ABCA8 Antibody

What is ABCA8 and why is it important in research?

ABCA8 (ATP-binding cassette, sub-family A, member 8) is a transmembrane transporter belonging to the ABC transporter superfamily. It plays crucial roles in transporting organic molecules, particularly cholesterol, and in drug efflux mechanisms. Research significance stems from its demonstrated roles in cholesterol metabolism and cancer biology. ABCA8 has been shown to facilitate cholesterol efflux and modulate high-density lipoprotein (HDL) levels in both humans and mice . Additionally, ABCA8 has emerged as a potential tumor suppressor, with downregulation observed in hepatocellular carcinoma (HCC) correlating with poor prognosis .

What are the best applications for ABCA8 antibody in laboratory research?

ABCA8 antibodies are primarily utilized in Western Blot (WB), Immunohistochemistry (IHC), and ELISA applications. Based on validation studies, these antibodies demonstrate specific reactivity with human and mouse samples . For Western Blot applications, ABCA8 antibody (such as 24351-1-AP) typically detects protein bands at 160-180 kDa, consistent with the calculated molecular weight of 179 kDa . In IHC applications, ABCA8 antibody shows positive detection in human liver tissue and mouse brain tissue, making these tissues excellent positive controls for antibody validation . Researchers should select application-specific dilutions: 1:200-1:1000 for WB and 1:50-1:500 for IHC applications .

How should ABCA8 antibody be stored and handled for optimal performance?

For optimal performance, ABCA8 antibody should be stored at -20°C where it remains stable for one year after shipment. The antibody is typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Unlike some antibodies that require aliquoting for long-term storage, aliquoting is unnecessary for -20°C storage of ABCA8 antibody . When handling, it's important to avoid repeated freeze-thaw cycles which can compromise antibody performance. For smaller quantity formats (20μl sizes), the solution contains 0.1% BSA as a stabilizer . Proper storage and handling are critical for maintaining antibody specificity and sensitivity across experimental applications.

What are the methodological considerations for using ABCA8 antibody in cancer research?

When applying ABCA8 antibody in cancer research, several methodological considerations become critical. First, researchers should implement a quantitative scoring system for immunohistochemical analysis. As demonstrated in HCC research, ABCA8 staining can be evaluated using a combined intensity and extent score system: intensity scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong), and extent scored based on percentage of positive cells (0 for negative, 1 for 0.01-25%, 2 for 25.01-50%, 3 for 50.01-75%, and 4 for 75.01-100%) . The histologic score (H-score) is calculated by multiplying the proportion score by the intensity score, resulting in total scores classified as negative/low (0-4) or positive/high (6-12) . This quantitative approach enables statistical correlation between ABCA8 expression and clinical parameters such as tumor stage, differentiation, and patient survival.

How can ABCA8 antibody be used to investigate cholesterol efflux mechanisms?

ABCA8 antibody serves as a crucial tool for investigating cholesterol efflux mechanisms through several sophisticated experimental approaches. Researchers can employ co-immunoprecipitation techniques to study ABCA8's interaction with other cholesterol transport proteins, particularly ABCA1, with which it colocalizes and functionally interacts to potentiate cholesterol efflux . For cholesterol efflux assays, researchers should establish cell lines with stable ABCA8 overexpression or knockdown, then measure cholesterol efflux to apolipoprotein AI using radiolabeled cholesterol . Comparative analysis between wild-type and mutant ABCA8 (such as P609R, IVS17-2 A>G, or T741X variants) will elucidate structure-function relationships, as wild-type ABCA8 overexpression increases cholesterol efflux approximately 1.8-fold compared to mutant variants .

What technical challenges exist in detecting ABCA8 expression in different tissue types?

Detecting ABCA8 expression across different tissue types presents several technical challenges requiring specific optimization strategies. For IHC applications in neural tissues, mouse brain samples serve as excellent positive controls, while human liver tissue provides another validated control tissue type . Antigen retrieval conditions critically impact detection sensitivity—ABCA8 antibody generally requires retrieval with TE buffer at pH 9.0, though an alternative approach using citrate buffer at pH 6.0 may be necessary for certain tissue types . Tissue-specific background staining may occur, necessitating careful titration of antibody dilutions (1:50-1:500 for IHC) for each specific tissue type being studied . When comparing expression across tissue types with varying baseline expression levels, quantitative PCR should complement protein detection to establish comprehensive expression profiles.

How is ABCA8 implicated in hepatocellular carcinoma progression?

ABCA8 plays a significant role in hepatocellular carcinoma (HCC) progression through multiple mechanisms. Research demonstrates that ABCA8 is frequently downregulated in HCC tissues compared to adjacent normal tissues, and this downregulation negatively correlates with patient prognosis . Functionally, ABCA8 acts as a tumor suppressor in HCC, with overexpression inhibiting cellular proliferation as demonstrated through Cell Counting Kit-8 (CCK-8) assays and colony formation experiments . Mechanistically, ABCA8 is regulated by miR-374b-5p, which is upregulated in HCC. This microRNA targets ABCA8's 3'-untranslated region (UTR), reducing its expression . The downstream pathway involves the ERK/ZEB1 signaling axis—ABCA8 downregulation induces epithelial-to-mesenchymal transition via this pathway, promoting HCC progression and metastasis . This mechanism provides potential targets for therapeutic intervention in HCC.

What is the relationship between ABCA8 genetic variants and HDL cholesterol levels?

ABCA8 genetic variants demonstrate a significant relationship with HDL cholesterol (HDLc) levels through direct impact on cholesterol efflux mechanisms. Sequencing studies have identified three predicted deleterious heterozygous ABCA8 mutations (p.Pro609Arg [P609R], IVS17-2 A>G, and p.Thr741Stop [T741X]) exclusively in probands with low HDLc levels . Carriers of these heterozygous mutations exhibit significantly lower HDLc levels compared to first-degree family controls (0.86±0.34 versus 1.17±0.26 mmol/L; P=0.005) . Animal models confirm this relationship, as Abca8b-/- mice on high-cholesterol diets show a significant 29% decrease in HDLc levels compared to wild-type mice . Conversely, hepatic overexpression of human ABCA8 in mice results in significant increases in plasma HDLc and enhanced macrophage-to-feces reverse cholesterol transport . These findings establish ABCA8 as an important modulator of HDL metabolism and potential therapeutic target for dyslipidemia.

How does ABCA8 interact with ABCA1 in cholesterol transport pathways?

ABCA8 interacts with ABCA1 through both physical association and functional cooperation in cholesterol transport pathways. Colocalization studies demonstrate that ABCA8 physically interacts with ABCA1 at the cellular level . This interaction appears mechanistically significant as ABCA8 potentiates ABCA1-mediated cholesterol efflux . Functional studies reveal that overexpression of wild-type ABCA8 results in a significant 1.8-fold increase in cholesterol efflux to apolipoprotein AI, while mutant ABCA8 fails to produce this effect . The synergistic relationship between these two ABC transporters suggests a coordinated system for cholesterol efflux, where ABCA8 may enhance or regulate ABCA1 function. This interaction has important implications for reverse cholesterol transport, the process by which excess cholesterol is removed from peripheral tissues and transported to the liver for excretion, a protective mechanism against atherosclerosis .

What are the optimal protocols for Western Blot detection of ABCA8?

For optimal Western Blot detection of ABCA8, researchers should implement a specialized protocol tailored to this high molecular weight protein (160-180 kDa observed size) . Sample preparation should include complete protein denaturation with SDS and reducing agents, with heating at 95°C for 5 minutes. For gel electrophoresis, use lower percentage (6-8%) SDS-PAGE gels to properly resolve the large ABCA8 protein. Extended transfer times (>2 hours) at lower voltage or overnight transfers at 4°C are recommended for complete transfer of large proteins to membranes. For primary antibody incubation, ABCA8 antibody should be used at dilutions between 1:200-1:1000, with overnight incubation at 4°C for maximum sensitivity . As ABCA8 shows tissue-specific expression, mouse brain tissue serves as an excellent positive control for Western Blot validation . Given the large size of ABCA8, verify transfer efficiency with protein ladders and consider using reversible membrane staining to confirm protein transfer prior to antibody incubation.

What controls should be included when studying ABCA8 expression in experimental models?

When studying ABCA8 expression in experimental models, comprehensive controls must be included to ensure valid and reproducible results. Positive tissue controls should include mouse brain tissue for murine studies and human liver tissue for human studies, as these have been validated for ABCA8 expression . For antibody validation, include negative controls by either omitting primary antibody or using pre-immune serum. When conducting knockdown or overexpression studies, three control groups should be included: untreated cells as blank groups (Bg), vector-only transfected controls, and scrambled shRNA controls for knockdown experiments . For functional studies like proliferation or colony formation assays, multiple time points should be assessed, with standardized cell numbers across experimental groups . When analyzing ABCA8 expression in pathological conditions such as HCC, paired samples of tumor and adjacent non-tumor tissues from the same patient provide optimal controlled comparisons .

How should researchers quantify and interpret ABCA8 expression in clinical samples?

For accurate quantification and interpretation of ABCA8 expression in clinical samples, researchers should employ a multi-modal approach combining molecular and histological techniques. For immunohistochemical analysis, implement the standardized H-score system that incorporates both staining intensity (0-3) and extent (0-4), with final scores categorized as negative/low (0-4) or positive/high (6-12) . This semi-quantitative approach facilitates statistical comparisons across patient cohorts. Complement protein-level assessment with mRNA quantification using qPCR, normalizing ABCA8 expression to stable housekeeping genes . For clinical relevance, correlate ABCA8 expression with patient clinicopathological parameters and survival outcomes using appropriate statistical tests such as Kaplan-Meier analysis for survival and chi-square tests for categorical variables . Validation through public databases like UALCAN (http://ualcan.path.uab.edu) and Kaplan-Meier plotter (http://kmplot.com) strengthens clinical findings and provides external confirmation of expression patterns and prognostic significance .

What are common issues when using ABCA8 antibody in IHC and how can they be resolved?

Several common issues may arise when using ABCA8 antibody in IHC applications, each requiring specific troubleshooting approaches. Background staining presents a frequent challenge, particularly in liver tissues which naturally express ABCA8. To reduce background, researchers should optimize blocking conditions (5-10% normal serum from the same species as the secondary antibody for 1-2 hours) and antibody dilutions (between 1:50-1:500 for IHC) . Weak or absent staining often results from inadequate antigen retrieval; ABCA8 detection specifically requires retrieval with TE buffer at pH 9.0, though citrate buffer at pH 6.0 presents an alternative when needed . Tissue-dependent variability can be addressed through tissue-specific optimization of antibody concentration and incubation times. For suspected non-specific binding, perform peptide competition assays using the immunogen peptide (ABCA8 fusion protein Ag18991) to confirm antibody specificity . Finally, include positive control tissues (human liver or mouse brain) in each experimental run to verify staining protocols and antibody performance .

How can researchers differentiate between ABCA8 isoforms or related ABC transporters?

Differentiating between ABCA8 isoforms or related ABC transporters requires careful experimental design utilizing multiple complementary techniques. First, select antibodies with known epitope information to target unique regions—the 24351-1-AP ABCA8 antibody is raised against a fusion protein immunogen (Ag18991) that enables specific detection . For mRNA analysis, design PCR primers that span unique exon junctions to distinguish between isoforms, and perform melt curve analysis to ensure amplification specificity. When studying ABCA subfamily members with high homology, conduct immunoprecipitation followed by mass spectrometry for definitive protein identification. For functional discrimination, implement selective knockdown of specific transporters using targeted siRNAs followed by functional assays like cholesterol efflux to apolipoprotein AI . Co-expression analysis can identify differences in tissue distribution patterns—while ABCA8 shows strong expression in brain and liver tissues, other ABC transporters may have distinct tissue expression profiles .

What strategies can improve detection of low ABCA8 expression in cancer tissues?

Detecting low ABCA8 expression in cancer tissues presents challenges that can be addressed through several signal amplification and sensitivity enhancement strategies. For IHC applications in tissues with low ABCA8 expression, implement tyramide signal amplification systems, which can increase detection sensitivity by 10-50 fold while maintaining specificity. Optimize antigen retrieval conditions, specifically using TE buffer at pH 9.0 as recommended for ABCA8 antibody, with extended retrieval times (20-30 minutes) . Consider using polymer-based detection systems rather than traditional avidin-biotin methods to reduce background and enhance specific signal. For Western blot detection of low abundance ABCA8, increase protein loading (50-100 μg), extend exposure times, and use enhanced chemiluminescence substrates with higher sensitivity . Complementary techniques like droplet digital PCR can detect low copy number transcripts with greater sensitivity than conventional qPCR. In HCC samples specifically, use the established H-score system with appropriate statistical analysis to detect subtle differences in expression levels that correlate with clinical outcomes .

How might ABCA8 serve as a therapeutic target in cancer and cardiovascular disease?

ABCA8 presents promising therapeutic potential in both cancer and cardiovascular disease through distinct mechanistic pathways. In HCC, where ABCA8 functions as a tumor suppressor, therapeutic strategies could focus on upregulating ABCA8 expression or activity . One approach involves targeting miR-374b-5p, which negatively regulates ABCA8, using antagomirs or locked nucleic acid inhibitors to restore ABCA8 expression . Small molecule activators of ABCA8 could potentially inhibit tumor growth and metastasis by enhancing its native tumor-suppressive functions. For cardiovascular applications, ABCA8's role in cholesterol efflux and HDL metabolism makes it a potential target for dyslipidemia and atherosclerosis treatment . Pharmacological enhancement of ABCA8 activity or expression could increase reverse cholesterol transport, thereby potentially reducing atherosclerotic plaque formation. Gene therapy approaches delivering functional ABCA8 to carriers of deleterious mutations could normalize HDL levels . Development of screening assays for ABCA8 functional modulators would enable high-throughput identification of therapeutic candidates for both disease contexts.

What is the relationship between ABCA8 and drug resistance mechanisms?

The relationship between ABCA8 and drug resistance mechanisms represents an emerging area of research with significant clinical implications. As a member of the ABC transporter superfamily known for drug efflux capabilities, ABCA8 potentially contributes to multidrug resistance in cancer and other diseases . Investigation of this relationship should focus on several experimental approaches: correlation of ABCA8 expression levels with drug response in patient-derived xenografts or clinical samples; functional studies measuring intracellular accumulation of chemotherapeutic agents in cells with modulated ABCA8 expression; and transport studies with radiolabeled or fluorescently-tagged drugs to determine if they serve as ABCA8 substrates. Research should also examine whether ABCA8's interaction with ABCA1 affects drug transport capabilities of either protein . Understanding specific drug substrates for ABCA8 would inform pharmaceutical development and potentially enable prediction of drug resistance mechanisms. Studies should evaluate whether ABCA8 genetic variants affect drug transport efficiency, potentially explaining individual variations in drug response.

How does ABCA8 interact with other lipid transport systems beyond ABCA1?

ABCA8's interaction with lipid transport systems beyond ABCA1 represents a complex network that merits systematic investigation. While ABCA8 has been shown to colocalize and interact with ABCA1 in cholesterol efflux pathways , its potential interactions with other lipid transporters remain largely unexplored. Research approaches should include co-immunoprecipitation studies with other ABC transporters (particularly ABCG1 and ABCG5/8) and non-ABC lipid transporters such as SR-BI and NPC1/2. Lipidomic analyses of cells with modulated ABCA8 expression would identify specific lipid species affected beyond cholesterol, potentially revealing broader roles in phospholipid or sphingolipid transport. Intracellular trafficking studies using fluorescently-tagged ABCA8 could identify subcellular compartments where ABCA8 operates and potential overlap with other transport systems. In vivo studies using tissue-specific Abca8 knockout models would elucidate tissue-dependent interactions with different lipid transport pathways. Understanding these broader interactions would provide insight into integrated lipid homeostasis mechanisms and potentially reveal new therapeutic targets for metabolic and cardiovascular diseases .