ABCC9 Antibody

What is ABCC9 and what are its primary functions in cellular physiology?

ABCC9, a 174 kDa protein of the ATP-binding cassette transporter superfamily, functions as a regulatory subunit of ATP-sensitive potassium channels (KATP). It forms cardiac and smooth muscle-type KATP channels by partnering with KCNJ11, where ABCC9 handles activation and regulation while KCNJ11 creates the channel pore . This protein plays critical roles in cellular energy homeostasis by linking metabolic status to membrane excitability. During electrophysiological studies, researchers have demonstrated that ABCC9 responds to intracellular ATP/ADP ratios, enabling cells to adjust electrical activity based on metabolic conditions .

Which applications are most suitable for ABCC9 antibody use in research?

ABCC9 antibodies have been validated for multiple research applications with varying tissue and species specificity. Western blotting (WB) is highly effective for quantifying ABCC9 protein expression, with recommended dilutions around 1/1000 for mouse monoclonal antibodies . Immunohistochemistry on paraffin-embedded sections (IHC-P) provides excellent visualization of ABCC9 distribution in tissue samples, while immunocytochemistry/immunofluorescence (ICC/IF) allows subcellular localization studies at concentrations of approximately 1/100 . ELISA applications are also suitable, particularly when quantitative measurements are required across multiple samples .

How should researchers select between different ABCC9 antibodies for specific experimental needs?

Selection of appropriate ABCC9 antibodies depends on target epitopes, species reactivity, and experimental applications. For C-terminal epitope recognition, mouse monoclonal antibodies like N319A/14 targeting amino acids from position 1500 to the C-terminus demonstrate robust performance in Western blot and IHC-P applications with cross-reactivity across human, mouse, and rat samples . For mid-protein epitope recognition, polyclonal antibodies targeting amino acids 640-669 offer an alternative binding profile with human and mouse reactivity . Researchers should consider whether their experimental design requires monoclonal specificity or the broader epitope recognition of polyclonal antibodies, based on whether they need to detect specific isoforms or total ABCC9 protein expression.

What are the optimal fixation and sample preparation methods for ABCC9 immunodetection?

For immunocytochemistry applications, 4% formaldehyde fixation for 15 minutes at room temperature has been validated with ABCC9 antibodies, followed by a one-hour primary antibody incubation . For tissue preparation in IHC-P applications, standard paraffin embedding following formalin fixation preserves ABCC9 epitopes effectively. For Western blotting, membrane-enriched lysates are recommended as ABCC9 is a membrane protein; for example, brain membrane lysates at 10 μg concentration have shown successful detection of the predicted 174 kDa band . Antigen retrieval techniques may be necessary for formalin-fixed tissues to expose epitopes masked during fixation.

How should researchers validate ABCC9 antibody specificity in genetic knockout or knockdown models?

Validating antibody specificity requires rigorous controls, particularly when investigating ABCC9 function. Researchers should consider:

• Genetic validation: Using tissues from Abcc9 knockout models as negative controls. The established methods for generating Abcc9-targeted alleles involve loxP site insertion near exon 5, with subsequent Cre-mediated excision . RT-PCR amplification between exons 2 and 6 can confirm deletion at the mRNA level.

• siRNA/shRNA knockdown: For cell culture systems, transfection with ABCC9-targeted siRNA followed by Western blot to confirm protein reduction provides a flexible validation approach.

• Peptide competition: Pre-incubation of the antibody with the immunizing peptide should abolish specific signals if the antibody is truly binding to ABCC9.

• Cross-validation: Using multiple antibodies targeting different ABCC9 epitopes to confirm consistent localization and expression patterns.

What are the critical considerations when investigating ABCC9 mutations with antibody-based methods?

ABCC9 mutations like V734I and S1402C have been identified in patients with cardiac conditions such as Early Repolarization Syndrome and Brugada Syndrome . When studying these mutations, researchers should:

• Determine whether the antibody's epitope encompasses or is affected by the mutation site. For instance, antibodies targeting amino acids 640-669 would be unaffected by the V734I mutation .

• Consider using heterologous expression systems to compare wild-type and mutant protein detection. HEK293 cells co-transfected with wild-type KCNJ11 and either wild-type or mutant ABCC9 constructs provide a controlled system for studying mutant protein expression and localization .

• Evaluate whether mutations affect protein stability, potentially altering antibody detection sensitivity compared to wild-type ABCC9.

• Combine antibody-based detection with functional patch-clamp studies to correlate protein expression with altered channel function, as mutations like V734I cause a 5-fold change in Mg-ATP IC50 .

How can researchers effectively use ABCC9 antibodies to investigate its role in tissue-specific pathologies?

ABCC9 has been implicated in various pathologies, including hippocampal sclerosis during aging and cardiac arrhythmias . For targeted pathology studies:

• Co-staining protocols: Combine ABCC9 antibodies with tissue-specific markers and pathology indicators. For example, in hippocampal sclerosis studies, co-staining with phosphorylated TDP-43 antibodies would help correlate ABCC9 expression with pathological protein aggregation .

• Comparative tissue arrays: Analyze ABCC9 expression across affected and unaffected tissues from the same patient to control for individual variation. This approach is particularly valuable in heterogeneous conditions like hippocampal sclerosis.

• Quantitative image analysis: Develop standardized protocols for measuring staining intensity and distribution patterns using digital image analysis software to detect subtle changes in ABCC9 expression or localization.

• Age-matched controls: Since ABCC9 polymorphisms have been associated with age-related pathologies, carefully match experimental and control samples by age to avoid confounding variables .

What techniques can be applied to study ABCC9 protein interactions with channel partners and regulatory molecules?

To investigate ABCC9's interactions with proteins like KCNJ11 and regulatory molecules:

• Co-immunoprecipitation: Using ABCC9 antibodies to pull down protein complexes, followed by Western blotting for potential interaction partners such as KCNJ11, can reveal physiologically relevant protein-protein interactions.

• Proximity ligation assays: This technique can visualize and quantify protein interactions in situ with spatial resolution below 40 nm, allowing detection of ABCC9 interactions with channel components in native tissue contexts.

• FRET/BRET approaches: For live-cell studies of protein interactions, fluorescence or bioluminescence resonance energy transfer techniques using tagged ABCC9 and potential partners can reveal dynamic interaction changes under different physiological conditions.

• Split-reporter complementation assays: These can be used to confirm direct protein-protein interactions when overexpressing ABCC9 with candidate interacting proteins.

How can researchers address non-specific binding when using ABCC9 antibodies in complex tissue samples?

When working with tissues that show high background or non-specific signals:

• Optimize blocking conditions: For Western blots containing ABCC9, which has a predicted size of 174 kDa, use 5% non-fat milk or BSA in TBS-T with extended blocking times (1-2 hours) to reduce non-specific binding .

• Titrate antibody concentration: For the mouse monoclonal anti-ABCC9 antibody, a dilution series from 1:500 to 1:2000 should be tested to determine optimal signal-to-noise ratio for each specific application .

• Include absorption controls: Pre-incubate the antibody with recombinant ABCC9 protein to confirm signal specificity.

• Consider tissue-specific validation: Since ABCC9 expression varies across tissues, validation in each new tissue type is essential. Brain membrane lysates have been successfully used as positive controls for Western blot applications .

What are the most effective strategies for studying low-abundance ABCC9 expression in research samples?

ABCC9 expression can vary significantly between tissues, requiring special approaches for low-abundance detection:

• Signal amplification systems: Tyramide signal amplification or polymer-based detection systems can enhance sensitivity for IHC-P applications.

• Membrane enrichment: For Western blotting, preparing membrane-enriched fractions through ultracentrifugation can concentrate ABCC9, improving detection of the 174 kDa band in tissues with low expression .

• Transcript analysis correlation: Combine protein detection with RT-qPCR to verify low protein expression results against mRNA levels using validated primers targeting regions between exons 2-5 .

• Extended exposure times: For Western blotting of low-abundance samples, longer exposure times with high-sensitivity chemiluminescent substrates may be necessary, with appropriate negative controls to distinguish specific from non-specific signals.

How can researchers accurately interpret ABCC9 isoform expression patterns in different experimental systems?

ABCC9 exists in multiple splice variants, complicating interpretation of antibody-based detection:

• Isoform-specific detection: Select antibodies that target regions common to all isoforms (such as the C-terminal region) for total ABCC9 detection, or use isoform-specific antibodies when studying particular variants .

• Molecular weight verification: Careful analysis of band patterns in Western blots, comparing to predicted molecular weights of known isoforms (approximately 174 kDa for the full-length protein) .

• RT-PCR analysis: Supplement protein detection with transcript analysis using primers spanning alternative splice junctions to confirm the presence of specific isoforms .

• Developmental context: Consider developmental and tissue-specific expression patterns, as Northern blot analyses have shown variations in ABCC9 expression across different developmental timepoints (e16, P1, P7, P14, and adult) .

How can ABCC9 antibody-based studies be integrated with genetic association studies in disease research?

ABCC9 genetic variants have been associated with conditions like Brugada syndrome and hippocampal sclerosis . To integrate antibody studies with genetic findings:

• Genotype-phenotype correlation: Analyze ABCC9 protein expression and localization in samples stratified by genotype at risk SNPs such as rs704178 .

• Allele-specific expression: Employ allele-specific antibodies (where available) to distinguish expression from different alleles in heterozygous samples.

• eQTL analysis framework: Correlate genetic variants with protein expression levels detected by antibody-based methods to identify expression quantitative trait loci.

• Tissue-specific effects: Given that ABCC9 polymorphisms show tissue-specific effects, compare antibody staining patterns across multiple tissue types from the same individual to detect differential effects of genetic variants .

What methodologies can combine electrophysiological studies with ABCC9 antibody-based protein detection?

To correlate ABCC9 expression with channel function:

• Patch-clamp with immunocytochemistry: Perform electrophysiological recordings followed by fixation and ICC/IF to correlate channel activity with protein expression in the same cells .

• Split-sample analysis: Divide tissue samples for parallel processing - one portion for patch-clamp studies and another for antibody-based protein quantification.

• Pharmacological manipulation: Study changes in ABCC9 expression following treatment with sulfonylurea drugs or KATP channel modulators, which have been shown to modify ABCC9 function and may affect expression patterns .

• Heterologous expression systems: For mutant studies, combine Western blotting and ICC/IF with patch-clamp analyses in tsA201 cells expressing wild-type or mutant ABCC9 to correlate expression levels with functional changes .

How should researchers approach ABCC9 antibody selection for multi-species comparative studies?

For evolutionary or translational studies spanning multiple species:

• Epitope conservation analysis: Select antibodies targeting highly conserved epitopes, such as those in the C-terminal region of ABCC9, which shows conservation across human, mouse, and rat species .

• Validation hierarchy: Start with species officially validated by manufacturers (human, mouse, and rat for many ABCC9 antibodies), then extend to closely related species with confirmation using positive and negative controls .

• Cross-reactivity prediction: For untested species, align the target epitope sequence across species to predict potential cross-reactivity. High sequence homology (>90%) suggests likely cross-reactivity, though experimental validation remains essential .

• Multiple antibody approach: When studying novel species, employ multiple antibodies targeting different ABCC9 epitopes to confirm consistent results and reduce the risk of species-specific epitope differences causing false negatives.

How can ABCC9 antibodies contribute to understanding the role of this protein in metabolic transition during development?

ABCC9 is required for the transition from glycolytic to oxidative metabolism in the developing heart . To study this developmental role:

• Temporal expression analysis: Use developmental tissue arrays with ABCC9 antibodies to track expression changes across key transitional timepoints from embryonic to postnatal stages .

• Co-localization with metabolic markers: Combine ABCC9 antibody staining with markers of glycolytic and oxidative metabolism to visualize the correlation between ABCC9 expression and metabolic phenotype shifts.

• Conditional knockout models: Apply ABCC9 antibodies to validate tissue-specific and temporal deletion in conditional knockout models, correlating protein absence with metabolic phenotypes .

• Single-cell approaches: Implement single-cell immunofluorescence analysis to detect heterogeneity in ABCC9 expression during developmental transitions, which may reveal subpopulations of cells at different stages of metabolic adaptation.

What considerations are important when using ABCC9 antibodies in research on cardiac channelopathies?

ABCC9 mutations have been identified in cardiac arrhythmia syndromes like Brugada Syndrome and Early Repolarization Syndrome :

• Mutation-specific considerations: For the V734I and S1402C mutations that cause gain-of-function effects, researchers should verify that antibody epitopes are unaffected by these mutations to ensure comparable detection of wild-type and mutant proteins .

• Tissue distribution analysis: Map ABCC9 expression across different cardiac regions (atria, ventricles, conduction system) using region-specific sampling and immunohistochemistry to correlate with arrhythmia vulnerability.

• Co-expression studies: Analyze ABCC9 expression in relation to other cardiac ion channels, particularly KCNJ11 and SCN5A, as interactions between mutations in these genes can produce severe arrhythmic phenotypes .

• Translational biomarkers: Investigate whether circulating ABCC9 levels or expression in accessible tissues correlate with cardiac pathology, potentially providing biomarkers for channelopathy risk.

How can researchers optimize ABCC9 antibody-based methods for investigating its role in neurodegenerative conditions?

ABCC9 polymorphisms have been associated with hippocampal sclerosis during aging :

• Brain region-specific protocols: Optimize immunohistochemistry protocols specifically for brain tissue, with attention to antigen retrieval methods that preserve neural architecture while exposing ABCC9 epitopes.

• Co-pathology analysis: Develop multiple immunofluorescence protocols combining ABCC9 antibodies with markers of neurodegeneration (phosphorylated TDP-43) to correlate expression with pathological features .

• Age-stratified studies: Implement systematic analysis of ABCC9 expression across different age groups, correlating with both genetic status at rs704178 and development of hippocampal pathology .

• Glia-neuron differentiation: Use cell-type specific markers in combination with ABCC9 antibodies to distinguish expression patterns between neurons and glial cells, which may reveal cell-type specific vulnerability associated with ABCC9 variants.