ABCC5 Antibody

What is ABCC5 and why is it significant in research?

ABCC5, also known as MRP5, MOAT-C, SMRP, and pABC11, belongs to the ABC transporter superfamily and functions as a multispecific organic anion pump that transports nucleotide analogs and contributes to the cellular export of cyclic nucleotides . Its significance stems from its role in multidrug resistance and cancer progression, particularly in prostate cancer and hepatocellular carcinoma . ABCC5 has been identified as a potential biomarker and therapeutic target for various cancers, as it promotes resistance to treatments such as enzalutamide and sorafenib through both drug-efflux and non-drug efflux mechanisms .

What are the key characteristics of ABCC5 protein that researchers should be aware of?

ABCC5 is a transmembrane protein with a calculated molecular weight of 161 kDa, though it is observed between 161-200 kDa in experimental conditions . The canonical human protein consists of 1437 amino acid residues with up to five different isoforms reported . ABCC5 is primarily localized in the cell membrane, Golgi apparatus, and cytoplasm . The protein is encoded by the ABCC5 gene (Gene ID: 10057, GenBank Accession: NM_005688) . Understanding these characteristics is essential for proper experimental design and interpretation of results when using ABCC5 antibodies.

What types of ABCC5 antibodies are available for research applications?

ABCC5 antibodies are available in various formats, including polyclonal antibodies such as Rabbit IgG . These antibodies are commonly used for applications including Western Blot (WB), Immunohistochemistry (IHC), and ELISA . Most commercial antibodies are supplied in liquid form, stored in PBS with stabilizers such as sodium azide and glycerol . Researchers should select antibodies validated for their specific application and sample type, as reactivity may vary between human and other species samples .

What are the recommended protocols for using ABCC5 antibodies in Western Blot applications?

For Western Blot applications, ABCC5 antibodies are typically used at dilutions ranging from 1:500 to 1:2000 . When working with ABCC5, researchers should note that some cell lysates (like HepG2 and MCF-7) yield optimal results when unboiled . This suggests that the protein's tertiary structure may be important for antibody recognition. The observed molecular weight on Western blots is between 161-200 kDa, which may vary depending on post-translational modifications or the detection of different isoforms . For optimal results, use antigen affinity-purified antibodies and include appropriate positive controls such as MCF-7 cells, which are known to express detectable levels of ABCC5 .

How should ABCC5 antibodies be used for immunohistochemical detection?

For immunohistochemistry applications, the recommended dilution range for ABCC5 antibodies is 1:400-1:1600 . Optimal antigen retrieval is crucial for successful IHC staining; TE buffer at pH 9.0 is generally recommended, though citrate buffer at pH 6.0 can be used as an alternative . ABCC5 has been successfully detected in human tissues including ovarian cancer tissue . When optimizing IHC protocols, researchers should perform titration experiments to determine the optimal concentration for their specific tissue samples, as the required dilution may be sample-dependent . Including positive tissue controls such as ovarian cancer samples can help validate staining specificity.

What considerations are important when using ABCC5 ELISA kits?

ABCC5 ELISA kits typically employ a double antibody-sandwich detection method with a standard assay time of approximately 4 hours . The detection range for these kits is generally between 78.125-5000 pg/ml with a sensitivity of around 46.875 pg/ml . When using ELISA kits, proper sample preparation is crucial; these kits can analyze various sample types including serum, plasma, cell culture supernatant, and cell or tissue lysates . Researchers should always generate a standard curve for each experiment and ensure samples fall within the linear range of detection. For accurate quantification, samples may need to be appropriately diluted to fall within the kit's detection range .

How can researchers address cross-reactivity concerns with ABCC5 antibodies?

Cross-reactivity is a significant concern when working with ABCC5 antibodies due to the presence of multiple family members in the ABC transporter superfamily. To address this issue, researchers should select antibodies specifically validated against ABCC5 with minimal cross-reactivity to other ABCC family members . Include appropriate negative controls in experiments, such as ABCC5 knockout cells or tissues. When analyzing Western blot results, be aware that ABCC1 and ABCC3 are also frequently upregulated in response to treatments like sorafenib, which might complicate interpretation . Performing validation experiments using siRNA knockdown of ABCC5 can confirm antibody specificity by demonstrating reduced signal intensity following ABCC5 depletion .

What are common pitfalls in detecting ABCC5 protein expression in cancer cells?

Several challenges exist when detecting ABCC5 in cancer cells. First, expression levels may vary significantly between different cancer cell lines and can be induced by drug treatments, as demonstrated in HuH7, HepG2, and Sk-Hep-1 hepatocellular carcinoma cells treated with sorafenib . Second, protein denaturation conditions can affect antibody recognition, as evidenced by the recommendation to use unboiled lysates from certain cell lines . Third, ABCC5 expression may be regulated by multiple signaling pathways, including PI3K/AKT/NRF2, which can complicate expression analysis . To overcome these challenges, researchers should include multiple cell lines as controls, carefully optimize sample preparation protocols, and consider analyzing both mRNA and protein levels to obtain a comprehensive view of ABCC5 expression .

How can researchers validate the specificity of ABCC5 antibody staining in tissue samples?

To validate ABCC5 antibody specificity in tissue samples, several approaches are recommended. First, employ multiple antibodies targeting different epitopes of ABCC5 to confirm consistent staining patterns . Second, include positive control tissues known to express ABCC5, such as human ovary cancer tissue . Third, perform parallel immunohistochemistry and RNA in situ hybridization to correlate protein expression with mRNA localization. Fourth, conduct blocking experiments with the immunizing peptide to confirm specific binding. Finally, consider utilizing single-cell analysis techniques like t-SNE to evaluate cell-type specific expression of ABCC5, which has been shown to be concentrated primarily in macrophages and hepatocytes in liver tissue .

How can ABCC5 antibodies be utilized to investigate drug resistance mechanisms in cancer?

ABCC5 antibodies are valuable tools for investigating both classical drug efflux and non-classical resistance mechanisms. For studying drug efflux, researchers can use ABCC5 antibodies in combination with fluorescent substrate accumulation assays to correlate ABCC5 expression with drug retention . For non-classical mechanisms, co-immunoprecipitation experiments using ABCC5 antibodies can identify protein interactions that contribute to resistance, such as the interaction between ABCC5 and the PI3K/AKT/NRF2 pathway in sorafenib resistance .

In prostate cancer research, ABCC5 antibodies have helped identify a novel resistance mechanism where ABCC5 promotes enzalutamide resistance through AR-V7 upregulation via the NF-κB pathway rather than through drug efflux . This was demonstrated using ChIP assays confirming that P65 regulates AR expression by binding to its promoter . To implement such advanced studies, researchers should combine ABCC5 knockdown or overexpression models with selective pathway inhibitors and monitor changes in downstream targets using validated ABCC5 antibodies .

What methodologies can be used to study the relationship between ABCC5 expression and immune cell infiltration?

Recent research has revealed associations between ABCC5 expression and immune cell infiltration in cancers like hepatocellular carcinoma . To investigate this relationship, researchers can employ several advanced methodologies. First, multiplex immunohistochemistry using ABCC5 antibodies alongside immune cell markers (for macrophages, neutrophils, and dendritic cells) can visualize spatial relationships between ABCC5-expressing cells and immune infiltrates .

Second, flow cytometry with ABCC5 antibodies can quantify expression levels across different immune cell populations. Third, single-cell RNA sequencing complemented by protein validation with ABCC5 antibodies can provide comprehensive insights into cell-specific expression patterns . The t-SNE algorithm has revealed that ABCC5 expression is most concentrated in macrophages in liver tissues, suggesting a potential role in immune regulation . For functional validation, co-culture experiments with ABCC5-manipulated cancer cells and immune cells, monitored with ABCC5 antibodies, can elucidate the mechanistic impact on immune function.

How can researchers employ ABCC5 antibodies to investigate its potential as a biomarker for cancer prognosis?

To evaluate ABCC5 as a prognostic biomarker, researchers should implement a multi-faceted approach. First, tissue microarray (TMA) analysis with ABCC5 antibodies can assess expression patterns across large patient cohorts, as demonstrated in studies of hepatocellular carcinoma where ABCC5 expression correlated with poor prognosis . Second, researchers should correlate immunohistochemical staining intensity with clinical outcomes using statistical methods like Kaplan-Meier survival analysis and Cox proportional hazards models, which have confirmed ABCC5 as an independent risk factor in liver cancer .

Third, multiplex protein analysis combining ABCC5 with other potential biomarkers can identify synergistic prognostic panels. Fourth, liquid biopsy approaches measuring circulating ABCC5 protein levels using sensitive ELISA methods can provide minimally invasive monitoring options . Finally, researchers should validate findings across independent patient cohorts from diverse databases such as The Cancer Genome Atlas and Gene Expression Omnibus, as was done in hepatocellular carcinoma research that identified ABCC5 as a potential prognostic biomarker .

What experimental approaches can determine whether ABCC5 contributes to cancer progression through mechanisms beyond drug efflux?

To investigate non-drug efflux functions of ABCC5, researchers should implement several sophisticated experimental approaches. First, compare the phenotypic effects of ABCC5 knockdown/overexpression in the presence and absence of drug treatments. In enzalutamide-resistant prostate cancer cells, ABCC5 depletion resensitized cells to treatment and impeded xenograft tumor proliferation, suggesting functions beyond drug efflux .

Second, identify downstream signaling pathways affected by ABCC5 modulation using phosphoprotein arrays and Western blot analysis with pathway-specific antibodies. Research has shown that ABCC5 can influence the NF-κB pathway and AR-V7 expression in prostate cancer . Third, investigate ABCC5's impact on metabolic processes, as demonstrated in hepatocellular carcinoma where ABCC5 increased intracellular glutathione and attenuated lipid peroxidation by stabilizing SLC7A11, thereby inhibiting ferroptosis . Fourth, perform xenograft studies with ABCC5-modulated cells to evaluate in vivo tumor growth and metastasis independent of drug treatment. These approaches can comprehensively elucidate ABCC5's multifaceted roles in cancer progression beyond its classical drug transporter function.

How can ABCC5 antibodies be optimized for use in patient-derived xenograft (PDX) models?

For optimal use of ABCC5 antibodies in PDX models, researchers should first validate antibody cross-reactivity between human ABCC5 and the host animal species (typically mouse) . This is particularly important as PDX models combine human tumor cells with murine stromal components. Antibodies should be tested on both human and mouse tissues to ensure specificity and minimize background. Implement dual-staining protocols using species-specific markers alongside ABCC5 antibodies to distinguish between tumor and stromal expression.

When analyzing PDX samples, researchers should optimize antigen retrieval methods, as formalin fixation in PDX tissues may require stronger conditions than cell lines . For quantitative analysis, use digital pathology approaches with standardized scoring systems to evaluate ABCC5 expression levels across different PDX models and treatment conditions. This approach has proven valuable in enzalutamide resistance studies, where ABCC5 depletion was shown to impede xenograft tumor proliferation, suggesting its potential as a therapeutic target for resistant tumors .

What methods can be used to correlate ABCC5 expression with drug sensitivity in personalized medicine approaches?

To correlate ABCC5 expression with drug sensitivity for personalized medicine applications, researchers should implement a multi-platform approach. First, establish patient-derived organoids or primary cultures from tumor biopsies and quantify baseline ABCC5 expression using validated antibodies in Western blot, IHC, or ELISA formats . Second, perform drug sensitivity assays across panels of compounds at clinically relevant concentrations and correlate responses with ABCC5 expression levels.

Third, implement genetic manipulation studies (siRNA, CRISPR) to modulate ABCC5 expression and determine whether this alters drug sensitivity profiles, as demonstrated in hepatocellular carcinoma where ABCC5 knockdown significantly reduced resistance to sorafenib . Fourth, utilize computational approaches by mining databases like GDSC (Genomics of Drug Sensitivity in Cancer) and CTRP (Cancer Therapeutics Response Portal) to identify correlations between ABCC5 expression and response to various drugs . Finally, monitor ABCC5 expression changes during treatment using sequential biopsies or liquid biopsy approaches to track potential resistance development. These methodologies collectively provide a comprehensive framework for incorporating ABCC5 assessment into precision oncology.

How should researchers interpret conflicting ABCC5 expression data between mRNA and protein levels?

Discrepancies between ABCC5 mRNA and protein expression are common and require careful interpretation. First, researchers should recognize that post-transcriptional and post-translational modifications may significantly impact ABCC5 protein levels without corresponding changes in mRNA. Second, evaluate the stability of both ABCC5 mRNA and protein in experimental systems, as differences in half-life can lead to temporal disconnects between transcript and protein levels.

Third, consider technical factors that might influence detection, such as antibody specificity, detection thresholds of different methodologies, and sample preparation techniques that may affect protein extraction efficiency . Fourth, implement comprehensive approaches that simultaneously measure both mRNA (via qRT-PCR) and protein (via Western blot or ELISA) from the same samples, as done in studies examining sorafenib-induced ABCC5 expression in hepatocellular carcinoma cell lines . Finally, evaluate whether discrepancies are biologically meaningful by correlating functional outcomes with either mRNA or protein levels to determine which better predicts the phenotype of interest.

What statistical approaches are most appropriate for analyzing ABCC5 expression data in relationship to clinical outcomes?

For robust analysis of ABCC5 expression in relation to clinical outcomes, several statistical methods are recommended. First, employ Kaplan-Meier survival analysis with log-rank tests to compare outcomes between patient groups stratified by ABCC5 expression levels, as this approach has demonstrated associations between ABCC5 and poor prognosis in hepatocellular carcinoma . Second, conduct univariate and multivariate Cox proportional hazards regression to determine whether ABCC5 serves as an independent prognostic factor while controlling for confounding variables such as age, gender, and tumor stage .

Third, implement principal component analysis (PCA) to identify patterns within ABCC5 expression data and potentially stratify patients into distinct molecular subgroups . Fourth, use receiver operating characteristic (ROC) curve analysis to determine optimal cutoff values for ABCC5 expression that maximize sensitivity and specificity for predicting clinical outcomes. Finally, validate findings through bootstrapping or cross-validation approaches and confirm results in independent validation cohorts to ensure reproducibility. These rigorous statistical methods collectively strengthen the clinical relevance of ABCC5 expression data.