Phospho-ACACA (Ser79) Antibody

What is Phospho-ACACA (Ser79) Antibody and what specifically does it detect?

Phospho-ACACA (Ser79) Antibody is a research tool that specifically detects endogenous levels of Acetyl-CoA Carboxylase (ACACA) only when phosphorylated at the serine 79 residue. This antibody recognizes both ACCα and ACCβ isoforms when phosphorylated at this specific site . The antibody is particularly valuable for monitoring the activation status of AMPK signaling pathways, as ACACA phosphorylation at Ser79 is a direct downstream consequence of AMPK activation, resulting in the inhibition of ACACA enzymatic activity .

The antibody is typically produced in rabbits and available in polyclonal format, enabling detection of phosphorylated ACACA across multiple species including human, mouse, and rat samples . The molecular weight of the detected protein is approximately 280 kDa, making it identifiable on western blots through its characteristic high molecular weight band.

What applications are validated for Phospho-ACACA (Ser79) Antibody?

Phospho-ACACA (Ser79) Antibody has been validated for several research applications with specific recommended protocols:

For Western blotting applications, researchers should note that ACACA is a high molecular weight protein (280 kDa), necessitating the use of lower percentage (5%) SDS-PAGE gels for proper separation . When performing Western blotting, cellular extracts should be prepared carefully to preserve phosphorylation status, typically using phosphatase inhibitors in lysis buffers.

What are recommended controls for validating antibody specificity?

When working with Phospho-ACACA (Ser79) Antibody, implementing appropriate controls is essential for result validation:

• Positive control: Treat cells with AMPK activators (e.g., AICAR, metformin, or energy stress conditions) to increase phosphorylation at Ser79.

• Negative control: Use untreated cells or AMPK inhibitors (e.g., Compound C) to reduce phosphorylation .

• Phosphatase treatment control: Treat some sample aliquots with lambda phosphatase to remove phosphorylation and confirm antibody specificity.

• Loading control: Include detection of total ACACA protein to normalize for total protein expression levels.

These controls help distinguish between specific and non-specific signals and provide confidence in experimental outcomes. In studies examining the dynamic phosphorylation during cell division, comparison between interphase and mitotic cells provides an internal control, as mitotic cells exhibit significantly increased phospho-ACACA Ser79 levels compared to interphase cells .

How should sample preparation be optimized for detecting phospho-ACACA Ser79?

Optimal sample preparation is critical for preserving the phosphorylation status of ACACA at Ser79:

• Cell lysis should be performed using ice-cold buffers containing phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate).

• For Western blotting, separate proteins using 5% SDS-PAGE gels due to ACACA's high molecular weight (280 kDa) .

• When analyzing mitotic cells, consider synchronization methods or mitotic shake-off techniques to enrich for dividing cells.

• For immunofluorescence studies, rapid fixation with 4% paraformaldehyde followed by permeabilization with 0.1-0.5% Triton X-100 preserves phospho-epitopes while maintaining cellular architecture.

• For adherent cells grown in tissue culture plates, in situ fixation and permeabilization in 96-well clear-bottom imaging plates facilitates high-resolution imaging of mitotic cells .

When investigating phospho-ACACA Ser79 localization during mitosis, co-staining with markers such as α-tubulin for microtubules, Hoechst for DNA, and Aurora A for centrosomes provides spatial reference points for accurate interpretation of localization patterns .

What factors influence phospho-ACACA Ser79 detection and how can they be controlled?

Several factors can affect the detection of phospho-ACACA Ser79 in experimental settings:

• Energy status: Cellular energy depletion activates AMPK, increasing ACACA phosphorylation. Control media conditions and glucose availability.

• Cell cycle phase: Phospho-ACACA Ser79 levels increase significantly during mitosis . Consider cell synchronization methods when studying cell cycle effects.

• Phosphatase activity: Rapid dephosphorylation can occur during sample processing. Always use fresh phosphatase inhibitors in buffers.

• Antibody specificity: Cross-reactivity with other phospho-epitopes may occur. Validate specificity using the controls described in section 1.3.

• PLK1 activity: Polo-like kinase 1 influences AMPK-mediated phosphorylation during mitosis . Consider PLK1 inhibitors (e.g., GW843682X) to evaluate this relationship.

For quantitative assays, establishing a standard curve using controls with known phosphorylation status helps normalize results across experiments. Additionally, when studying mitotic cells, identifying cells in specific mitotic phases through morphological characteristics or mitotic markers ensures accurate interpretation of phosphorylation patterns.

How can immunofluorescence protocols be optimized for studying phospho-ACACA Ser79 localization?

For high-resolution imaging of phospho-ACACA Ser79 during mitosis and cell division:

• Use asynchronously growing cells to capture various mitotic stages in a single experiment.

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilize with 0.2% Triton X-100 for 10 minutes.

• Block with 1-3% BSA in PBS for 1 hour.

• Incubate with primary antibodies against phospho-ACACA Ser79 and co-staining markers (e.g., Aurora A for centrosomes, α-tubulin for microtubules).

• Use appropriate secondary antibodies conjugated to distinct fluorophores (e.g., Alexa Fluor 488, Alexa Fluor 594).

• Counterstain DNA with Hoechst 33258 or DAPI.

• Mount slides with anti-fade mounting medium to prevent photobleaching.

• Image using confocal microscopy for optimal spatial resolution .

For automated imaging applications, cells can be grown directly in 96-well clear-bottom imaging tissue culture plates and processed for immunofluorescence. This approach facilitates high-throughput analysis of mitotic cells using systems like the BD Pathway™ 855 Bioimager System .

What is the spatio-temporal dynamics of phospho-ACACA Ser79 during mitosis and cytokinesis?

Phospho-ACACA Ser79 displays a highly dynamic localization pattern throughout cell division:

• Prophase to Metaphase: Distinct punctuate staining is observed during chromosome condensation, with notable localization to centrosomes .

• Early Anaphase to Late Telophase: The phospho-ACACA Ser79 signal almost disappears during chromatid separation .

• Cytokinesis: The signal reappears at the constriction ring until the completion of the furrowing process and cytokinesis .

At the completion of telophase, phospho-ACACA Ser79 appears as a doublet-like structure on either side of the midbody within the intercellular cytokinetic bridge, similar to the pattern observed with phospho-AMPKα Thr172 .

How does the relationship between AMPK and ACACA function during mitosis?

The relationship between AMPK and ACACA during mitosis reveals a complex regulatory mechanism:

• Mitotic cells exhibit significantly increased levels of phospho-ACACA Ser79 compared to interphase cells .

• AMPK is known to phosphorylate ACACA at Ser79, causing inhibition of ACACA enzymatic activity in response to cellular energy status .

• The mitotic enhancement of phospho-ACACA Ser79 is attenuated by Compound C (an AMPK inhibitor), suggesting that AMPK phosphorylates ACACA when cells enter mitosis .

• Activated AMPKα (phospho-AMPKα Thr172) displays a dynamic localization during cell division, associating with centrosomes, spindle poles, the central spindle midzone, and the midbody throughout mitosis and cytokinesis .

• Both phospho-AMPKα Thr172 and phospho-ACACA Ser79 localize to mitotic spindle poles and increase when cells enter mitosis .

These findings suggest that AMPK-mediated phosphorylation of ACACA during mitosis may serve functions beyond metabolic regulation. The similar localization patterns of phospho-AMPK and its substrate phospho-ACACA indicate a coordinated role in proper spindle orientation and efficient progression through mitosis .

What is the potential functional significance of phospho-ACACA Ser79 at mitotic structures?

The localization of phospho-ACACA Ser79 to specific mitotic structures suggests functions beyond its canonical role in fatty acid metabolism:

• Cell Division Regulation: Studies in yeast have shown that disruption of the ACACA gene impairs nuclear division, resulting in large undivided nuclei, abnormally shortened mitotic spindles, aberrant mitosis, and cell cycle arrest at G2/M . This suggests an essential role for ACACA in cell division progression.

• Spindle Pole Function: The co-localization of phospho-ACACA Ser79 with Aurora A at centrosomes and spindle poles indicates a potential role in spindle organization or function .

• Cytokinesis Completion: The reappearance of phospho-ACACA Ser79 at the constriction ring during cytokinesis suggests involvement in the completion of cell division .

• Independent of Lipogenesis: The failure of long-chain fatty acids to overcome ACACA-defective cell cycle arrest suggests that ACACA's role in cell division may be independent of its biosynthetic function, or alternatively, indicates a strict coupling between lipogenesis and cell cycle progression .

• PLK1 Interaction: The potential regulation of phospho-ACACA Ser79 by Polo-like kinase 1 (PLK1) during mitosis suggests integration with core mitotic regulatory mechanisms .

These observations point to a specialized function of phosphorylated ACACA during mitosis that may involve structural roles in the mitotic apparatus or localized regulation of metabolic processes necessary for cell division.

How can inconsistent phospho-ACACA Ser79 detection be addressed?

Researchers facing challenges with phospho-ACACA Ser79 detection should consider the following troubleshooting approaches:

• Phosphorylation preservation: Ensure rapid sample processing with freshly prepared phosphatase inhibitors. Consider using commercial phosphatase inhibitor cocktails for consistency.

• Antibody validation: Verify antibody specificity using positive controls (AMPK activator treatment) and negative controls (AMPK inhibitor treatment) .

• Protocol optimization: For Western blotting, use 5% SDS-PAGE gels for proper separation of high molecular weight ACACA (280 kDa) . Optimize transfer conditions for large proteins.

• Signal enhancement: Consider using enhanced chemiluminescence (ECL) substrates with higher sensitivity for Western blotting. For immunofluorescence, use signal amplification systems if necessary.

• Cell cycle considerations: When studying mitotic cells, phospho-ACACA Ser79 levels vary dramatically throughout different mitotic phases . Ensure proper identification of mitotic stages.

• Antibody dilution optimization: Test different antibody dilutions (1:500 to 1:1000 for Western blotting) to determine optimal conditions for specific experimental setups .

For immunofluorescence studies specifically, optimizing fixation methods (paraformaldehyde vs. methanol), permeabilization conditions, and blocking reagents can significantly improve detection quality and specificity.

How can researchers distinguish between specific phospho-ACACA Ser79 staining and background?

Distinguishing specific phospho-ACACA Ser79 signals from background requires methodical validation:

• Parallel negative controls: Include samples treated with lambda phosphatase to remove phosphorylation or cells treated with AMPK inhibitors.

• Peptide competition: Pre-incubate the antibody with phospho-ACACA Ser79 peptide to block specific binding.

• Mitotic enrichment: Compare interphase versus mitotic cells, as mitotic cells show enhanced phospho-ACACA Ser79 signal .

• Pattern recognition: True phospho-ACACA Ser79 staining follows specific localization patterns during mitosis - centrosomal in prophase and metaphase, reduced in anaphase, and present at the cytokinetic furrow during telophase .

• Co-localization analysis: Validate subcellular localization through co-staining with established markers (e.g., Aurora A for centrosomes) .

• Genetic validation: When possible, use ACACA knockdown/knockout cells or Ser79-to-Ala mutants as additional specificity controls.

In image analysis, setting appropriate thresholds based on negative controls helps distinguish specific staining from background. For automated imaging platforms, consistent acquisition parameters and background subtraction algorithms improve quantitative analysis accuracy.

What is the relationship between PLK1 activity and ACACA phosphorylation during mitosis?

Recent research has uncovered intriguing connections between Polo-like kinase 1 (PLK1) and the phosphorylation of ACACA during mitosis:

• PLK1 activity has been linked to the mitotic phosphorylation of AMPKα, which in turn phosphorylates ACACA at Ser79 .

• PLK1 and phospho-AMPKα Thr172 exhibit significant spatio-temporal overlap at centrosomes from prophase to anaphase and at the midbody during telophase and cytokinesis .

• Short-term treatment with GW843682X (a thiophene benzimidazole ATP-competitive inhibitor of PLK1) largely prevents the localization of phospho-AMPKα Thr172 to mitotic and cytokinetic structures, independent of glucose availability .

• This suggests that PLK1 may indirectly influence ACACA phosphorylation by regulating AMPK activation during mitosis.

• The relationship may indicate a mitosis-specific activation pathway that operates independently of the canonical energy status-dependent regulation of AMPK and ACACA .

This emerging research area suggests a more complex regulatory network governing metabolism during cell division, where mitotic kinases like PLK1 may coordinate with metabolic regulators like AMPK to ensure proper cell division through the phosphorylation of downstream targets including ACACA.

What are the implications of ACACA phosphorylation for cancer research and therapeutic development?

The phosphorylation status of ACACA at Ser79 has significant implications for cancer research:

• Metabolic reprogramming: Cancer cells often exhibit altered fatty acid metabolism, with ACACA serving as a key regulatory node that could be influenced by its phosphorylation status.

• Cell division regulation: The involvement of phospho-ACACA Ser79 in mitosis suggests potential roles in cancer cell proliferation, with localization to critical mitotic structures .

• AMPK pathway targeting: Many cancer therapeutics aim to activate AMPK (e.g., metformin), which would increase ACACA phosphorylation and inhibit its lipogenic activity.

• Biomarker potential: Changes in phospho-ACACA Ser79 levels or localization patterns might serve as biomarkers for specific cancer types or stages.

• Therapeutic vulnerability: The dual role of ACACA in metabolism and cell division presents a potential vulnerability that could be exploited therapeutically.

Research examining phospho-ACACA Ser79 in various cancer models could reveal novel insights into how metabolism and cell division are coordinated in malignant cells. Furthermore, understanding the relationship between PLK1, AMPK, and ACACA phosphorylation might identify new combination therapy approaches that simultaneously target cell division and metabolic vulnerabilities in cancer cells .

What techniques are emerging for studying dynamic phosphorylation events during cell division?

Advanced technologies for investigating phospho-ACACA Ser79 dynamics include:

• Live-cell imaging: Development of phospho-specific fluorescent biosensors could enable real-time monitoring of ACACA phosphorylation during mitosis.

• Super-resolution microscopy: Techniques like STORM, PALM, or STED microscopy provide nanoscale resolution to precisely localize phospho-ACACA Ser79 relative to mitotic structures.

• Mass spectrometry-based phosphoproteomics: Quantitative analysis of phosphorylation changes across the cell cycle with single-cell resolution.

• CRISPR-based genetic engineering: Creation of endogenously tagged ACACA or phospho-mimetic/phospho-dead mutants to study functional consequences.

• Proximity labeling: BioID or APEX2-based approaches to identify proteins interacting with phospho-ACACA Ser79 during specific mitotic phases.

• Automated high-content imaging: Systems like the BD Pathway™ 855 Bioimager enable high-throughput analysis of phospho-ACACA Ser79 localization across many cells and conditions .

These emerging approaches will help address outstanding questions regarding the temporal dynamics, molecular interactions, and functional significance of ACACA phosphorylation during cell division.

How might the dual roles of ACACA in metabolism and cell division be integrated?

The emerging dual functionality of ACACA in both metabolism and cell division raises intriguing questions about cellular coordination of these processes:

• Metabolic checkpoints: Phospho-ACACA Ser79 might serve as a metabolic checkpoint during mitosis, ensuring adequate energy reserves before commitment to cell division.

• Localized lipid synthesis: Phosphorylated ACACA at specific mitotic structures might regulate localized lipid synthesis required for membrane dynamics during cytokinesis.

• Structural roles: Beyond its enzymatic function, phosphorylated ACACA might serve structural roles in organizing the mitotic apparatus, similar to other metabolic enzymes with moonlighting functions.

• Nutrient sensing: The AMPK-ACACA axis could integrate nutrient availability with cell cycle progression, potentially explaining why disruption of ACACA leads to G2/M arrest .

• Evolutionary conservation: The essential role of ACACA in yeast nuclear division suggests evolutionary conservation of this dual functionality .

Future research exploring these possibilities will likely reveal new paradigms for understanding how cells coordinate metabolic status with proliferative decisions, with implications for normal development, stem cell biology, and diseases characterized by dysregulated cell division.

Best practices for Phospho-ACACA (Ser79) Antibody applications

Based on the collective evidence, researchers working with Phospho-ACACA (Ser79) Antibody should:

• Use freshly prepared buffers with phosphatase inhibitors for all sample preparation steps.

• Include appropriate positive and negative controls to validate antibody specificity.

• For Western blotting, use 5% SDS-PAGE gels with optimized transfer conditions for high molecular weight proteins (280 kDa) .

• For immunofluorescence studies of mitotic cells, co-stain with markers like Aurora A (centrosomes), α-tubulin (microtubules), and DNA stains to accurately identify mitotic phases .

• Consider cell synchronization or mitotic shake-off techniques when studying cell cycle-dependent phosphorylation.

• Be aware that phospho-ACACA Ser79 levels and localization patterns change dramatically throughout mitosis .

• When quantifying phosphorylation levels, normalize to total ACACA protein expression.

• For high-resolution imaging, consider automated confocal systems that enable 3D reconstructions of mitotic cells .