MRTO4 Antibody

What is MRTO4 and what cellular functions does it regulate?

MRTO4 (mRNA turnover 4 homolog) is a protein involved in multiple cellular processes including mRNA turnover and ribosome assembly. The protein, also known by synonyms C1orf33, MRT4, and dJ657E11.4, functions in the regulation of mRNA translation and cell growth, highlighting its importance in cellular homeostasis and proliferation . Recent research indicates that MRTO4 is essential for the functioning of mitochondrial ribosomes and protein synthesis within mitochondria, making it a crucial component of cellular energy metabolism pathways . The protein has a calculated molecular weight of 239 amino acids (28 kDa), though its observed molecular weight in experimental conditions typically ranges between 28-30 kDa .

What are the recommended applications for MRTO4 antibodies?

MRTO4 antibodies are primarily validated for Western Blot (WB) and ELISA applications. For Western Blot applications, a dilution range of 1:500-1:2400 is generally recommended, though researchers should optimize this range for their specific experimental systems . Positive Western Blot detection has been confirmed in human skeletal muscle tissue, mouse heart tissue, mouse brain tissue, and HEK-293 cells . When designing experiments, it's important to note that MRTO4 antibodies demonstrate reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across these species .

What are the optimal storage and handling conditions for MRTO4 antibodies?

MRTO4 antibodies are typically supplied in liquid form with storage buffers containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . These antibodies should be stored at -20°C, where they remain stable for approximately one year after shipment. Repeated freeze-thaw cycles should be avoided to maintain antibody integrity and performance . For the 20194-1-AP antibody specifically, aliquoting is unnecessary for -20°C storage, and 20μL sizes contain 0.1% BSA for additional stability . When handling the antibody, researchers should follow standard laboratory safety protocols, particularly due to the presence of sodium azide in the storage buffer.

How can MRTO4 antibodies be optimized for detecting low protein expression levels?

For detecting low MRTO4 expression levels, several methodological optimizations can enhance sensitivity. First, increase the antibody concentration from the standard 1:500-1:2400 range to 1:250-1:500, while simultaneously extending primary antibody incubation times to overnight at 4°C . Second, employ enhanced chemiluminescence (ECL) detection systems with longer exposure times, potentially utilizing signal amplification methods such as biotin-streptavidin systems. Third, increase the total protein loaded per well (50-100 μg) while ensuring even loading with validated housekeeping protein controls. Additionally, incorporate signal enhancement techniques such as tyramide signal amplification for immunohistochemistry applications or use concentrated protein samples through immunoprecipitation before Western blotting to improve detection of low abundance targets.

What experimental approaches can effectively investigate MRTO4's role in ribosome assembly?

Investigating MRTO4's function in ribosome assembly requires multi-faceted experimental approaches. Begin with polysome profiling using sucrose gradient centrifugation to fractionate ribosomes and analyze MRTO4 distribution across monosomal and polysomal fractions using the MRTO4 antibody (20194-1-AP) at 1:500 dilution . Complement this with ribosome affinity purification techniques such as Translating Ribosome Affinity Purification (TRAP) combined with mass spectrometry to identify MRTO4 interaction partners within ribosomal complexes. For functional studies, employ CRISPR-Cas9 mediated MRTO4 knockout or knockdown, followed by ribosome biogenesis assessment through nucleolar visualization and rRNA processing analysis. Proximity-dependent biotin identification (BioID) can further map the spatial organization of MRTO4 within ribosomal assembly complexes, while cryo-electron microscopy provides structural insights into MRTO4's positioning within nascent ribosomes.

How can researchers effectively validate MRTO4 antibody specificity for their experimental system?

Comprehensive validation of MRTO4 antibody specificity requires multiple complementary approaches. First, perform comparative Western blot analysis using different MRTO4 antibodies (such as PACO10618 and 20194-1-AP) to verify consistent band patterns at the expected 28-30 kDa molecular weight . Second, include essential controls: positive controls using tissues known to express MRTO4 (human skeletal muscle, mouse heart and brain tissues), negative controls utilizing MRTO4 knockout/knockdown samples, and peptide competition assays where pre-incubation with immunogen peptide should abolish specific binding . Third, employ orthogonal validation through immunoprecipitation followed by mass spectrometry identification of the pulled-down protein. Additionally, for cell/tissue staining applications, correlation between protein expression levels detected via Western blot and staining intensity provides further validation. Finally, cross-species reactivity testing can confirm antibody performance across human, mouse, and rat samples as claimed in the specifications .

How is MRTO4 expression associated with hepatocellular carcinoma prognosis and tumor microenvironment?

MRTO4 overexpression demonstrates significant correlation with poor prognosis in hepatocellular carcinoma (HCC), with expression levels positively associated with both tumor stage and grade in HCC patients . Analysis of tumor microenvironment (TME) characteristics reveals that low MRTO4 expression correlates with significantly higher TME scores compared to high MRTO4 expression groups, a difference primarily attributed to variations in stromal scores rather than immune scores . Regarding immune cell infiltration, MRTO4 expression shows positive correlation specifically with M0 macrophages (P<0.001), while demonstrating negative correlations with CD4+ memory resting T cells, naive B cells, monocytes (all P<0.001), and resting mast cells (P<0.05) . These findings suggest MRTO4 functions as an independent prognostic biomarker with significant implications for understanding the immunological landscape of HCC.

What is the relationship between MRTO4 expression and immunotherapy response prediction in HCC?

MRTO4 expression demonstrates significant potential as a predictor of immunotherapy response in HCC through multiple immunological correlates. Patients with low MRTO4 expression exhibit significantly higher immunophenoscores (IPS) compared to high expression groups, particularly in response categories involving CTLA4 and PD1 inhibition: ips-(ctla4–, pd1–) (P<0.001), ips-(ctla4+, pd1–) (P<0.05), and ips-(ctla4+, pd1+) (P<0.05) . MRTO4 expression positively correlates with tumor mutation burden (TMB) (R=0.27, P=2.0e-07), an established biomarker for immunotherapy response prediction . Additionally, MRTO4 demonstrates positive associations with nearly all major immune checkpoint genes, including CTLA4, LAG3, PDCD1, TIGIT, CD274, and TNFRSF9, further supporting its utility in predicting immune therapy responses . These correlations provide a multifaceted basis for employing MRTO4 expression as a biomarker for immunotherapy response prediction in HCC patients.

How does MRTO4 expression relate to drug sensitivity in HCC treatment?

Drug sensitivity analysis reveals significant correlations between MRTO4 expression levels and chemotherapeutic efficacy in HCC. Patients with high MRTO4 expression demonstrate significantly lower IC50 values for several key HCC therapeutic agents compared to those with low expression, including 5-fluorouracil (P=8.4e-07), gemcitabine (P=2.2e-06), and sorafenib (P=5.6e-08) . This inverse relationship between MRTO4 expression and IC50 values indicates that high MRTO4-expressing tumors may exhibit enhanced sensitivity to these standard chemotherapeutic agents. These findings suggest MRTO4 could serve as a predictive biomarker for therapeutic response, potentially enabling more personalized treatment selection for HCC patients. Further exploration of the molecular mechanisms underlying this correlation could identify novel therapeutic targets or combination strategies to overcome drug resistance in HCC treatment regimens.

What are the recommended protocols for detecting MRTO4 using Western blot analysis?

For optimal Western blot detection of MRTO4, follow this comprehensive protocol: Begin with sample preparation by lysing cells/tissues in RIPA buffer supplemented with protease inhibitors, sonicating briefly, and centrifuging at 14,000g for 15 minutes at 4°C. Quantify protein concentration using BCA assay and load 20-40 μg per lane on a 12% SDS-PAGE gel . Transfer proteins to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer containing 20% methanol. Block membranes with 5% non-fat milk in TBST for 1 hour at room temperature. Incubate with primary MRTO4 antibody at 1:500-1:2400 dilution overnight at 4°C . After washing three times with TBST, incubate with HRP-conjugated secondary antibody at 1:5000 for 1 hour at room temperature. Develop using enhanced chemiluminescence detection. Expected MRTO4 band should appear at 28-30 kDa, with positive signals confirmed in human skeletal muscle tissue, mouse heart and brain tissues, and HEK-293 cells .

What experimental controls should be included when studying MRTO4 in tumor samples?

Comprehensive experimental design for MRTO4 analysis in tumor samples requires multiple control types. First, include matched adjacent non-tumor tissue from the same patient to establish baseline expression levels. Second, incorporate positive control tissues known to express MRTO4 (skeletal muscle, heart, brain) and cell lines with verified MRTO4 expression (HEK-293) . Third, employ technical controls including loading controls (β-actin, GAPDH) for protein normalization and IgG controls for immunoprecipitation experiments. Fourth, include biological gradient controls representing different tumor stages and grades to correlate MRTO4 expression with disease progression . Fifth, utilize functional controls through MRTO4 knockdown/overexpression models to validate antibody specificity and biological effects. Finally, when analyzing tumor microenvironment correlations, incorporate immune cell markers and stromal markers to contextualize MRTO4 expression patterns within the heterogeneous tumor landscape .

How can researchers accurately quantify MRTO4 protein levels across different experimental systems?

Accurate quantification of MRTO4 protein levels across experimental systems requires a multi-method approach. First, employ densitometric analysis of Western blot signals using ImageJ or similar software, normalizing MRTO4 band intensity to validated housekeeping proteins such as β-actin or GAPDH . Second, develop a standard curve using recombinant MRTO4 protein at known concentrations to establish absolute quantification parameters. Third, validate Western blot findings with quantitative ELISA assays, which can provide more precise numerical data on protein concentration. Fourth, for tissue-specific analysis, complement protein quantification with immunohistochemistry using tissue microarrays with standardized staining protocols and automated image analysis for consistent scoring . Fifth, when comparing across species or cell types, account for potential cross-reactivity variations by validating antibody performance in each system. Finally, employ spike-in controls of known MRTO4 concentrations to assess recovery rates and quantification accuracy across different sample matrices.

How might MRTO4 function as a therapeutic target in cancer treatment strategies?

MRTO4's potential as a therapeutic target in cancer stems from its multifaceted roles in cellular processes and disease progression. First, its overexpression in hepatocellular carcinoma correlates with poor prognosis, suggesting inhibition could provide clinical benefit . Second, MRTO4's involvement in ribosome biogenesis represents a targetable vulnerability, as cancer cells often exhibit heightened dependence on protein synthesis machinery. Third, the correlation between MRTO4 expression and drug sensitivity provides a foundation for combination therapies—inhibiting MRTO4 might enhance responsiveness to established drugs like sorafenib, 5-fluorouracil, and gemcitabine . Fourth, given MRTO4's association with immune checkpoint expression, targeting it could potentially modulate the tumor immune microenvironment to enhance immunotherapy efficacy. Therapeutic approaches could include small molecule inhibitors disrupting MRTO4's interaction with ribosomal assembly factors, antisense oligonucleotides to reduce expression, or antibody-drug conjugates targeting MRTO4-overexpressing cells in tumors with accessible vasculature.

What techniques can effectively assess MRTO4's interaction with the immune microenvironment in tumors?

Investigating MRTO4's interactions with the tumor immune microenvironment requires sophisticated methodological approaches. First, employ multiplex immunofluorescence or imaging mass cytometry to simultaneously visualize MRTO4 expression alongside multiple immune cell markers, enabling spatial relationship analysis between MRTO4-expressing cells and immune infiltrates . Second, utilize single-cell RNA sequencing of tumor samples to correlate MRTO4 expression with transcriptional profiles of various immune cell populations. Third, perform co-culture experiments with MRTO4-modulated tumor cells and immune cell subsets (particularly M0 macrophages, CD4+ memory resting T cells, naive B cells, monocytes, and resting mast cells) to assess functional interactions . Fourth, develop syngeneic mouse models with MRTO4 knockout or overexpression to evaluate in vivo immune infiltration dynamics. Fifth, employ chromatin immunoprecipitation sequencing (ChIP-seq) to identify potential transcriptional regulation of immune-related genes by MRTO4 or its downstream effectors. Additionally, cytokine profiling of MRTO4-modulated systems can reveal immunomodulatory signaling networks influenced by MRTO4 expression.

How can researchers integrate MRTO4 expression data with other molecular profiling to develop comprehensive cancer biomarker panels?

Developing comprehensive cancer biomarker panels incorporating MRTO4 requires systematic integration of multi-omics data. First, combine MRTO4 protein expression data from immunohistochemistry and Western blot analyses with transcriptomic profiling to identify co-expressed gene networks . Second, integrate MRTO4 expression with mutational landscape analysis, particularly given its positive correlation with tumor mutation burden (TMB) in HCC . Third, perform pathway enrichment analysis to contextualize MRTO4 within functional biological processes, identifying potential synergistic biomarkers. Fourth, correlate MRTO4 expression with clinical parameters including tumor stage, grade, and patient survival data to establish prognostic significance thresholds . Fifth, employ machine learning algorithms to develop predictive models combining MRTO4 with other molecular features to optimize sensitivity and specificity for clinical outcomes. Finally, validate integrated biomarker panels through retrospective analysis of large patient cohorts with complete clinical follow-up data, preferably from multiple institutions to ensure robust generalizability. This comprehensive approach can transform MRTO4 from a single biomarker into a component of clinically actionable multi-parameter prognostic and predictive tools.