HMGCL Antibody, HRP conjugated

What is HMGCL and why is it important in metabolic research?

HMGCL is a mitochondrial enzyme that catalyzes the cation-dependent cleavage of (S)-3-hydroxy-3-methylglutaryl-CoA into acetyl-CoA and acetoacetate, representing a key step in ketogenesis and the final step in leucine degradation . Ketone bodies (β-hydroxybutyrate, acetoacetate, and acetone) serve as alternative energy sources to glucose during fasting conditions and function as lipid precursors and metabolic regulators . Recent research has revealed HMGCL's unexpected role in cancer metabolism, where its expression is downregulated in certain cancers, suggesting potential tumor-suppressive properties .

What are the optimal dilutions for using HMGCL antibodies with HRP detection systems?

For optimal results when using HMGCL primary antibodies with HRP-conjugated secondary antibodies, follow these validated dilution ranges:

For Western blot applications specifically, a 1:1000 dilution of the primary HMGCL antibody paired with HRP-conjugated secondary antibody at 1:50,000-1:100,000 is recommended for optimal signal-to-noise ratio . It is crucial to empirically determine the optimal concentration for each specific experimental system .

What is the expected molecular weight for HMGCL detection and how should I validate specificity?

The calculated molecular weight for human HMGCL is approximately 34 kDa (corresponding to a protein of 325 amino acids), which matches the observed molecular weight in Western blot applications . For validation of specificity:

• Run appropriate positive control tissues: mouse liver, mouse brain, human heart, or rat liver tissues have been validated to express detectable levels of HMGCL .

• Include a negative control using either HMGCL-knockout cells or tissues (where available) or primary antibody omission.

• Check for a single band at the expected 34 kDa molecular weight.

• For HRP-conjugated detection systems, include controls for potential non-specific binding of the secondary antibody .

What are the recommended storage conditions for HMGCL antibodies?

Most HMGCL antibodies maintain optimal activity when stored at -20°C, where they remain stable for approximately one year after shipment . Typical formulations include PBS buffer with 0.02% sodium azide and 50% glycerol at pH 7.3. Avoid freeze/thaw cycles, as these significantly reduce antibody performance. For HRP-conjugated secondary antibodies used in HMGCL detection systems, aliquoting is recommended to minimize freeze/thaw cycles that can diminish enzymatic activity .

Which tissue types show the highest expression of HMGCL for positive controls?

HMGCL is primarily expressed in tissues where ketogenesis is active. Based on validated testing data, the following tissues serve as reliable positive controls:

For IHC applications, antigen retrieval with TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative method .

How can I distinguish between mitochondrial HMGCL and the extramitochondrial isoform HMGCLL1?

Discriminating between HMGCL and its extramitochondrial isoform HMGCLL1 requires careful antibody selection and experimental design:

• Use antibodies raised against non-conserved regions: Researchers have successfully developed antibodies targeting residues 19-37 of human HMGCLL1, a region not conserved in mitochondrial HMGCL .

• Implement subcellular fractionation followed by Western blotting with HRP-conjugated detection systems to separate mitochondrial and non-mitochondrial fractions.

• Include appropriate subcellular markers in co-immunostaining experiments:

  • For mitochondria: mitochondrial markers

  • For HMGCLL1 localization: markers such as Golgin 58, GM-130 (Golgi markers), or PEX-14 (peroxisomal marker)

• For immunofluorescence applications, use purified antibodies at dilutions of approximately 1:200 and observe distinct subcellular localization patterns .

The ability to differentiate between these isoforms is critical as they may play distinct roles in ketone body metabolism in different cellular compartments .

What are the optimal protocols for studying HMGCL phosphorylation and degradation?

Research has identified that HMGCL undergoes phosphorylation by IKKβ at Ser258, which promotes its degradation via the ubiquitin-proteasome pathway . To study this regulatory mechanism:

• For phosphorylation detection:

  • Stimulate cells with TNFα in a dose-dependent (varying concentrations) and time-dependent manner to activate IKKβ.

  • Immunoprecipitate HMGCL using specific antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate).

  • Detect phosphorylation using Western blot with phospho-specific antibodies and HRP-conjugated secondary antibodies.

  • Include the HMGCL S258A mutant as a negative control for phosphorylation.

• For degradation analysis:

  • Perform cycloheximide chase assays to measure HMGCL protein half-life (with and without TNFα treatment).

  • For ubiquitination studies, co-express HMGCL with NEDD4 (the E3 ligase) and detect using anti-ubiquitin antibodies with emphasis on K48-linked ubiquitination.

This approach revealed that TNFα treatment shortened the half-life of HMGCL protein in A549 and H1299 cells, and the S258A mutation stabilized HMGCL protein upon TNFα treatment .

What experimental design is recommended for investigating HMGCL's role in cancer metabolism?

HMGCL has been identified as a potential tumor suppressor in lung cancer, where its expression is downregulated . For comprehensive investigation:

• Expression analysis:

  • Compare HMGCL levels between paired tumor and normal tissues using immunohistochemistry with HRP detection (1:50-1:200 dilution).

  • Analyze correlation with clinical parameters such as tumor size, stage, and patient survival.

• Functional studies:

  • Generate stable cell lines with HMGCL overexpression (LV-HMGCL) or knockdown (shHMGCL).

  • Measure β-hydroxybutyrate (β-HB) levels as a functional readout of HMGCL activity.

  • Assess growth in vitro and anchorage-independent growth using soft agar assays.

  • Investigate tumor growth in vivo using xenograft models where tumor volume is calculated as V = L× W²/2.

• Mechanistic studies:

  • Monitor mTOR-P70S6K pathway activation by assessing P70S6K phosphorylation (p-Thr389).

  • Investigate the effects of exogenous β-HB supplementation on reversing phenotypes caused by HMGCL knockdown.

Research has demonstrated that HMGCL overexpression increased β-HB content and inhibited the tumorigenicity of lung cancer cells, while HMGCL knockdown promoted cell growth and anchorage-independent growth .

How should I optimize antigen retrieval for HMGCL detection in different tissue samples?

Optimal detection of HMGCL in tissue samples requires specific fixation and antigen retrieval protocols:

• For paraffin-embedded sections:

  • Deparaffinize sections in xylene followed by rehydration through a graded ethanol series.

  • For antigen retrieval, recommended conditions include:

    • Primary option: TE buffer at pH 9.0

    • Alternative option: Citrate buffer at pH 6.0

  • Block endogenous peroxidase activity using appropriate blocking solutions.

  • Permeabilize with PBS containing 0.3% TritonX-100 to enhance antibody penetration .

• For frozen sections:

  • Fix in paraformaldehyde (typical concentration 4%) before immunostaining.

  • Permeabilization with detergent is essential for accessing intracellular antigens.

• Troubleshooting weak signals:

  • Increase antibody concentration (within recommended ranges)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Ensure fresh DAB substrate when using HRP-conjugated detection systems

These optimized protocols have been validated for detecting HMGCL in human liver, ovary, spleen, and testis tissues .

What are the best methods for studying HMGCL's role in ketogenesis and autophagy?

Recent research has uncovered a connection between HMGCL, ketogenesis, and autophagy regulation . For investigating this relationship:

• Cell culture models:

  • Select appropriate cell lines: osteosarcoma lines (HOS, 143B, U2OS, MG63) and control osteoblast lines (hFOB1.19).

  • Manipulate HMGCL expression using overexpression vectors (LV-HMGCL) or knockdown constructs (shHMGCL).

  • Culture cells in DMEM with 10% FBS at 37°C and 5% CO₂.

• Metabolic analysis:

  • Measure β-hydroxybutyrate levels as a direct readout of ketogenesis activity.

  • For autophagy studies, include treatments with:

    • Autophagy inhibitors: PI3K inhibitor 3-MA

    • mTOR activators: 3BDO

    • Chloroquine for assessing autophagic flux

• Autophagy assessment:

  • Monitor LC3 conversion by Western blot with HRP-conjugated detection.

  • Assess ULK1 activity, which can be targeted with specific siRNAs.

  • For visualizing autophagosomes, use fluorescence microscopy with LC3 antibodies.

• In vivo models:

  • Implement mouse xenograft models with HMGCL-overexpressing cells.

  • Measure tumor volume weekly and harvest after 5 weeks for analysis.

  • Analyze β-HB content in tumor tissues as a marker of ketogenesis.

This experimental approach revealed that HMGCL activates autophagy in osteosarcoma through β-HB, potentially explaining its tumor-suppressive effects .

What insights do structural studies provide about HMGCL's enzymatic mechanism?

Crystal structures of human HMGCL have revealed key insights into its enzymatic mechanism:

• HMGCL adopts a β/α-barrel fold with the catalytic site positioned at the C-terminal end of the barrel .

• Substrate binding studies using both the competitive inhibitor 3-hydroxyglutaryl-CoA (HG-CoA) and the authentic substrate HMG-CoA have identified critical interactions:

  • The R41 residue is essential for catalytic activity, as demonstrated by the catalytically deficient R41M mutant .

  • Mg²⁺ serves as an activator cation with specific coordination geometry in the active site .

• Crystal structures of ternary complexes have been determined at high resolution:

  • WT HMGCL with inhibitor HG-CoA: 2.4 Å resolution

  • R41M mutant with substrate HMG-CoA: 2.2 Å resolution

These structural insights are valuable for understanding disease-causing mutations and developing potential modulators of HMGCL activity.

How can I design experiments to study the effects of HMGCL mutations associated with metabolic disorders?

Human mutations in the HMGCL gene are associated with HMG-CoA lyase deficiency, a potentially lethal inherited metabolic disorder . To study these mutations:

• Structure-based analysis:

  • Map mutations onto the known crystal structure to predict their impact on enzyme function.

  • Focus on mutations affecting the active site, cation binding, or protein stability.

• Functional characterization:

  • Generate site-directed mutants of HMGCL in expression vectors.

  • Express and purify mutant proteins using affinity chromatography.

  • Assess enzymatic activity through spectrophotometric assays measuring acetoacetate formation.

  • Determine kinetic parameters (Km, Vmax) for mutant enzymes compared to wild-type.

• Cellular studies:

  • Introduce mutations into cellular models using CRISPR/Cas9 or overexpression of mutant forms.

  • Measure ketone body production as a functional readout.

  • Assess protein stability through cycloheximide chase assays.

  • Evaluate mitochondrial localization of mutant proteins.

These approaches enable correlation of specific mutations with enzyme deficiency, providing insights into the molecular basis of the disease.

What methods are best for studying HMGCL interactions with other proteins in the ketogenic pathway?

To investigate HMGCL's interactions within the broader ketogenic pathway:

• Co-immunoprecipitation studies:

  • Use HMGCL antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) to pull down protein complexes.

  • Detect interacting partners by Western blot with HRP-conjugated secondary antibodies.

  • Known interactions include NEDD4 (E3 ubiquitin ligase) and IKKβ (kinase) .

• Proximity labeling approaches:

  • Express HMGCL fused to BioID or APEX2 in relevant cell types.

  • After biotin labeling, purify biotinylated proteins and identify by mass spectrometry.

• Functional validation:

  • Confirm direct interactions using purified proteins in GST pulldown assays.

  • For example, GST-HMGCL fusion protein has been shown to pull down endogenous IKKβ and NEDD4 from cell lysates .

• Visualization:

  • Implement co-localization studies using immunofluorescence microscopy.

  • Consider super-resolution techniques for detailed subcellular localization.

These methods have successfully identified regulatory partners of HMGCL, including the revelation that NEDD4 promotes K48-linked ubiquitination of phosphorylated HMGCL, leading to its degradation .

How do extramitochondrial HMGCL isoforms contribute to cellular metabolism?

Recent research has identified HMGCLL1, an extramitochondrial isoform of HMGCL, which may participate in cytosolic or peroxisomal ketone body synthesis . To investigate this emerging area:

• Expression pattern analysis:

  • Compare tissue distribution of HMGCL versus HMGCLL1 using isoform-specific antibodies.

  • HMGCLL1 haplotypes have been implicated as genetic biomarkers for tyrosine kinase inhibitor therapy response in chronic myeloid leukemia .

• Subcellular localization:

  • Use co-localization studies with organelle markers to determine precise cellular distribution.

  • Antibodies raised against non-conserved regions (residues 19-37 of HMGCLL1) have successfully discriminated between the isoforms .

• Functional differentiation:

  • Selectively knock down each isoform using specific siRNAs.

  • Measure compartment-specific ketone body production.

  • Investigate potential non-canonical functions in signaling or gene regulation.

This research direction promises to clarify the potential importance of ketone body synthesis in cytosol or peroxisomes, expanding our understanding beyond traditional mitochondrial ketogenesis pathways .

What are the most sensitive detection methods for measuring HMGCL levels in clinical samples?

For clinical and translational research applications requiring precise quantification of HMGCL:

• Immunohistochemistry optimization:

  • Use antigen retrieval with TE buffer pH 9.0 for formalin-fixed paraffin-embedded (FFPE) samples .

  • Employ polymer-based HRP detection systems for enhanced sensitivity.

  • Score staining using established systems (e.g., H-score or percentage of positive cells).

• Advanced protein quantification:

  • Implement digital pathology with automated image analysis for consistent scoring.

  • Consider multiplexed immunofluorescence to simultaneously detect HMGCL and relevant markers.

  • For serum or plasma samples, develop ELISA-based approaches using validated antibodies.

• Correlation with clinical parameters:

  • HMGCL expression has shown negative correlation with tumor size and positive correlation with survival in lung cancer patients .

  • In tissue arrays (84 samples of cancerous tissues and 84 paired adjacent noncancerous tissues), HMGCL protein levels were significantly reduced in lung cancer tissues .

These methods enable translational studies correlating HMGCL expression with disease progression and treatment response.

How can I investigate the relationship between HMGCL activity and the mTOR signaling pathway?

HMGCL has been linked to mTOR pathway regulation, which is central to cellular growth control:

• Experimental setup:

  • Manipulate HMGCL expression (overexpression or knockdown) in relevant cell lines.

  • Monitor mTOR pathway activity through phosphorylation of downstream targets:

    • P70S6K phosphorylation at Thr389 is decreased when HMGCL is overexpressed .

  • Investigate the role of branched-chain amino acid (BCAA) metabolism, as HMGCL is involved in leucine degradation.

• Mechanistic investigation:

  • Supplement media with β-hydroxybutyrate to determine if ketone bodies directly modulate mTOR signaling.

  • Use pharmacological modulators:

    • mTOR inhibitors (rapamycin)

    • mTOR activators (3BDO)

  • Assess autophagy induction as a readout of mTOR inhibition.

• In vivo validation:

  • Implement xenograft models with altered HMGCL expression.

  • Analyze tumor tissues for mTOR pathway activation markers.

  • Correlate with β-hydroxybutyrate levels in the tumor microenvironment.

This approach can elucidate how metabolic enzymes like HMGCL contribute to signaling networks controlling cell growth and survival.

What techniques can be used to study HMGCL's role in different subcellular compartments?

To investigate the compartment-specific functions of HMGCL:

• Subcellular fractionation:

  • Separate mitochondrial, cytosolic, and nuclear fractions using differential centrifugation.

  • Verify fraction purity using specific markers:

    • Mitochondria: cytochrome c oxidase

    • Cytosol: GAPDH

    • Nucleus: histone H3

  • Detect HMGCL in different fractions by Western blot using HRP-conjugated detection systems.

• Engineered localization variants:

  • Create HMGCL constructs with altered localization signals:

    • Mitochondrial targeting sequence mutations

    • Addition of nuclear localization signals

    • Peroxisomal targeting signals

  • Express these variants and assess functional consequences.

• Super-resolution microscopy:

  • Implement techniques such as STORM or PALM for nanoscale localization.

  • Perform co-localization studies with organelle-specific markers.

• Proximity-based labeling:

  • Express HMGCL fused to compartment-specific BioID variants.

  • Identify compartment-specific interaction partners.

These approaches can reveal non-canonical functions of HMGCL beyond its established role in mitochondrial ketogenesis.

How do I design experiments to study the epigenetic effects of HMGCL-derived metabolites?

Ketone bodies produced through HMGCL activity may function as epigenetic regulators:

• Metabolite supplementation studies:

  • Treat cells with physiologically relevant concentrations of β-hydroxybutyrate.

  • Assess histone acetylation levels by Western blot with specific antibodies against acetylated histones.

  • Perform chromatin immunoprecipitation (ChIP) to identify affected genomic regions.

• HMGCL manipulation approaches:

  • Compare epigenetic landscapes between cells with altered HMGCL expression.

  • Techniques to consider:

    • ATAC-seq for chromatin accessibility

    • ChIP-seq for histone modification patterns

    • RNA-seq for transcriptional consequences

• Mass spectrometry analysis:

  • Implement metabolomics approaches to quantify ketone bodies and related metabolites.

  • Perform protein mass spectrometry to identify acetylation or other modifications on histones and transcription factors.

• Functional validation:

  • Use epigenetic inhibitors in combination with HMGCL modulation.

  • Assess specific gene expression changes through quantitative PCR with reverse transcription.