ACACA (Ab-79) Antibody

What is ACACA (Ab-79) Antibody and what epitope does it recognize?

ACACA (Ab-79) Antibody specifically recognizes Acetyl-CoA Carboxylase, a crucial enzyme in fatty acid metabolism. There are two main types available: phospho-specific antibodies that detect ACACA only when phosphorylated at Serine 79, and total ACACA antibodies that recognize the protein regardless of phosphorylation status. The phospho-specific monoclonal antibody (such as Clone#RM270) is designed to detect human Acetyl-CoA Carboxylase specifically when phosphorylated at Ser79 and shows no cross-reactivity with the unphosphorylated form . The polyclonal variant targets a peptide sequence around amino acids 78-82 (S-M-S-G-L) derived from human Acetyl-CoA Carboxylase . This region is significant because Ser79 phosphorylation by AMPK inhibits ACC1 activity, making it an important regulatory site for studying metabolic regulation.

What applications are ACACA (Ab-79) antibodies validated for?

ACACA (Ab-79) antibodies have been validated for multiple research applications, with different variants showing specific performance characteristics:

For Western blot applications, these antibodies detect a protein of approximately 280kD (SDS-PAGE MW) . When designing experiments, researchers should consider that validation has been conducted primarily in human samples. The application scope determines experimental design parameters including sample preparation methods, blocking reagents, and detection systems appropriate for the specific technique.

How can I optimize storage conditions to maintain ACACA (Ab-79) antibody activity?

For optimal activity retention, store ACACA (Ab-79) antibodies at -20°C . The monoclonal antibody is typically supplied in 50% Glycerol/PBS with 1% BSA and 0.09% sodium azide to maintain stability during storage . The polyclonal variant is generally formulated in phosphate buffered saline without Mg²⁺ and Ca²⁺ (pH 7.4, 150mM NaCl) containing 0.02% sodium azide and 50% glycerol . To preserve antibody functionality:

• Aliquot upon first thaw to minimize freeze-thaw cycles

• Avoid repeated freeze-thaw cycles as they can significantly reduce antibody activity

• For working solutions, store at 4°C for up to one week

• When retrieving from storage, thaw on ice rather than at room temperature

• Centrifuge briefly after thawing to collect all material at the bottom of the tube

Following these guidelines will ensure consistent antibody performance across experiments and maximize the one-year shelf life typically specified by manufacturers .

What is the difference between monoclonal and polyclonal ACACA antibodies?

Monoclonal and polyclonal ACACA antibodies differ in several key aspects that impact experimental design and interpretation:

When designing experiments studying ACACA phosphorylation dynamics, monoclonal antibodies provide greater specificity for monitoring the phosphorylation state at Ser79. For total protein expression studies or applications requiring detection of multiple epitopes, polyclonal antibodies may offer advantages. The choice between these antibody types should align with your specific research question and experimental needs.

How can I validate the specificity of ACACA (Ab-79) antibody in my experimental system?

Rigorous validation of ACACA (Ab-79) antibody specificity in your specific experimental system is crucial for reliable results. Implement the following comprehensive validation strategy:

• Phosphatase treatment control: Split your samples and treat half with lambda phosphatase before immunoblotting with phospho-specific ACACA (Ab-79) antibody. The signal should diminish or disappear in treated samples while remaining in untreated controls.

• AMPK activation/inhibition: Treat cells with AMPK activators (e.g., AICAR, metformin) or inhibitors (e.g., Compound C) to modulate ACACA phosphorylation status. The phospho-ACACA (Ser79) signal should increase with AMPK activation and decrease with inhibition.

• Knockdown/knockout validation: Use siRNA-mediated knockdown or CRISPR/Cas9 knockout of ACACA to confirm antibody specificity. The signal should be reduced proportionally to knockdown efficiency or eliminated in knockout samples.

• Peptide competition assay: Pre-incubate the antibody with the immunizing phosphopeptide before applying to your samples. A specific antibody will show diminished or absent signal when the detecting epitope is blocked by the competing peptide.

• Cross-species reactivity assessment: Although the antibody is specified for human ACACA , if working with other species, validate cross-reactivity by comparing signals between human and target species samples under identical conditions, particularly if the epitope sequence is conserved.

• Phosphorylation-specific controls: For phospho-specific antibodies, include samples from conditions known to alter ACACA phosphorylation status (e.g., starvation, insulin stimulation) as physiological controls.

• Dual antibody approach: Compare results using both phospho-specific and total ACACA antibodies on the same samples to establish the ratio of phosphorylated to total protein.

Document all validation results thoroughly to establish confidence in your antibody's performance in your specific experimental system.

What are the critical considerations for troubleshooting inconsistent results with ACACA (Ab-79) antibody?

When encountering inconsistent results with ACACA (Ab-79) antibody, systematically evaluate these key factors:

• Sample preparation protocol:

  • Ensure rapid sample collection and immediate denaturation to preserve phosphorylation status

  • Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) in lysis buffers

  • Standardize protein extraction methods across experiments

  • Verify protein concentration measurement consistency

• Antibody dilution optimization:

  • Perform a dilution series (1:500 to 1:5000) to identify optimal signal-to-noise ratio

  • For Western blots, optimize both primary and secondary antibody concentrations

  • Document the optimal dilution for each new lot of antibody

• Blocking and washing stringency:

  • Compare different blocking agents (BSA vs. non-fat dry milk)

  • Note that milk contains phosphatases that may interfere with phospho-specific detection

  • Increase washing stringency (more washes, higher detergent concentration) to reduce background

• Detection system sensitivity:

  • For low abundance targets, consider using enhanced chemiluminescence substrates or fluorescent detection

  • Compare results using different detection methods to identify optimal sensitivity threshold

• Antibody cross-reactivity assessment:

  • Evaluate potential cross-reactivity with ACC2 (the other ACC isoform)

  • Perform immunoprecipitation followed by mass spectrometry to verify antibody targets

• Protein loading controls:

  • Include phosphorylation-insensitive controls (total protein stains)

  • Verify equal protein loading across all lanes

• Antibody storage and handling:

  • Check for antibody precipitation or contamination

  • Ensure proper storage at -20°C and minimize freeze-thaw cycles

• Positive and negative controls:

  • Include lysates from cells treated with AMPK activators (positive control)

  • Include samples treated with phosphatases (negative control)

Creating a troubleshooting decision tree based on these factors can systematically guide resolution of inconsistent results.

How should I interpret phosphorylation status data when using phospho-specific ACACA antibodies?

Interpreting phosphorylation status data requires nuanced analysis beyond simple signal detection. Consider these methodological approaches:

• Normalization strategy selection:

  • Always normalize phospho-ACACA (Ser79) signal to total ACACA to account for variations in total protein expression

  • Avoid normalizing to housekeeping proteins alone, as they don't account for changes in total ACACA levels

  • Consider dual normalization: first to total ACACA, then to loading controls

• Signal quantification methods:

  • Use densitometry software with defined background subtraction protocols

  • Apply multiple measurement methods (peak area vs. intensity) to confirm quantification

  • Report phospho-to-total ACACA ratios rather than absolute phospho-signal values

• Biological context interpretation:

  • Remember that Ser79 phosphorylation by AMPK inhibits ACC1 activity

  • Increased phospho-ACACA typically correlates with decreased fatty acid synthesis

  • Consider parallel measurement of downstream metabolites (malonyl-CoA, fatty acids) to confirm biological activity

• Temporal dynamics consideration:

  • ACACA phosphorylation can change rapidly (minutes) in response to metabolic signals

  • Document precise timing of sample collection relative to treatments

  • Consider time-course experiments to capture phosphorylation dynamics

• Physiological state influence:

  • Interpret results in context of feeding/fasting status, energy state, or stress conditions

  • Account for cell confluence and growth phase when using cultured cells

  • Consider that basal phosphorylation levels vary by tissue and cell type

• Signal detection threshold determination:

  • Establish the linear range of detection for your system

  • Identify the lower limit of detection through dilution series

  • Be cautious interpreting small changes (<20%) in phosphorylation status

• Multi-site phosphorylation integration:

  • Consider that ACACA is regulated by phosphorylation at multiple sites (not just Ser79)

  • Interpret Ser79 phosphorylation in context of other modifications when possible

A comprehensive interpretation requires integration of phosphorylation data with appropriate controls and contextual biological information.

What methodological considerations are important when using ACACA antibodies to study the AMPK-ACC pathway?

When investigating the AMPK-ACC regulatory pathway, several methodological considerations are critical for generating reliable data:

• Sample preparation timing:

  • AMPK-mediated phosphorylation of ACACA at Ser79 occurs rapidly (within minutes)

  • Use rapid sample collection methods (e.g., direct lysis in SDS buffer) to preserve phosphorylation state

  • Document precise timing between treatments and sample collection

• Physiological activator selection:

  • Choose AMPK activators based on research question: AMP-mimetics (AICAR), biguanides (metformin), or energy stress inducers (glucose deprivation)

  • Include both acute and chronic activation timepoints to distinguish immediate signaling from adaptive responses

  • Consider dose-response curves to identify optimal activation conditions

• Pathway validation approach:

  • Monitor multiple nodes in the pathway simultaneously (AMPK-Thr172 phosphorylation, ACC-Ser79 phosphorylation)

  • Include measurement of upstream activators (LKB1, CaMKK) and downstream effects (malonyl-CoA levels)

  • Apply AMPK inhibitors (Compound C) or activators (A-769662) as pathway controls

• Isoform-specific analysis:

  • Consider that ACACA/ACC1 (cytosolic) and ACACB/ACC2 (mitochondrial) have different cellular localizations and functions

  • Design experiments to distinguish between isoforms through subcellular fractionation

  • Verify antibody specificity for ACC1 vs. ACC2 in your experimental system

• Cell type and tissue considerations:

  • AMPK-ACC pathway activity varies significantly between tissues (liver, muscle, adipose)

  • Document cell type-specific baseline phosphorylation levels

  • Consider tissue-specific pathway interactions (e.g., liver: de novo lipogenesis; muscle: fatty acid oxidation)

• Metabolic state standardization:

  • Standardize feeding/fasting conditions before sample collection

  • Document glucose levels, insulin status, and other metabolic parameters

  • Consider time of day effects on metabolic signaling (circadian regulation)

• Complementary techniques integration:

  • Combine antibody-based detection with functional assays (ACC activity, fatty acid synthesis rates)

  • Consider metabolomics approaches to measure pathway outputs (malonyl-CoA, fatty acids)

  • Use genetic models (AMPK knockout, ACC knock-in with S79A mutation) to validate pathway regulation

These considerations ensure that antibody-based detection of ACACA phosphorylation status accurately reflects the biological regulation of the AMPK-ACC pathway.

How can I optimize sample preparation to preserve ACACA phosphorylation status for antibody detection?

Preserving ACACA phosphorylation status during sample preparation requires specific protocols to inhibit phosphatase activity and maintain phosphoepitope integrity:

• Immediate sample processing protocol:

  • Process tissues or cells immediately after collection

  • For tissues, flash-freeze in liquid nitrogen immediately after dissection

  • For cultured cells, avoid PBS washing steps that activate phosphatases; directly add lysis buffer to the plate

• Optimized lysis buffer formulation:

  • Include multiple phosphatase inhibitors: sodium fluoride (50mM), sodium pyrophosphate (10mM), β-glycerophosphate (25mM), sodium orthovanadate (2mM)

  • Add protease inhibitor cocktail to prevent degradation

  • Use RIPA or modified RIPA buffer for better solubilization of membrane-associated proteins

  • Maintain cold temperature (4°C) throughout extraction

• Physical disruption method selection:

  • For tissues: Pulverize frozen tissue under liquid nitrogen before adding to lysis buffer

  • For cells: Scrape cells directly into lysis buffer rather than trypsinizing

  • Avoid excessive sonication which can generate heat and activate phosphatases

• Temperature control throughout processing:

  • Maintain samples on ice at all times during processing

  • Pre-chill all equipment (homogenizers, centrifuges)

  • Add ice-cold acetone for protein precipitation if concentrated samples are needed

• Phosphatase inhibitor strategy:

  • Use fresh phosphatase inhibitors in all buffers

  • Consider adding okadaic acid (PP2A inhibitor) and calyculin A (PP1 inhibitor) for enhanced phosphatase inhibition

  • Include EDTA (1-2mM) to inhibit metal-dependent phosphatases

• Sample storage considerations:

  • Add 2X Laemmli buffer and heat immediately after quantification for Western blot samples

  • For immunoprecipitation, maintain phosphatase inhibitors in all wash buffers

  • For long-term storage, add glycerol (final 10-20%) and store at -80°C

• Validation of phosphorylation preservation:

  • Process aliquots of the same sample with and without phosphatase inhibitors as controls

  • Include positive controls (AMPK activator-treated samples) in each experiment

  • Monitor other phosphoproteins (e.g., phospho-AMPK) as internal controls for phosphatase inhibition efficacy

These optimized sample preparation protocols ensure that the phosphorylation status of ACACA at Ser79 accurately reflects the biological state at the time of sample collection.

What cross-reactivity considerations are important when using ACACA (Ab-79) antibody in non-human samples?

When applying ACACA (Ab-79) antibodies to non-human samples, systematic assessment of cross-reactivity is essential for valid data interpretation:

• Sequence homology analysis:

  • Analyze sequence conservation around the Ser79 phosphorylation site across species

  • The human ACACA sequence targeted by the antibody contains the motif S-M-S-G-L around amino acids 78-82

  • Higher sequence conservation suggests greater probability of cross-reactivity

• Epitope conservation evaluation:

  • Focus specifically on the phosphorylation site microenvironment

  • Even single amino acid differences can significantly affect antibody binding

  • Assess both primary sequence and potential structural conservation

• Species validation strategy:

  • When considering bovine tissue applications, as noted in customer inquiries , systematic validation is required

  • Use paired samples from human (positive control) and target species under identical conditions

  • Include phosphatase-treated controls from both species to confirm phospho-specificity

• Graduated cross-reactivity testing:

  • Start with closely related species (primates, then other mammals)

  • Use increasing antibody concentrations to detect lower-affinity binding

  • Document species-specific optimal antibody dilutions

• Multiple antibody comparison approach:

  • Test multiple antibodies targeting different epitopes of ACACA

  • Compare results between phospho-specific and total protein antibodies

  • Consider using species-specific secondary antibodies to reduce background

• Positive and negative control selection:

  • Obtain species-specific positive controls (AMPK activator-treated samples)

  • Include samples from ACACA knockout models or siRNA-treated cells when available

  • Consider using recombinant proteins from target species as standards

• Cross-validation with orthogonal methods:

  • Confirm antibody results with mass spectrometry-based phosphoproteomics

  • Use metabolic labeling to track ACC activity independent of antibody detection

  • Apply gene expression analysis to correlate protein detection with transcript levels

This methodical cross-reactivity assessment creates confidence in applying ACACA (Ab-79) antibodies beyond their validated human reactivity, potentially expanding their research utility while maintaining data reliability.

How can I design experiments to study the relationship between ACACA phosphorylation and metabolic regulation?

Designing experiments to investigate the relationship between ACACA phosphorylation and metabolic regulation requires a multi-faceted approach:

• Stimulus-response experimental design:

  • Apply metabolic stimuli known to affect ACACA: insulin (decreases Ser79 phosphorylation), AMPK activators like AICAR or metformin (increase phosphorylation)

  • Design time-course experiments (5min, 15min, 30min, 1h, 3h, 24h) to capture both acute signaling and adaptive responses

  • Include dose-response studies to establish threshold effects

• Cell/tissue model selection strategy:

  • Choose models based on metabolic relevance: hepatocytes (lipogenesis), adipocytes (fat storage), myocytes (fatty acid oxidation)

  • Consider primary cells vs. cell lines (primary cells offer more physiological responses)

  • For in vivo studies, target metabolically active tissues (liver, muscle, adipose) with appropriate controls

• Pathway integration analysis:

  • Simultaneously monitor related pathways: insulin/AKT/mTOR (anabolic) and AMPK (catabolic)

  • Track multiple phosphorylation sites on ACACA (not just Ser79)

  • Measure upstream regulators (AMPK-Thr172 phosphorylation) and downstream metabolites (malonyl-CoA)

• Genetic modification approach:

  • Use phospho-mutant models: ACACA-S79A (non-phosphorylatable) or S79D (phosphomimetic)

  • Apply CRISPR/Cas9 to generate endogenous mutations at the Ser79 site

  • Consider inducible systems to distinguish developmental from acute effects

• Functional output measurement:

  • Directly measure ACACA enzymatic activity using radiometric or coupled assays

  • Track de novo lipogenesis using labeled substrates (¹⁴C-acetate incorporation)

  • Monitor fatty acid oxidation rates in parallel to assess metabolic switching

• Nutrient manipulation design:

  • Apply glucose deprivation/refeeding protocols to modulate AMPK activity

  • Test high-fat vs. high-carbohydrate conditions to observe differential regulation

  • Include fasting/feeding cycles in in vivo studies to capture physiological regulation

• Multi-omics integration:

  • Combine phosphoproteomics, metabolomics, and transcriptomics

  • Correlate ACACA phosphorylation with global metabolic profiles

  • Use metabolic flux analysis to determine the functional impact of phosphorylation changes

This comprehensive experimental design strategy enables researchers to establish causal relationships between ACACA phosphorylation status and metabolic outcomes, providing mechanistic insights into this critical regulatory node.

What are the optimal conditions for using ACACA (Ab-79) antibody in Western blotting?

Optimizing Western blot conditions for ACACA (Ab-79) antibody detection requires attention to several technical parameters:

• Sample preparation specifications:

  • Use RIPA buffer with phosphatase inhibitors for protein extraction

  • Load 20-50μg total protein per lane (ACACA is moderately abundant in metabolic tissues)

  • Include reducing agents (β-mercaptoethanol or DTT) in sample buffer

  • Heat samples at 70°C for 10 minutes rather than boiling to preserve phosphoepitopes

• Gel selection and separation parameters:

  • Use low percentage gels (6-8% acrylamide) to resolve the high molecular weight ACACA (280kD)

  • Consider gradient gels (4-15%) for better resolution

  • Extend running time to ensure adequate separation of high molecular weight proteins

  • Include visible molecular weight markers spanning 75-300kD range

• Transfer optimization protocol:

  • For large proteins like ACACA (280kD), use wet transfer rather than semi-dry

  • Add 0.1% SDS to transfer buffer to improve high MW protein transfer

  • Extend transfer time (overnight at low voltage or 2-3 hours at higher voltage)

  • Use PVDF membrane (0.45μm pore size) rather than nitrocellulose for better protein retention

• Blocking strategy selection:

  • For phospho-specific detection, use 5% BSA in TBST rather than milk (milk contains phosphatases)

  • For total ACACA detection, 5% non-fat dry milk in TBST is suitable

  • Block for 1-2 hours at room temperature or overnight at 4°C

• Antibody dilution optimization:

  • Start with manufacturer's recommended dilution (typically 1:1000)

  • Perform a dilution series to determine optimal signal-to-noise ratio

  • Dilute antibodies in the same buffer used for blocking

  • Incubate primary antibody overnight at 4°C with gentle agitation

• Washing protocol enhancement:

  • Perform 4-5 washes with TBST, 5-10 minutes each

  • Increase washing stringency (higher Tween-20 concentration, up to 0.1%) if background is high

  • Maintain consistent washing protocols between experiments

• Detection system selection:

  • For phospho-specific detection, enhanced chemiluminescence (ECL) offers good sensitivity

  • Consider fluorescent secondary antibodies for multiplex detection and better quantification

  • Optimize exposure times to ensure signal is within linear range

• Controls inclusion:

  • Run phosphatase-treated samples as negative controls

  • Include AMPK activator-treated samples as positive controls

  • Consider using recombinant ACACA protein as reference standard

Following these optimized conditions will maximize detection specificity and sensitivity when using ACACA (Ab-79) antibodies in Western blotting applications.

Can ACACA (Ab-79) antibody be used effectively in immunoprecipitation studies?

While immunoprecipitation (IP) is not listed among the validated applications for the ACACA (Ab-79) antibodies in the provided data , researchers can develop and optimize IP protocols with these considerations:

• Antibody selection criteria:

  • Polyclonal antibodies targeting total ACACA may be more effective for IP than phospho-specific antibodies

  • If using phospho-specific antibodies, maintain phosphatase inhibitors throughout all steps

  • Consider using antibodies specifically validated for IP applications when available

• Lysis buffer formulation:

  • Use mild non-denaturing buffers to preserve protein conformation and complexes

  • Standard IP buffer: 50mM Tris-HCl pH 7.5, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate with protease inhibitors

  • For phospho-IP, add phosphatase inhibitors: 50mM NaF, 10mM Na₄P₂O₇, 2mM Na₃VO₄

• Pre-clearing strategy:

  • Pre-clear lysates with appropriate control IgG and protein A/G beads

  • Perform for 1 hour at 4°C to reduce non-specific binding

  • Remove any precipitated material by centrifugation before adding the specific antibody

• Antibody binding optimization:

  • Use 2-5μg antibody per 500μg-1mg total protein

  • Incubate overnight at 4°C with gentle rotation

  • Consider a crosslinking step to covalently attach antibody to beads (prevents antibody co-elution)

• Bead selection and handling:

  • For rabbit antibodies, use protein A or protein A/G beads

  • Add pre-washed beads after antibody incubation

  • Use magnetic beads for gentler washing and better recovery

• Washing stringency titration:

  • Perform sequential washes with increasing stringency

  • Start with lysis buffer, then add higher salt concentration (300mM NaCl)

  • Final washes with buffer without detergents

  • Keep all steps at 4°C to maintain complex integrity

• Elution method selection:

  • For Western blot analysis: elute directly in 2X Laemmli buffer at 70°C

  • For activity assays: use peptide competition or mild elution conditions

  • For mass spectrometry: consider on-bead digestion to reduce contamination

• Validation approaches:

  • Perform reciprocal IP with antibodies against known ACACA-interacting proteins

  • Include IgG control IP processed identically to experimental samples

  • Verify specificity by IP from ACACA-depleted or knockout samples when available

By adapting these methodological considerations, researchers can develop effective IP protocols using ACACA (Ab-79) antibodies for studying protein interactions and complexes involving ACACA.

How can I use ACACA (Ab-79) antibody to analyze the effects of metabolic disease models?

ACACA (Ab-79) antibody can be powerful for investigating metabolic disease models when applied with these methodological considerations:

• Disease model selection strategy:

  • Choose models relevant to ACACA function: insulin resistance, obesity, non-alcoholic fatty liver disease, cancer

  • Include appropriate controls matched for age, sex, and genetic background

  • Consider both genetic models (db/db, ob/ob mice) and diet-induced models (high-fat diet)

• Tissue sampling prioritization:

  • Prioritize metabolically active tissues: liver (major site of de novo lipogenesis), adipose tissue, skeletal muscle

  • Consider tissue-specific differences in ACACA expression and regulation

  • Sample multiple adipose depots separately (subcutaneous, visceral, brown) as they have distinct metabolic profiles

• Analytical approach development:

  • Measure both phospho-ACACA (Ser79) and total ACACA levels

  • Calculate phospho-to-total ratio to normalize for expression changes

  • Correlate with upstream regulators (phospho-AMPK) and downstream effects (fatty acid synthesis rates)

• Context-specific interpretation framework:

  • In insulin resistance: expect decreased ACACA phosphorylation at baseline, with impaired response to AMPK activators

  • In obesity: analyze both basal phosphorylation and dynamic responses to feeding/fasting

  • In cancer models: assess metabolic reprogramming through altered ACACA regulation

• Intervention response assessment:

  • Use ACACA phosphorylation as a biomarker for drug efficacy (e.g., metformin, AMPK activators)

  • Track acute vs. chronic changes in phosphorylation status

  • Correlate phosphorylation changes with physiological outcomes (glucose tolerance, insulin sensitivity)

• Multi-parameter data integration:

  • Combine ACACA phosphorylation data with metabolic parameters (glucose, insulin, lipids)

  • Correlate with histological assessment of tissue lipid accumulation

  • Integrate with gene expression data for key lipogenic enzymes

• Translational relevance establishment:

  • Compare findings between animal models and human samples when available

  • Consider species differences in metabolic regulation

  • Develop standardized protocols that can be applied across model systems

This methodological framework enables researchers to effectively use ACACA (Ab-79) antibody to gain mechanistic insights into metabolic disease pathophysiology and potential therapeutic interventions.

What are the best approaches for quantifying ACACA phosphorylation in complex tissue samples?

Quantifying ACACA phosphorylation in complex tissue samples requires specialized approaches to ensure accurate and reproducible results:

• Sample preparation method selection:

  • Rapidly collect and process tissues to prevent post-collection changes in phosphorylation

  • Consider tissue-specific extraction protocols (e.g., liver requires different detergent composition than adipose)

  • Perform subcellular fractionation to enrich for cytosolic fraction where ACACA is predominantly located

  • Include phosphatase inhibitors at all stages of sample preparation

• Normalization strategy optimization:

  • Implement a two-tier normalization approach:

    • First normalize phospho-ACACA to total ACACA

    • Then normalize to an appropriate loading control or total protein stain

  • Avoid exclusive reliance on single housekeeping proteins, which may vary in expression

  • Consider using total protein stains (SYPRO Ruby, Ponceau S) for robust normalization

• Quantification method selection:

  • For Western blot analysis:

    • Use digital image capture systems with wide dynamic range

    • Ensure exposures fall within the linear range of detection

    • Apply consistent background subtraction methods

  • For ELISA-based detection:

    • Develop sandwich ELISA with capture antibody against total ACACA and detection antibody against phospho-Ser79

    • Include standard curves with recombinant phosphorylated and non-phosphorylated proteins

• Statistical approach development:

  • Account for intra-tissue heterogeneity by analyzing multiple samples per tissue

  • Apply appropriate statistical tests based on data distribution

  • Consider power analysis to determine required sample size for detecting meaningful differences

  • Report both absolute and relative changes in phosphorylation

• Alternative technology consideration:

  • Mass spectrometry-based phosphoproteomics for absolute quantification

  • Capillary Western systems (Wes, Jess) for improved reproducibility and lower sample requirements

  • Phospho-flow cytometry for cell-specific analysis in mixed cell populations

  • Proximity ligation assay for in situ detection of phosphorylated proteins

• Validation controls inclusion:

  • Process paired samples with and without phosphatase inhibitors

  • Include calibrator samples on each gel/blot for inter-assay normalization

  • Run pooled reference samples across multiple experiments to assess reproducibility

• Reporting standards implementation:

  • Document all sample processing steps with precise timing

  • Report antibody dilutions, exposure times, and image processing parameters

  • Present both representative images and quantitative data with appropriate statistical analysis

These methodological approaches enhance the accuracy and reliability of ACACA phosphorylation quantification in complex tissue samples, yielding more robust insights into metabolic regulation.

How can ACACA (Ab-79) antibody be used to study the intersection of metabolism and cancer?

ACACA (Ab-79) antibody offers valuable tools for investigating the complex relationship between metabolism and cancer, with these methodological considerations:

• Cancer model selection strategy:

  • Focus on cancers with known metabolic reprogramming (breast, prostate, colorectal, hepatocellular carcinoma)

  • Include both in vitro cancer cell lines and in vivo tumor models

  • Compare metabolic tissues (liver, adipose) with cancers arising from these tissues

• Experimental design considerations:

  • Analyze ACACA phosphorylation across cancer progression stages

  • Compare cancer cells with normal cells of the same tissue origin

  • Assess changes in ACACA regulation under hypoxia, nutrient restriction, and other tumor microenvironment conditions

• Mechanistic pathway analysis approach:

  • Investigate AMPK-ACC axis regulation in cancer contexts

  • Explore how oncogenic signaling (PI3K/AKT/mTOR) affects ACACA phosphorylation

  • Assess how altered ACACA regulation contributes to lipid synthesis required for cancer cell proliferation

• Therapeutic intervention assessment:

  • Use ACACA phosphorylation as a pharmacodynamic marker for drugs targeting cancer metabolism

  • Test combination therapies targeting both ACACA and related metabolic enzymes

  • Monitor changes in ACACA phosphorylation in response to standard chemotherapies

• Metabolic flux integration:

  • Correlate ACACA phosphorylation with de novo lipogenesis rates using isotope tracing

  • Assess how ACACA phosphorylation affects fatty acid oxidation in cancer cells

  • Monitor changes in lipid composition associated with altered ACACA activity

• Single-cell level analysis:

  • Apply immunofluorescence with phospho-ACACA antibodies to assess heterogeneity within tumors

  • Combine with markers of proliferation, hypoxia, or stem-like properties

  • Develop protocols for phospho-flow cytometry to quantify ACACA phosphorylation at single-cell resolution

• Translational research applications:

  • Evaluate ACACA phosphorylation in patient-derived xenografts or organoids

  • Assess potential as a biomarker for stratifying patients for metabolism-targeting therapies

  • Correlate ACACA phosphorylation patterns with patient outcomes and treatment responses

This research framework enables investigators to leverage ACACA (Ab-79) antibody for gaining insights into the role of altered metabolic regulation in cancer development, progression, and treatment response.

What are the considerations for using ACACA (Ab-79) antibody in multi-parameter immunofluorescence studies?

Implementing ACACA (Ab-79) antibody in multi-parameter immunofluorescence requires specific technical considerations:

• Antibody compatibility assessment:

  • Test for cross-reactivity between antibodies from different host species

  • Ensure secondary antibodies do not cross-react with other primaries in the panel

  • Consider using directly conjugated primary antibodies to reduce species limitations

• Signal amplification strategy selection:

  • For phospho-specific detection, implement tyramide signal amplification (TSA)

  • Consider quantum dots for narrow emission spectra and minimal overlap

  • Use zenon labeling technology for same-species antibody combinations

• Protocol optimization approach:

  • Determine optimal fixation method (paraformaldehyde concentration, duration)

  • Optimize antigen retrieval conditions (heat-induced vs. enzymatic)

  • Test different permeabilization methods for accessing intracellular epitopes

  • Establish appropriate blocking conditions to minimize background

• Sequential staining design:

  • Start with the weakest signal (often phospho-epitopes)

  • Implement sequential rather than simultaneous staining for challenging combinations

  • Consider antibody stripping and reprobing methods with appropriate controls

• Controls implementation:

  • Include single-stained controls for each antibody to assess bleed-through

  • Use phosphatase-treated sections as negative controls for phospho-antibodies

  • Implement fluorescence-minus-one (FMO) controls to set thresholds

• Image acquisition optimization:

  • Use spectral imaging to separate overlapping fluorophores

  • Apply consistent exposure settings across experimental groups

  • Establish threshold settings based on negative controls

  • Implement z-stack imaging for thick specimens

• Quantification strategy development:

  • Develop automated image analysis workflows for unbiased quantification

  • Use nuclear counterstains for cell identification and normalization

  • Implement cell segmentation algorithms for single-cell analysis

  • Quantify both signal intensity and subcellular localization

• Subcellular localization analysis:

  • Co-stain with markers for specific subcellular compartments

  • Assess ACACA distribution between cytosolic and membrane-associated pools

  • Examine co-localization with other metabolic enzymes or signaling molecules

These methodological considerations enable researchers to effectively incorporate ACACA (Ab-79) antibody into multi-parameter immunofluorescence studies, providing spatial context to ACACA regulation and function within cellular architecture.