HMGCS2 Antibody, FITC conjugated

What is HMGCS2 and what is its biological significance?

HMGCS2 (Hydroxymethylglutaryl-CoA synthase, mitochondrial) is a crucial mitochondrial enzyme (EC 2.3.3.10) that catalyzes the first reaction of ketogenesis, a metabolic pathway that provides lipid-derived energy during carbohydrate deprivation, such as during fasting . This protein belongs to the HMG-CoA synthase family and has significant importance in energy metabolism, particularly in facilitating the use of fatty acids for energy production when glucose is limited . HMGCS2 functions primarily in the mitochondria and has a calculated molecular weight between 50-56 kDa, with observed weight typically around 50 kDa in Western blot analyses . The importance of HMGCS2 extends beyond basic metabolism, as it has been implicated in various research areas including cancer, cardiovascular disease, and signal transduction . Recent studies have also demonstrated that mutations in the HMGCS2 gene are associated with HMG-CoA synthase deficiency, underscoring its clinical relevance in metabolic disorders .

What are the key specifications of HMGCS2 antibody FITC conjugated?

The HMGCS2 Antibody, FITC conjugated, is a polyclonal antibody raised in rabbit hosts against recombinant human HMGCS2 protein . Specifically, the immunogen used for production is a recombinant Human Hydroxymethylglutaryl-CoA synthase mitochondrial protein (amino acids 426-508) . The antibody has an IgG isotype and is conjugated with FITC (Fluorescein Isothiocyanate) for fluorescence-based applications . The antibody preparation is supplied in liquid form with a buffer containing 50% glycerol, 0.01M PBS at pH 7.4, and 0.03% Proclin 300 as a preservative . This antibody is highly purified (>95%) using protein G purification methods . For storage considerations, the antibody should be stored at -20°C or -80°C, and repeated freeze-thaw cycles should be avoided to maintain its reactivity and performance . These specifications are essential for researchers to consider when planning experiments utilizing this antibody.

What model systems can be studied with HMGCS2 antibody?

The HMGCS2 antibody demonstrates reactivity across multiple species, making it versatile for comparative studies across different research models. According to specification data, the antibody effectively recognizes HMGCS2 in human, mouse, and rat samples . This cross-reactivity has been verified in specific tissue types including:

• Western Blot applications: Verified in mouse heart, mouse liver, and rat kidney tissues

• Immunohistochemistry (IHC): Verified in human thyroid cancer and human colon carcinoma samples

• Immunofluorescence (IF): Verified in rat liver, human liver, and mouse liver tissues

Beyond these standard model systems, the antibody has also been successfully employed in intestinal epithelial cancer cell lines and normal intestinal organoids as demonstrated in published research . Researchers have utilized the antibody to study HMGCS2 expression in transgenic mice lacking intestinal expression of Hmgcs2 (Hmgcs2ΔIEC), highlighting its application in specialized knockout models . The ability to detect HMGCS2 across these diverse biological systems makes this antibody particularly valuable for comparative studies of HMGCS2 regulation and function across species and in different disease states.

How should I optimize HMGCS2 antibody for Western blotting experiments?

For optimal Western blotting results with HMGCS2 antibody, follow this methodological approach:

Sample Preparation:

• Extract proteins from target tissues (verified samples include mouse heart/liver and rat kidney)

• Use a buffer containing protease inhibitors to prevent protein degradation

• For mitochondrial proteins like HMGCS2, consider using a mitochondrial isolation protocol before general protein extraction

Protocol Optimization:

• Use a dilution range of 1:500-1:2000 for the primary antibody, as recommended in the specifications

• Run samples on 10-12% SDS-PAGE gels, optimal for the 50 kDa size range of HMGCS2

• Transfer proteins to PVDF or nitrocellulose membranes

• Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

Controls and Validation:

• Include positive control tissues (mouse liver is strongly recommended based on verification data)

• Consider including samples from HMGCS2-knockout models as negative controls when available

• Validate expected band size (note: the observed MW is typically 50 kDa, but multiple bands may be observed due to post-translational modifications)

Troubleshooting Considerations:

• If signal is weak, decrease antibody dilution (use more concentrated antibody)

• If background is high, increase dilution and optimize blocking conditions

• If multiple bands appear, consider the presence of different modified forms of the protein as indicated in the specifications

For densitometric analysis, use ImageJ software for quantification as demonstrated in published research methodologies . This approach allows for standardized comparison of HMGCS2 expression levels across different experimental conditions.

What are the recommended protocols for immunofluorescence with FITC-conjugated HMGCS2 antibody?

For optimal immunofluorescence staining with FITC-conjugated HMGCS2 antibody, implement the following detailed protocol:

Sample Preparation:

• Fix tissue sections or cultured cells with 4% paraformaldehyde for 15-20 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 in PBS for 10 minutes

• Perform antigen retrieval if necessary (particularly important for formalin-fixed tissues)

Staining Protocol:

• Block non-specific binding with 5% normal serum from the same species as the secondary antibody

• Apply FITC-conjugated HMGCS2 antibody at a dilution of 1:50-1:100 as recommended in specifications

• Incubate in a humid chamber overnight at 4°C or 1-2 hours at room temperature

• Wash thoroughly with PBS (3 x 5 minutes)

• For nuclear counterstaining, use DAPI (1 μg/mL) for 5 minutes

• Mount with anti-fade mounting medium

Optimization Considerations:

• Since HMGCS2 is a mitochondrial protein, consider co-staining with mitochondrial markers (e.g., MitoTracker) to confirm subcellular localization

• Adjust antibody concentration if signal intensity is too weak or too strong

• Include control slides without primary antibody to assess background autofluorescence

• For verified samples, prioritize liver tissues from human, mouse, or rat models

Visualization and Analysis:

• Use appropriate excitation/emission filters for FITC (typically 495 nm/519 nm)

• Capture images at consistent exposure settings across experimental and control samples

• Analyze fluorescence intensity using software like ImageJ with standardized thresholding

• For quantitative analysis, measure fluorescence intensity relative to appropriate housekeeping markers

This protocol has been optimized based on the recommended applications for this antibody and can be adjusted depending on specific experimental requirements and sample types.

How does the FITC conjugation affect the application and sensitivity of HMGCS2 antibody?

The FITC conjugation of HMGCS2 antibody provides distinct advantages and considerations that affect its application and sensitivity in various experimental contexts:

Impact on Detection Sensitivity:

• Direct conjugation eliminates the need for secondary antibodies, reducing potential background signal and non-specific binding

• FITC has a relatively high quantum yield (0.85) providing good brightness, but is more susceptible to photobleaching compared to newer fluorophores

• The conjugation ratio (fluorophore:antibody) may affect sensitivity; optimal ratio preserves antibody binding while maximizing fluorescence

Methodological Advantages:

• Enables direct detection in flow cytometry without secondary antibody incubation steps

• Allows for multiplexing with antibodies from the same host species (since no species-specific secondary antibodies are needed)

• Reduces protocol time and complexity for immunofluorescence and flow cytometry applications

• Particularly useful for co-localization studies with antibodies from the same species

Technical Considerations:

• FITC has an optimal excitation at 495 nm and emission at 519 nm (green spectrum)

• FITC is sensitive to pH fluctuations; maintain samples at physiological pH (7.2-7.4) for optimal fluorescence

• The buffer containing 50% glycerol and 0.01M PBS at pH 7.4 is specifically formulated to maintain FITC stability

• FITC conjugation may slightly affect antibody shelf-life; store at -20°C or -80°C and avoid repeated freeze-thaw cycles as recommended

Application-Specific Adjustments:

• For immunofluorescence: Direct conjugation allows for simplified protocols, but may require higher antibody concentrations (1:50-1:100) compared to indirect methods

• For flow cytometry: Titration experiments are recommended to determine optimal antibody concentration

• For ELISA applications: The FITC conjugation allows for direct detection without enzyme-conjugated secondary antibodies

When planning experiments, consider that while FITC conjugation offers workflow advantages, it may not provide the signal amplification achieved through indirect detection methods (primary + secondary antibody). In cases where target protein expression is low, signal enhancement techniques may be necessary.

How is HMGCS2 regulated by the Wnt/β-catenin pathway in intestinal cells?

HMGCS2 expression is negatively regulated by the Wnt/β-catenin pathway in intestinal cells through a complex signaling mechanism. This regulatory relationship has significant implications for metabolism and disease:

Molecular Mechanism of Regulation:

• Inhibition of Wnt/β-catenin signaling (using iCRT3) results in a dose-dependent increase in HMGCS2 protein and mRNA expression in intestinal cancer cell lines (LS174T and Caco2)

• Conversely, activation of Wnt/β-catenin signaling through Wnt3a treatment suppresses HMGCS2 expression at both protein and mRNA levels

• This suppression occurs in conjunction with increased expression of Wnt target genes such as Axin2, confirming pathway activation

• The regulation appears to be mediated through PPARγ (Peroxisome Proliferator-Activated Receptor gamma), suggesting a Wnt/β-catenin/PPARγ signaling axis

Experimental Evidence from Different Models:

• Cell lines: LS174T and Caco2 intestinal cancer cells show consistent HMGCS2 suppression upon Wnt activation

• Primary cells: Mouse small intestinal organoids cultured in Matrigel demonstrate suppression of HMGCS2 protein expression when treated with Wnt3a (100 ng/mL), accompanied by increased β-catenin levels

• The consistent findings across cancer cell lines and normal intestinal organoids suggest this regulatory mechanism is conserved in both normal and transformed intestinal cells

Methodological Approaches to Study This Regulation:

• Western blot analysis to detect HMGCS2 and Wnt pathway proteins (β-catenin, Axin2)

• Real-time RT-PCR to quantify HMGCS2 mRNA expression changes

• Chromatin immunoprecipitation (ChIP) assays to examine PPARγ binding to the HMGCS2 promoter region containing PPRE (PPAR Response Element)

• Treatment with pathway modulators: Wnt3a (100 ng/mL) for activation and iCRT3 for inhibition of Wnt/β-catenin signaling

This regulatory relationship between Wnt/β-catenin signaling and HMGCS2 expression provides insight into the metabolic programming of intestinal cells and may have implications for understanding diseases involving dysregulated Wnt signaling, including colorectal cancer and inflammatory bowel diseases.

What is known about HMGCS2's role in ketogenesis and metabolic disorders?

HMGCS2 plays a pivotal role in ketogenesis and metabolism, with significant implications for metabolic disorders:

Fundamental Role in Ketogenesis:

• HMGCS2 catalyzes the first and rate-limiting reaction of ketogenesis, converting acetyl-CoA and acetoacetyl-CoA to HMG-CoA

• This mitochondrial enzyme is critical for producing ketone bodies (primarily β-hydroxybutyrate or βHB) that serve as alternative energy sources during carbohydrate limitation

• The product βHB can be measured using specialized assay kits (e.g., Beta-Hydroxybutyrate Assay Kit) to quantify ketogenesis in experimental settings

Tissue-Specific Expression and Function:

• While classically associated with liver metabolism, HMGCS2 expression has been identified in intestinal epithelial cells with functional significance

• Research with transgenic mice lacking intestinal HMGCS2 expression (Hmgcs2ΔIEC) demonstrates the importance of tissue-specific ketogenesis

• Studies show intestinal HMGCS2-derived βHB affects local immune responses without significantly altering systemic βHB levels

Implications in Disease Models:

• In experimental autoimmune encephalomyelitis (EAE) models, intestinal HMGCS2 expression correlates with disease severity

• Mice lacking intestinal HMGCS2 (Hmgcs2ΔIEC) show heightened disease severity compared to wildtype (Hmgcs2WT) controls

• The connection between HMGCS2-derived ketone bodies and immune modulation is evidenced by increased IL-17a+ CD4+ Th17 cells in Hmgcs2ΔIEC mice

• Ketogenic diet intervention can rescue disease phenotypes, highlighting potential therapeutic applications

Microbial Interactions:

• Intestinal HMGCS2 expression appears to shape the gut microbiota composition

• Fecal microbiota transplantation (FMT) experiments demonstrate that microbiota from Hmgcs2ΔIEC mice transmit disease susceptibility regardless of recipient genotype

• This suggests a bidirectional relationship between host ketogenesis and microbial communities with implications for immune regulation

These findings collectively highlight HMGCS2's multifaceted role beyond simple energy metabolism, positioning it at the intersection of metabolic programming, immune regulation, and host-microbiome interactions. The enzyme represents a potential therapeutic target for metabolic disorders and immune-mediated diseases through its impact on ketone body production.

How can I design experiments to investigate HMGCS2's role in specific tissues?

Designing comprehensive experiments to investigate tissue-specific roles of HMGCS2 requires a multi-faceted approach:

Tissue-Specific Expression Analysis:

• Western Blot Analysis:

  • Use validated HMGCS2 antibody at recommended dilutions (1:500-1:2000)

  • Compare expression across tissues (verified tissues include liver, heart, kidney)

  • Include mitochondrial markers (e.g., VDAC) for normalization

  • Quantify using densitometric analysis via ImageJ

• Immunohistochemistry/Immunofluorescence:

  • Apply HMGCS2 antibody at 1:50-1:100 dilution

  • Co-stain with tissue-specific markers to identify expressing cell types

  • Verify subcellular localization with mitochondrial markers

  • Compare expression patterns across normal and disease states (e.g., cancer tissues)

Functional Studies Design:

• Genetic Manipulation Approaches:

  • Generate tissue-specific Hmgcs2 knockout models using Cre-loxP system (e.g., Hmgcs2ΔIEC for intestine-specific deletion)

  • Design floxed Hmgcs2 constructs targeting critical exons

  • Verify knockout efficiency via Western blot and qPCR

  • Assess phenotypes under various metabolic challenges (fasting, ketogenic diet)

• Metabolic Output Assessment:

  • Measure tissue-specific ketone body production using Beta-Hydroxybutyrate Assay Kit

  • Normalize values to cell number or total protein amount

  • Compare βHB levels between wild-type and tissue-specific knockout models

  • Assess both local tissue and systemic circulating ketone levels

Regulatory Pathway Analysis:

• Signaling Pathway Interrogation:

  • Manipulate Wnt/β-catenin signaling using activators (Wnt3a) or inhibitors (iCRT3)

  • Assess HMGCS2 expression changes via Western blot and qPCR

  • Perform ChIP assays using primers for HMGCS2 promoter region (forward: 5′-CAGCCATTCCCACACATGCTCA-3′, reverse: 5′-GACTTTATAAAGCCCCAAGACT-3′)

  • Compare regulatory patterns across different tissue types

• Transcriptional Control Investigation:

  • Analyze PPARγ binding to HMGCS2 promoter via ChIP

  • Use reporter assays with HMGCS2 promoter constructs to assess tissue-specific transcriptional regulation

  • Compare regulation between different cell types (e.g., intestinal vs. liver cells)

Physiological Impact Studies:

• Disease Model Applications:

  • Apply tissue-specific knockout models to relevant disease contexts (e.g., EAE for neuroinflammation)

  • Track disease progression using standardized scoring systems

  • Analyze immune cell populations (e.g., IL-17a+ CD4+ Th17 cells) by flow cytometry

  • Investigate rescue strategies using ketogenic diet or exogenous ketone esters

This comprehensive experimental design approach allows for detailed characterization of HMGCS2 function in specific tissues while connecting molecular mechanisms to physiological outcomes in normal and disease states.

What are the considerations for studying HMGCS2 in transgenic mouse models?

When studying HMGCS2 in transgenic mouse models, several critical considerations must be addressed for robust experimental design and data interpretation:

Model Generation and Validation:

• Targeting Strategy:

  • For tissue-specific knockouts, carefully select the appropriate Cre driver line (e.g., Villin-Cre for intestinal epithelium)

  • Design floxed alleles that ensure complete functional disruption without affecting neighboring genes

  • Consider potential compensatory mechanisms from HMGCS1 (cytosolic isoform)

• Validation Requirements:

  • Confirm genotype by PCR using specific primers

  • Verify protein expression changes by Western blot across relevant tissues

  • Assess functional consequences through measurement of tissue and circulating βHB levels

  • Document any developmental or fertility issues that might complicate breeding

Experimental Design Considerations:

• Dietary Manipulations:

  • Standard chow versus ketogenic diet comparisons are essential as HMGCS2 function is highly diet-dependent

  • Define diet composition precisely (e.g., ketogenic diet formulation with specific fat:protein:carbohydrate ratios)

  • Document diet-induced changes in circulating βHB levels using appropriate assays

  • Consider time-course experiments to capture acute versus chronic adaptations

• Control Groups:

  • Use littermate controls whenever possible

  • Include both floxed non-Cre and wild-type controls to rule out effects of the floxed allele itself

  • Age- and sex-match experimental groups, as metabolic phenotypes can be highly sex-dependent

  • Consider housing controls and experimental animals in the same cage to minimize microbiome differences

Phenotypic Analysis Approaches:

• Metabolic Phenotyping:

  • Measure multiple ketone bodies beyond βHB (acetoacetate, acetone)

  • Conduct fasting response studies (12-24 hours) to reveal functional deficits

  • Perform glucose and insulin tolerance tests to assess metabolic flexibility

  • Consider metabolomic profiling to identify broader metabolic alterations

• Tissue-Specific Consequences:

  • For intestinal models (Hmgcs2ΔIEC), examine both local intestinal effects and systemic consequences

  • Investigate cross-talk between affected and non-affected tissues

  • Analyze immune cell populations in relevant tissues (e.g., IL-17a+ CD4+ Th17 cells) by flow cytometry

  • Consider microbiome analysis as intestinal HMGCS2 can shape gut microbiota composition

Interventional Studies:

• Rescue Experiments:

  • Test supplementation with ketone esters to bypass HMGCS2 deficiency

  • Document dose-dependent effects on circulating βHB and disease parameters

  • Consider timing of intervention (preventative versus therapeutic)

  • Validate mechanism through molecular and cellular readouts

• Microbiome Manipulations:

  • Use antibiotics (e.g., AVNM cocktail) to deplete gut microbiota before fecal microbiota transplantation (FMT)

  • Perform bidirectional FMT between wild-type and knockout mice to establish causality

  • Track disease parameters and immune responses following microbiota transfer

By systematically addressing these considerations, researchers can develop robust experimental paradigms for investigating HMGCS2 function in transgenic mouse models while minimizing confounding factors and strengthening the translational relevance of their findings.

Why might there be discrepancies between expected and observed molecular weights for HMGCS2?

Discrepancies between the calculated (expected) and observed molecular weights of HMGCS2 on Western blots are common and can be attributed to several biological and technical factors:

Biological Factors:

• Post-translational Modifications:

  • HMGCS2 may undergo various modifications (phosphorylation, acetylation, ubiquitination) that alter its migration pattern

  • Multiple bands may appear if a protein sample contains different modified forms simultaneously

  • Enzymatically active HMGCS2 may have different migration properties than inactive forms

• Protein Processing:

  • As a mitochondrial protein, HMGCS2 contains a targeting sequence that may be cleaved upon mitochondrial import

  • The calculated MW (50-56 kDa) versus observed MW (50 kDa) difference may reflect this processing

  • Alternative splicing can generate isoforms with different molecular weights

Technical Considerations:

• SDS-PAGE Conditions:

  • Protein mobility is affected by gel percentage, running buffer composition, and voltage

  • Highly charged or hydrophobic regions in HMGCS2 may bind SDS irregularly

  • Use gradient gels (4-15%) to improve resolution around the expected MW range

• Sample Preparation Effects:

  • Incomplete denaturation can result in partially folded structures with altered mobility

  • Ensure complete denaturation by heating samples at 95°C for 5 minutes in sample buffer

  • Reducing agent concentration (β-mercaptoethanol or DTT) can affect protein migration

Verification Approaches:

• Validation Methods:

  • Run recombinant HMGCS2 protein alongside samples as a size reference

  • Include samples from HMGCS2-knockout tissues as negative controls

  • Perform immunoprecipitation followed by mass spectrometry to confirm protein identity

• Reporting Considerations:

  • Document both calculated (50-56 kDa) and observed (typically 50 kDa) molecular weights

  • If multiple bands appear, consider immunoprecipitation followed by phosphatase treatment to determine if phosphorylation causes the pattern

  • When publishing, clearly indicate which band corresponds to HMGCS2 and provide justification

Experimental Adaptations:

• Optimized Detection Protocol:

  • Use fresh samples and avoid repeated freeze-thaw cycles

  • Include protease and phosphatase inhibitors in lysis buffers

  • Consider native PAGE for applications where preserving protein complexes is important

• Results Interpretation:

  • When observed MW is consistently 50 kDa rather than the calculated 52-56 kDa, document this known discrepancy

  • Interpret multiple bands carefully, considering the possibility of degradation products versus modified forms

  • Verify critical findings with alternative detection methods (e.g., mass spectrometry)

Understanding these factors helps researchers correctly interpret Western blot results and avoid misidentification of HMGCS2, particularly when studying its expression in different experimental contexts.

What are the optimal conditions for storing and handling HMGCS2 antibody?

For maximum performance and longevity of HMGCS2 antibody, proper storage and handling conditions are critical:

Storage Temperature and Conditions:

• Long-term Storage:

  • Store at -20°C or -80°C as recommended by manufacturers

  • The liquid antibody formulation contains 50% glycerol specifically to prevent freezing damage

  • Validate shelf life (typically 12 months under proper storage conditions)

• Working Stock Management:

  • For frequent use, small aliquots (10-20 μL) can be maintained at 4°C for up to 2 weeks

  • Avoid storing diluted antibody solutions for extended periods

  • Maintain sterile conditions to prevent microbial contamination

Handling Practices:

• Freeze-Thaw Considerations:

  • Minimize freeze-thaw cycles as explicitly warned in product specifications

  • Quick-thaw frozen antibody on ice rather than at room temperature

  • If multiple freeze-thaw cycles are unavoidable, monitor performance with positive controls

• Temperature Transitions:

  • Allow refrigerated antibody to equilibrate to room temperature before opening to prevent condensation

  • Immediately return to appropriate storage after use

  • Transport on ice when moving between laboratory locations

Buffer Compatibility:

• Dilution Buffer Selection:

  • For immunoassays, dilute in buffers compatible with the supplied formulation (phosphate buffered solution, pH 7.4)

  • For FITC-conjugated antibodies, avoid buffers containing sodium azide which can quench fluorescence

  • Consider adding protein stabilizers (BSA 0.1-1%) to diluted antibody solutions

• pH Considerations:

  • Maintain pH between 7.2-7.4 for optimal antibody performance and FITC fluorescence

  • The supplied buffer (0.01M PBS, pH 7.4) is optimized for antibody stability

  • Avoid exposing FITC-conjugated antibodies to strong light or extreme pH conditions

Quality Control Practices:

• Performance Monitoring:

  • Periodically verify antibody performance using positive control samples

  • Document lot numbers and correlate with experimental outcomes

  • Consider performing titration experiments if sensitivity appears to decrease

• Contamination Prevention:

  • Use sterile technique when handling antibody stocks

  • The preservative (0.03% Proclin 300) helps prevent microbial growth but proper handling remains important

  • Avoid introducing foreign materials into the antibody vial

Shipping and Receipt Protocols:

• Upon Receipt:

  • Inspect for any signs of damage or thawing

  • The product is shipped with ice packs; store immediately at recommended temperature

  • Document date of receipt to track shelf life

Adherence to these storage and handling guidelines will help maintain antibody performance and ensure reproducible results across experiments with the HMGCS2 antibody.

How can I address background issues in immunostaining with FITC-conjugated HMGCS2 antibody?

Addressing background issues in immunostaining with FITC-conjugated HMGCS2 antibody requires a systematic approach:

Sample Preparation Optimization:

• Fixation Improvements:

  • Optimize fixative concentration and duration (typically 4% paraformaldehyde for 15-20 minutes)

  • Over-fixation can create autofluorescence, particularly in tissues with high lipid content

  • Consider alternative fixatives (methanol/acetone) for specific applications

• Autofluorescence Reduction:

  • Treat tissue sections with 0.1-1% sodium borohydride for 5-10 minutes before blocking

  • For tissues with high natural autofluorescence (liver, kidney), consider Sudan Black B treatment (0.1-0.3% in 70% ethanol)

  • Use confocal microscopy with narrow bandpass filters to discriminate between specific signal and autofluorescence

Blocking Protocol Optimization:

• Enhanced Blocking Methods:

  • Extend blocking time to 1-2 hours at room temperature

  • Use a combination of 5-10% normal serum with 1-3% BSA

  • Add 0.1-0.3% Triton X-100 to blocking buffer for improved penetration

  • Consider specialized blocking reagents for tissues with high background (mouse-on-mouse blocking for mouse tissues)

• Antibody Diluent Optimization:

  • Prepare diluent with 1-2% of the same serum used for blocking

  • Add 0.05-0.1% Tween-20 to reduce non-specific binding

  • Maintain proper pH (7.2-7.4) for optimal FITC fluorescence

Antibody Incubation Adjustments:

• Concentration Optimization:

  • Perform titration experiments starting with the recommended 1:50-1:100 dilution

  • Test both shorter incubations at room temperature and overnight at 4°C

  • Consider signal-to-noise ratio rather than absolute signal intensity when optimizing

• Washing Protocol Enhancement:

  • Increase number and duration of washes (4-6 washes of 10 minutes each)

  • Use PBS with 0.05-0.1% Tween-20 for more effective removal of unbound antibody

  • Perform washes with gentle agitation to improve removal of non-specific binding

Counterstaining and Mounting Considerations:

• Nuclear Counterstain Selection:

  • Use DAPI at appropriate concentration (0.1-1 μg/mL) to avoid spillover into FITC channel

  • Consider alternative counterstains with minimal spectral overlap with FITC

  • Optimize counterstain concentration to provide context without overwhelming the FITC signal

• Mounting Medium Selection:

  • Use anti-fade mounting medium specifically formulated for fluorescence preservation

  • Avoid mounting media with high autofluorescence in the FITC channel

  • Consider pH-stable mounting media to maintain FITC fluorescence intensity

Control Implementations:

• Essential Controls:

  • No primary antibody control to assess secondary antibody non-specific binding

  • Isotype control at equivalent concentration to evaluate non-specific binding

  • Positive control tissue (verified samples like liver) to confirm staining pattern

  • Negative control tissue (non-expressing tissue) to confirm specificity

• Advanced Control Methods:

  • Absorption controls (pre-incubate antibody with immunizing peptide)

  • Competing unlabeled primary antibody control

  • Tissue from HMGCS2 knockout models when available

By systematically addressing these aspects, researchers can significantly reduce background and enhance specific detection of HMGCS2 using FITC-conjugated antibodies in immunofluorescence applications.

How can I design experiments to validate HMGCS2 antibody specificity?

Designing robust experiments to validate HMGCS2 antibody specificity is essential for reliable research outcomes:

Multi-technique Validation Approach:

• Western Blot Validation:

  • Compare staining pattern across multiple tissues with known HMGCS2 expression (liver, heart, kidney)

  • Verify single band at expected molecular weight (approximately 50 kDa)

  • Include positive controls (recombinant HMGCS2) and negative controls (non-expressing tissues)

  • Perform peptide competition assay by pre-incubating antibody with immunizing peptide (aa 426-508 of human HMGCS2)

• Immunoprecipitation-Mass Spectrometry:

  • Immunoprecipitate HMGCS2 from tissue lysates using the antibody

  • Confirm protein identity by mass spectrometry analysis

  • Compare detected peptides with known HMGCS2 sequence (UniprotID: P54868)

  • Document peptide coverage across different regions of the protein

Genetic Manipulation Approaches:

• Knockdown/Knockout Validation:

  • Perform siRNA knockdown of HMGCS2 in appropriate cell lines

  • Use CRISPR-Cas9 to generate HMGCS2 knockout cell lines

  • Compare antibody staining in wild-type versus knockdown/knockout samples

  • Validate knockdown efficiency by RT-qPCR in parallel

• Overexpression Studies:

  • Express tagged HMGCS2 in cell lines with low endogenous expression

  • Perform dual labeling with anti-tag and anti-HMGCS2 antibodies

  • Confirm co-localization to verify antibody recognition of the overexpressed protein

  • Include untagged HMGCS2 overexpression to rule out tag interference

Cross-reactivity Assessment:

• Species Cross-reactivity Testing:

  • Test across human, mouse, and rat samples as specified in the antibody information

  • Document differences in staining patterns or intensity between species

  • Align protein sequences across species to identify conserved epitopes

• Isoform Specificity Verification:

  • Test potential cross-reactivity with HMGCS1 (cytosolic homolog)

  • Compare staining patterns in tissues with differential expression of HMGCS1 versus HMGCS2

  • Consider targeted overexpression of each isoform separately to assess antibody discrimination

Application-specific Validation:

• Immunohistochemistry Validation:

  • Compare staining patterns with published literature on HMGCS2 expression

  • Verify mitochondrial localization through co-staining with established mitochondrial markers

  • Test antibody performance in different fixation conditions (formalin, methanol, acetone)

  • Include human thyroid cancer and colon carcinoma tissues as verified samples

• Immunofluorescence Cross-validation:

  • Perform parallel staining with multiple HMGCS2 antibodies targeting different epitopes

  • Confirm subcellular localization is consistent with mitochondrial distribution

  • Document performance across various tissue preparations (frozen sections, paraffin sections)

  • Verify performance in rat and mouse liver tissues as specified in antibody information

Antibody Performance Documentation:

• Standardized Reporting:

  • Record lot number, concentration, and storage conditions

  • Document all validation experiments with appropriate controls

  • Maintain detailed protocols for reproducibility

  • Consider publishing validation data as supplementary material in research articles

By implementing this comprehensive validation approach, researchers can establish high confidence in antibody specificity before proceeding with critical experiments using HMGCS2 antibody.

How does HMGCS2 expression correlate with disease states and what are the research implications?

HMGCS2 expression correlates with various disease states, providing valuable insights for both basic and translational research:

Cancer Research Applications:

• Expression Patterns in Cancer:

  • HMGCS2 expression has been investigated in thyroid cancer and colon carcinoma samples

  • Altered expression may correlate with cancer progression and metabolic reprogramming

  • The relationship between Wnt/β-catenin pathway activation (common in many cancers) and HMGCS2 suppression suggests potential mechanistic links

• Methodological Approaches:

  • Immunohistochemical analysis of tumor versus adjacent normal tissue

  • Correlation of expression levels with patient outcomes and tumor characteristics

  • Investigation of HMGCS2 as a potential metabolic biomarker in cancer

Inflammatory and Autoimmune Diseases:

• Role in Experimental Autoimmune Encephalomyelitis (EAE):

  • HMGCS2 expression in intestinal epithelial cells influences EAE disease progression

  • Tissue-specific knockout (Hmgcs2ΔIEC) mice show increased disease severity and heightened inflammatory responses

  • Higher levels of pathogenic IL-17a+ CD4+ Th17 cells are observed in Hmgcs2ΔIEC mice

• Therapeutic Implications:

  • Ketogenic diets rescue disease phenotypes in EAE models, suggesting metabolic intervention potential

  • Supplementation with ketone esters can compensate for HMGCS2 deficiency, offering a targeted approach

  • Microbiome modulation presents another intervention avenue based on the interaction between intestinal HMGCS2 and gut microbiota

Metabolic Disorders:

• HMGCS2 Deficiency Syndrome:

  • Mutations in the HMGCS2 gene are associated with HMG-CoA synthase deficiency

  • The condition presents as hypoketotic hypoglycemia during periods of fasting

  • Research applications include developing screening methods and therapeutic strategies

• Metabolic Adaptation Studies:

  • HMGCS2 expression is crucial during carbohydrate deprivation and fasting

  • Research can explore the therapeutic potential of modulating HMGCS2 activity in metabolic disorders

  • Tissue-specific roles expand our understanding beyond liver metabolism to intestinal functions

Host-Microbiome Interactions:

• Microbiota Influence:

  • Intestinal HMGCS2 expression shapes gut microbiota composition

  • Fecal microbiota transplantation (FMT) experiments demonstrate that microbiota from Hmgcs2ΔIEC mice can transmit disease susceptibility

  • This suggests bidirectional communication between host metabolism and microbial communities

• Research Methodologies:

  • Use of antibiotics (AVNM) to deplete gut microbiota before FMT experiments

  • Tracking disease parameters following microbiota transfer between wild-type and knockout mice

  • Metagenomic analysis to identify specific microbial taxa influenced by HMGCS2 expression

Future Research Directions:

• Mechanistic Investigations:

  • Further elucidation of the Wnt/β-catenin/PPARγ signaling axis in regulating HMGCS2

  • Exploration of epigenetic regulation of HMGCS2 expression in different tissues

  • Investigation of post-translational modifications affecting HMGCS2 activity

• Translational Applications:

  • Development of tissue-specific HMGCS2 modulators as potential therapeutics

  • Exploration of HMGCS2 as a biomarker for disease progression or treatment response

  • Integration of HMGCS2 biology into precision medicine approaches for metabolic and inflammatory conditions

These diverse research applications demonstrate HMGCS2's significance beyond its enzymatic function, positioning it at the intersection of metabolism, inflammation, and host-microbiome interactions.

What emerging techniques can enhance HMGCS2 protein research?

Emerging techniques are revolutionizing HMGCS2 protein research, offering unprecedented insights into its function, regulation, and therapeutic potential:

Advanced Imaging Approaches:

• Super-Resolution Microscopy:

  • Techniques like STORM, PALM, or STED can resolve HMGCS2 subcellular localization below the diffraction limit

  • Multi-color super-resolution imaging can reveal co-localization with other mitochondrial proteins at nanometer scale

  • Live-cell super-resolution microscopy can track dynamic changes in HMGCS2 distribution during metabolic shifts

• Correlative Light and Electron Microscopy (CLEM):

  • Combines fluorescence microscopy of FITC-conjugated HMGCS2 antibody with electron microscopy

  • Provides ultrastructural context of HMGCS2 localization within mitochondrial compartments

  • Especially valuable for understanding spatial organization of ketogenesis enzymes

Proteomics and Protein Interaction Analysis:

• Proximity Labeling Techniques:

  • BioID or APEX2-based approaches can identify proteins in close proximity to HMGCS2

  • Reveals potential regulatory partners and multi-protein complexes

  • Can be performed in different metabolic states to identify context-specific interactions

• Cross-linking Mass Spectrometry (XL-MS):

  • Maps protein-protein interaction interfaces at amino acid resolution

  • Identifies structural changes in HMGCS2 under different metabolic conditions

  • Helps understand how post-translational modifications affect protein interactions

Genetic Engineering Approaches:

• CRISPR-Cas9 Genomic Tagging:

  • Endogenous tagging of HMGCS2 with fluorescent proteins for live-cell imaging

  • Introduction of specific mutations to study structure-function relationships

  • Creation of reporter systems to monitor HMGCS2 expression in real-time

• Base Editing and Prime Editing:

  • Precise introduction of disease-associated HMGCS2 mutations

  • Correction of pathogenic variants to establish causality

  • Creation of allelic series to study dosage effects in cellular models

Single-Cell Analysis Techniques:

• Single-Cell RNA Sequencing:

  • Maps HMGCS2 expression heterogeneity across cell populations

  • Reveals co-expression patterns with regulatory factors

  • Identifies specific cell types responsible for tissue-level HMGCS2 activity

• Single-Cell Proteomics:

  • Quantifies HMGCS2 protein levels in individual cells

  • Correlates protein expression with functional states

  • Reveals post-transcriptional regulation not captured by RNA analysis

Metabolic Flux Analysis:

• Stable Isotope Tracing:

  • Uses 13C-labeled substrates to track carbon flow through the ketogenesis pathway

  • Quantifies HMGCS2 activity in living cells under different conditions

  • Reveals metabolic rewiring in disease states or upon therapeutic intervention

• Real-time Metabolite Imaging:

  • Genetically encoded biosensors for ketone bodies

  • Visualization of spatial and temporal dynamics of HMGCS2 products

  • Correlation with cellular events such as signaling pathway activation

Organoid and Microphysiological Systems:

• Advanced Intestinal Organoids:

  • 3D cultures that recapitulate intestinal epithelium for studying tissue-specific HMGCS2 function

  • Co-culture systems with immune cells to study HMGCS2-mediated immunomodulation

  • Patient-derived organoids to study disease-specific HMGCS2 dysregulation

• Organ-on-Chip Technologies:

  • Microfluidic devices that model tissue-tissue interactions influenced by HMGCS2

  • Integration of biosensors for real-time monitoring of ketone body production

  • Testing of therapeutic compounds targeting HMGCS2 or its regulatory pathways

These emerging techniques offer complementary approaches to traditional methods, enabling researchers to address increasingly sophisticated questions about HMGCS2 biology and its implications in health and disease.

What are the key considerations for researchers working with HMGCS2 antibodies?

Researchers working with HMGCS2 antibodies should consider several critical factors to ensure experimental success and reliable data interpretation. First and foremost, proper storage and handling conditions are essential, including storage at -20°C or -80°C, avoiding repeated freeze-thaw cycles, and working with aliquots to maintain antibody integrity . The choice of antibody format should align with experimental goals, with FITC-conjugated versions offering advantages for direct detection but potentially different sensitivity profiles compared to unconjugated alternatives .

For application-specific considerations, researchers should recognize that HMGCS2 antibodies have been validated in multiple experimental contexts, including Western blot (1:500-1:2000 dilution), immunohistochemistry, and immunofluorescence (1:50-1:100 dilution) . Particular attention should be paid to the mitochondrial localization of HMGCS2, which may require specialized sample preparation techniques and co-staining with mitochondrial markers for proper interpretation . Additionally, researchers should be aware of potential discrepancies between the calculated (50-56 kDa) and observed (typically 50 kDa) molecular weights when performing Western blot analysis .

Validation of antibody specificity remains paramount, ideally incorporating multiple approaches including positive and negative controls, peptide competition assays, and genetic approaches like knockdown or knockout validation when possible. When publishing results, researchers should clearly document the antibody source, catalog number, dilution, and validation methodology to ensure reproducibility. By carefully considering these factors, researchers can maximize the utility of HMGCS2 antibodies while minimizing potential pitfalls in their experimental approaches.

How might future research on HMGCS2 evolve based on current knowledge?

The future of HMGCS2 research is poised for significant evolution based on our current understanding of this multifaceted enzyme. Beyond its classical role in hepatic ketogenesis, recent discoveries highlighting tissue-specific functions, particularly in intestinal epithelial cells, will likely drive expanded investigation into organ-specific metabolic programming . The demonstrated connection between intestinal HMGCS2 expression and immune modulation opens promising avenues for exploring metabolic-immune crosstalk in various disease contexts beyond the current focus on experimental autoimmune encephalomyelitis .

The regulatory relationship between HMGCS2 and the Wnt/β-catenin/PPARγ signaling axis presents fertile ground for mechanistic studies of metabolic reprogramming in cancer and other contexts where Wnt signaling is dysregulated . This may lead to novel therapeutic strategies targeting this regulatory pathway in metabolic disorders and malignancies. Additionally, the emerging understanding of how HMGCS2-derived ketone bodies shape the gut microbiome composition suggests future research will increasingly focus on host-microbiome interactions and their therapeutic implications .