavidin conjugate selection is crucial to avoid spectral overlap with other fluorophores. If signal amplification is needed, tyramide signal amplification systems can be employed but require careful optimization to prevent oversaturation and maintain quantitative relationships.

How can discrepancies between RBM38 protein and mRNA expression be interpreted in research studies?

Discrepancies between RBM38 protein and mRNA levels require careful interpretation as they may reveal important regulatory mechanisms. Post-transcriptional regulation is a common cause of such discrepancies, particularly relevant for RBM38 which itself functions as a post-transcriptional regulator . Researchers should consider protein stability factors, as changes in ubiquitination or other protein degradation pathways might affect RBM38 protein levels without altering mRNA expression. Translational efficiency can be evaluated using polysome profiling to determine if RBM38 mRNA recruitment to ribosomes is altered under experimental conditions. The presence of alternative RBM38 isoforms should be investigated, as different isoforms may have distinct functions, such as isoform 1's ability to induce cell cycle arrest while isoform 2 lacks this function . Technical considerations include antibody specificity for particular isoforms and primer design for mRNA detection. Temporal dynamics are also important—protein level changes often lag behind mRNA changes, necessitating time-course studies for accurate correlation analysis. When discrepancies are observed, integrating multiple detection methods provides more reliable interpretations of RBM38 regulation.

What approaches can resolve contradictory results when studying RBM38's effect on target mRNA stability?

When faced with contradictory results regarding RBM38's effect on target mRNA stability, researchers should implement systematic troubleshooting approaches. First, experimental conditions should be standardized, controlling for cell confluence, passage number, and serum conditions that may affect mRNA decay rates. The actinomycin-D concentration and exposure time should be optimized, as insufficient transcription inhibition can mask stability effects . Different quantification methods should be compared, including qRT-PCR, Northern blotting, and RNA-seq, as each has distinct sensitivity and bias profiles. The presence of competing RNA-binding proteins that may antagonize or synergize with RBM38 should be evaluated in different cell types or conditions. The specific regions of target mRNAs being analyzed are crucial, as RBM38 binds to specific motifs like AREs in the 3'-UTR ; using reporter constructs with different regions of the target mRNA can map the precise requirements for RBM38-mediated stabilization. Cell-type specific factors should be considered, as the RBM38 effect observed in breast cancer cells might differ in other cellular contexts due to varying cofactor availability . Finally, post-translational modifications of RBM38 itself might alter its RNA-binding capacity and should be investigated when contradictory results are observed.

What emerging applications of RBM38 antibodies show promise for translational research?

Biotin-conjugated RBM38 antibodies are increasingly valuable in translational research applications that bridge basic science and clinical medicine. Their potential in tissue microarray analysis enables high-throughput screening of RBM38 expression across large patient cohorts, helping identify its prognostic significance in various cancers beyond the established breast cancer connections . In liquid biopsy development, these antibodies may help detect circulating tumor cells undergoing EMT by monitoring RBM38 expression changes. Companion diagnostic development represents another promising application, as RBM38 expression levels might predict response to TGF-β pathway inhibitors in cancer treatment . These antibodies also facilitate mechanistic studies of treatment resistance, particularly how alterations in post-transcriptional regulation contribute to therapy evasion. By establishing the relationship between RBM38 and ZO-1, these antibodies contribute to understanding epithelial barrier disruption in pathological conditions . As research tools become more sophisticated, the integration of RBM38 antibodies with spatial transcriptomics will provide unprecedented insights into the localized function of this RNA-binding protein within the tumor microenvironment.

How might technological advances improve the utility of RBM38 antibodies in research?

Emerging technologies promise to enhance the research applications of RBM38 antibodies significantly. Single-cell antibody-based techniques will allow for heterogeneity analysis of RBM38 expression within tumors, potentially revealing subpopulations with distinct EMT states and invasive potential . Advanced proximity ligation assays can be developed to visualize and quantify interactions between RBM38 and its protein partners directly in tissue sections, providing spatial context for protein-protein interactions. Mass cytometry (CyTOF) incorporation of metal-tagged RBM38 antibodies will enable high-dimensional analysis of up to 40 parameters simultaneously, creating comprehensive cellular profiles related to RBM38 function. Nanobody and recombinant antibody technologies may yield smaller binding molecules with improved tissue penetration and reduced background. For live-cell imaging, membrane-permeable RBM38 antibody fragments conjugated to fluorophores could track dynamic changes in RBM38 localization during cellular processes like EMT. Integration with CRISPR screens will facilitate systematic identification of genes that modulate RBM38 function. Additionally, antibody engineering to create bispecific antibodies that simultaneously target RBM38 and its binding partners could provide novel tools for studying protein complexes involved in post-transcriptional regulation of EMT-related genes.

What open questions remain about RBM38's role in cancer progression that antibody-based research could address?

Despite significant advances, crucial questions about RBM38's role in cancer remain amenable to antibody-based investigations. The molecular mechanisms by which TGF-β signaling suppresses RBM38 expression can be explored through chromatin immunoprecipitation studies using antibodies against transcription factors potentially binding the RBM38 promoter . The complete repertoire of RBM38 mRNA targets beyond the established ZO-1, p21, and HuR requires systematic identification through techniques like eCLIP using RBM38 antibodies . The potential isoform-specific functions of RBM38 remain poorly understood; isoform-specific antibodies could reveal distinct localization patterns and binding partners . The role of post-translational modifications in regulating RBM38 function during cancer progression can be investigated using modification-specific antibodies. The therapeutic potential of targeting the RBM38 pathway demands exploration of how RBM38 expression correlates with response to existing cancer therapies using IHC in patient samples . Additionally, the contribution of RBM38 to cancer stem cell properties and therapy resistance requires investigation through antibody-based isolation of cancer cell subpopulations with varying RBM38 levels. These studies would significantly advance our understanding of how this RNA-binding protein influences cancer progression and treatment response.

Technical Specifications Table