D2HGDH Antibody

What is D2HGDH and what is its primary function in cellular metabolism?

D2HGDH (D-2-hydroxyglutarate dehydrogenase) is a 521 amino acid mitochondrial enzyme belonging to the FAD-binding oxidoreductase/transferase type 4 protein family . Its primary function is catalyzing the conversion of D-2-hydroxyglutarate (D-2-HG) to alpha-ketoglutarate (α-KG), a critical intermediate in the tricarboxylic acid cycle . This enzymatic activity is essential for maintaining proper metabolic balance within the mitochondria . Beyond its primary substrate, D2HGDH also catalyzes the oxidation of other D-2-hydroxyacids, including D-malate (D-MAL) and D-lactate (D-LAC), though with varying degrees of enzymatic efficiency - exhibiting high activities toward D-2-HG and D-MAL but only weak activity toward D-LAC .

What types of D2HGDH antibodies are currently available for research?

Several types of D2HGDH antibodies are available for research applications, each with specific characteristics and applications:

• Rabbit polyclonal antibodies (e.g., ab233516) - Suitable for Western blot (WB) and immunohistochemistry-paraffin (IHC-P) applications, with demonstrated reactivity against human and rat samples .

• Mouse monoclonal antibodies (e.g., E-6) - Available as IgG2a kappa light chain antibodies for detecting human D2HGDH protein via western blotting, immunoprecipitation, immunofluorescence, and ELISA techniques .

• Various conjugated forms - Including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates, offering versatility for different experimental approaches .

What are the common applications for D2HGDH antibodies in research?

D2HGDH antibodies serve several critical research applications:

• Western blotting - For detecting and quantifying D2HGDH protein in tissue or cell lysates, with typical predicted band size of approximately 56 kDa .

• Immunohistochemistry - For visualizing D2HGDH distribution in formalin-fixed, paraffin-embedded tissues, including normal tissues and cancer samples .

• Immunoprecipitation - For isolating D2HGDH protein complexes to study protein-protein interactions .

• Immunofluorescence - For examining subcellular localization and expression patterns .

• Validation studies - For confirming pathogenicity of D2HGDH variants identified in clinical samples .

How should I optimize Western blot protocols for D2HGDH detection?

For optimal Western blot performance with D2HGDH antibodies, consider the following methodological approach:

• Sample preparation:

  • For cell lines: Use whole cell lysates (e.g., HEK-293T) with standard lysis buffers containing protease inhibitors .

  • For tissues: Rat kidney and liver tissue lysates have been successfully used for D2HGDH detection .

• Loading and separation:

  • Load sufficient protein (typically 20-50 μg total protein).

  • Use gels that provide good resolution in the 50-60 kDa range where D2HGDH (predicted size: 56 kDa) migrates .

• Antibody concentration:

  • Optimal concentration for rabbit polyclonal antibodies: 1-2 μg/mL .

  • For mouse monoclonal antibodies: Follow manufacturer recommendations, typically 1:100-1:1000 dilution .

• Detection system:

  • HRP-linked secondary antibodies (e.g., Goat anti-Rabbit IgG) at approximately 0.2 μg/mL have demonstrated good results .

  • Enhanced chemiluminescent substrates provide sensitive detection.

• Controls:

  • Include recombinant D2HGDH protein as positive control when possible .

  • Negative controls using lysates from cell lines with known low D2HGDH expression or CRISPR knockout lines are recommended.

What are the critical considerations for immunohistochemical analysis with D2HGDH antibodies?

When performing immunohistochemistry with D2HGDH antibodies, researchers should consider:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues have been successfully used with D2HGDH antibodies .

  • Both normal tissues (liver, cerebrum, testis) and cancer tissues (prostate, breast, colorectal, glioma) have shown compatibility .

• Antibody concentration:

  • Recommended concentration for IHC-P: 10 μg/ml for rabbit polyclonal antibodies .

  • Titrate antibody concentration if background staining is problematic.

• Detection system:

  • HRP-conjugated secondary antibodies (e.g., HRP-Linked Goat anti-Rabbit IgG at 2 μg/ml) with DAB staining have shown good results .

  • Consider signal amplification systems for detecting low-abundance expression.

• Antigen retrieval:

  • Heat-induced epitope retrieval methods are typically suitable for D2HGDH detection in FFPE tissues.

  • Standard citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) protocols should be tested and optimized.

• Controls:

  • Include tissues with known D2HGDH expression patterns as positive controls.

  • Use isotype control antibodies at equivalent concentrations to assess non-specific binding.

How can I verify the specificity of my D2HGDH antibody?

Verifying antibody specificity is critical for reliable research data. Implement these approaches:

• Multiple detection methods:

  • Confirm results using different techniques (Western blot, IHC, IF) when possible .

  • Concordant results across methods increase confidence in specificity.

• Positive and negative controls:

  • Use recombinant D2HGDH protein as positive control .

  • Tissues or cells with genetic knockout or knockdown of D2HGDH serve as negative controls.

  • Compare reactivity in tissues known to express high vs. low levels of D2HGDH.

• Peptide competition assays:

  • Pre-incubate antibody with the immunizing peptide or recombinant D2HGDH.

  • Specific staining or bands should be significantly reduced or eliminated.

• Molecular weight verification:

  • Confirm that detected bands match the predicted molecular weight of D2HGDH (approximately 56 kDa) .

  • Be aware of potential post-translational modifications that may alter migration patterns.

• Cross-validation with different antibodies:

  • Compare results using antibodies from different sources or those recognizing different epitopes of D2HGDH .

How can D2HGDH antibodies be utilized in studying the role of D2HGDH in cancer metabolism?

D2HGDH antibodies provide valuable tools for investigating D2HGDH's emerging role in cancer metabolism:

• Expression profiling:

  • Use IHC to compare D2HGDH expression across cancer types and correlate with clinical parameters .

  • Analyze D2HGDH levels in diffuse large B-cell lymphoma, where enzymatically inert D2HGDH mutants have been identified .

• Functional studies:

  • Combine D2HGDH detection with measurements of α-KG levels to understand metabolic dependencies.

  • Investigate the relationship between D2HGDH expression and IDH2 activity in cancer cells .

• Epigenetic regulation:

  • Use D2HGDH antibodies alongside histone and DNA methylation markers to elucidate how D2HGDH-mediated α-KG regulation influences epigenetic programming in cancer .

  • Study how D2HGDH affects HIF1α hydroxylation and subsequent hypoxia signaling pathways .

• Therapeutic target assessment:

  • Monitor D2HGDH expression and localization changes in response to metabolic or epigenetic therapies.

  • Evaluate potential compensatory mechanisms in response to therapies targeting related metabolic pathways.

• Cancer subtype stratification:

  • Develop IHC protocols for D2HGDH as a potential biomarker to stratify cancer subtypes with distinct metabolic profiles.

What experimental approaches are recommended for studying D2HGDH variants and their functional consequences?

Researchers investigating D2HGDH variants can implement these approaches:

• Overexpression systems:

  • Express wild-type and variant D2HGDH in appropriate cell models (e.g., HEK293 cells) .

  • Verify expression by Western blot using anti-D2HGDH antibodies .

• Enzymatic activity assays:

  • Measure conversion of D-2-HG to α-KG in cellular or biochemical assays.

  • Quantify substrate and product levels using techniques like LC-MS/MS .

  • Compare residual activities of variants to wild-type enzyme function.

• Structural analysis:

  • Use conservation analysis across D2HGDH orthologs to assess the potential impact of variants on protein structure and function .

  • Compare functional outcomes with in silico predictions from tools like PolyPhen-2, SIFT, and Mutation Taster .

• Subcellular localization studies:

  • Employ immunofluorescence with anti-D2HGDH antibodies to assess whether variants affect mitochondrial localization .

  • Use mitochondrial co-markers to confirm proper compartmentalization.

• Metabolic profiling:

  • Measure cellular metabolites, particularly D-2-HG, α-KG, and related TCA cycle intermediates, to assess the metabolic impact of D2HGDH variants.

How does D2HGDH regulate α-KG levels and how can this be experimentally investigated?

D2HGDH's regulation of α-KG levels involves complex mechanisms that can be investigated using these approaches:

• Metabolite quantification:

  • Measure α-KG levels using LC-MS/MS in contexts of D2HGDH overexpression, knockdown, or knockout.

  • Compare wild-type D2HGDH effects with enzymatically inactive mutants .

• IDH2 interaction studies:

  • Investigate the relationship between D2HGDH and IDH2 expression using co-immunoprecipitation with D2HGDH antibodies .

  • Perform genetic depletion of IDH2 to assess its requirement for D2HGDH-mediated effects on α-KG levels .

  • Use ectopic IDH2 expression to rescue D2HGDH-deficient cells .

• Epigenetic readouts:

  • Analyze histone and DNA methylation patterns as functional readouts of α-KG-dependent dioxygenase activity .

  • Employ ChIP-seq or reduced representation bisulfite sequencing to assess genome-wide epigenetic changes.

• HIF1α hydroxylation:

  • Measure HIF1α hydroxylation as a direct readout of α-KG-dependent prolyl hydroxylase activity .

  • Assess downstream hypoxia-responsive gene expression patterns.

• D2-HG supplementation experiments:

  • Test whether D2-HG supplementation can modulate mitochondrial IDH activity and IDH2 expression .

  • Compare the effects of D2-HG supplementation in the presence and absence of functional D2HGDH.

What are common issues in D2HGDH antibody applications and how can they be resolved?

Researchers may encounter several challenges when working with D2HGDH antibodies:

• Weak signal in Western blot:

  • Increase protein loading (up to 50-60 μg).

  • Optimize antibody concentration: consider using 1-2 μg/mL for polyclonal antibodies .

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

  • Use signal enhancement systems compatible with your detection method.

• High background in IHC:

  • Decrease antibody concentration below the recommended 10 μg/ml .

  • Extend blocking time with 5% BSA or 5% normal serum.

  • Include 0.1-0.3% Triton X-100 in antibody diluent to reduce non-specific membrane binding.

  • Use more stringent washing procedures between antibody incubations.

• Multiple bands in Western blot:

  • Verify predicted band size (56 kDa for D2HGDH) .

  • Consider potential post-translational modifications or splice variants.

  • Compare with recombinant D2HGDH protein control .

  • Test antibody in samples with D2HGDH knockdown or knockout.

• Cross-reactivity issues:

  • Validate antibody specificity with appropriate positive and negative controls.

  • Consider using monoclonal antibodies for higher specificity in systems with closely related proteins.

• Inconsistent results between experiments:

  • Standardize sample preparation protocols.

  • Prepare aliquots of antibody dilutions to ensure consistency.

  • Include internal controls in each experiment to normalize results.

How should D2HGDH antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of D2HGDH antibodies is critical for maintaining their performance over time:

• Storage conditions:

  • Store unconjugated antibodies at -20°C for long-term storage.

  • For conjugated antibodies (HRP, FITC, etc.), follow manufacturer-specific recommendations .

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots.

• Working dilution preparation:

  • Prepare fresh working dilutions before each experiment.

  • Use high-quality, filtered antibody diluents with appropriate protein carrier (typically 1-5% BSA).

  • For Western blot applications, consider adding 0.02% sodium azide to antibody solutions for reuse.

• Temperature considerations:

  • Always bring antibody aliquots to room temperature before opening to prevent condensation.

  • Return unconjugated antibodies to -20°C promptly after use.

  • Most antibody incubations are optimal at 4°C (overnight) or room temperature (1-2 hours).

• Quality control measures:

  • Document lot numbers and performance characteristics.

  • Include positive controls in each experiment to monitor antibody performance over time.

  • Consider validating new lots against previous lots if lot-to-lot variation is a concern.

• Contamination prevention:

  • Use clean pipette tips for each antibody retrieval.

  • Monitor for microbial contamination, particularly in antibody solutions stored at 4°C.

  • Consider adding antimicrobial agents to antibody dilutions stored for multiple uses.

What controls should be included when using D2HGDH antibodies for functional studies?

Comprehensive controls are essential for reliable interpretation of D2HGDH antibody results:

• Positive controls:

  • Recombinant human D2HGDH protein for Western blot applications .

  • Tissues with known D2HGDH expression (e.g., liver, kidney) for IHC and Western blot .

  • Cell lines with confirmed D2HGDH expression (e.g., HEK-293T) .

• Negative controls:

  • Primary antibody omission controls to assess secondary antibody specificity.

  • Isotype controls at equivalent concentrations to evaluate non-specific binding.

  • Ideally, D2HGDH knockout/knockdown samples when available.

• Technical controls:

  • Loading controls (β-actin, GAPDH) for Western blot normalization .

  • For overexpression studies, GFP or other tags to confirm transfection efficiency .

• Functional controls:

  • Wild-type D2HGDH alongside enzymatically inactive mutants when studying variant effects .

  • Metabolic pathway controls (e.g., IDH2 expression) when studying D2HGDH's metabolic effects .

• Specificity controls:

  • Peptide competition assays with immunizing peptide.

  • Validation with alternative antibodies targeting different epitopes of D2HGDH.

How can D2HGDH antibodies contribute to understanding D-2-hydroxyglutaric aciduria?

D2HGDH antibodies provide valuable tools for investigating D-2-hydroxyglutaric aciduria (D2HGA), a rare neurometabolic disorder:

• Functional validation of variants:

  • Use D2HGDH antibodies to verify expression of missense variants identified in patients .

  • Combine with enzymatic activity assays to determine pathogenicity .

  • Compare residual enzyme activities of different variants with clinical severity.

• Tissue expression studies:

  • Investigate D2HGDH expression patterns in relevant tissues from control and patient samples.

  • Focus on neurological tissues given the predominant neurological symptoms in D2HGA.

• Cellular pathology:

  • Examine subcellular localization of mutant D2HGDH proteins to determine if mislocalization contributes to pathology.

  • Study mitochondrial morphology and function in relation to D2HGDH deficiency.

• Diagnostic applications:

  • Develop protocols for immunohistochemical detection of D2HGDH in patient samples.

  • Create assays to assess D2HGDH protein levels in accessible patient specimens.

• Therapeutic monitoring:

  • Use D2HGDH antibodies to monitor protein expression in experimental therapeutic approaches.

  • Assess normalization of D2HGDH levels or localization following interventions.

What methodological approaches are recommended for analyzing D2HGDH in patient-derived samples?

When working with patient-derived samples, researchers should consider these methodological approaches:

• Sample types and preparation:

  • Fibroblasts: Primary culture from patient skin biopsies provides accessible material for functional studies.

  • Blood lymphocytes: Can be transformed with Epstein-Barr virus for sustained cell lines.

  • FFPE tissues: Compatible with IHC analysis using D2HGDH antibodies at 10 μg/ml concentration .

• D2HGDH protein analysis:

  • Western blot: Use 1-2 μg/ml antibody concentration with 20-50 μg total protein .

  • Immunofluorescence: Assess subcellular localization with co-staining for mitochondrial markers.

• Functional assessments:

  • Enzymatic activity: Measure conversion of D-2-HG to α-KG.

  • Metabolite analysis: Quantify D-2-HG levels using LC-MS/MS .

• Genetic correlation:

  • Compare D2HGDH protein expression and function with genotype information.

  • Create reference data for variant classification based on protein expression patterns.

• Standardization considerations:

  • Include age and sex-matched control samples.

  • Standardize sample collection, processing, and storage procedures.

  • Document clinical parameters for correlation with biochemical findings.

How should quantitative data from D2HGDH antibody experiments be analyzed?

Proper analysis of quantitative data from D2HGDH antibody experiments requires:

• Western blot quantification:

  • Use appropriate software (ImageJ, Image Lab, etc.) for densitometric analysis.

  • Normalize D2HGDH band intensity to loading controls (β-actin, GAPDH).

  • Present data as relative expression compared to control samples.

  • Apply statistical analysis appropriate for sample size and distribution.

• IHC scoring methods:

  • Develop standardized scoring system based on staining intensity and distribution.

  • Consider automated image analysis for quantitative assessment.

  • Use blinded assessment by multiple observers for reproducibility.

• Enzyme activity correlation:

  • Create standard curves relating D2HGDH protein levels to enzymatic activity.

  • Present data as percentage of wild-type activity for variant analysis .

  • Consider the following activity classification scale based on research findings:

• Multi-parameter analysis:

  • Correlate D2HGDH protein levels with metabolite measurements (D-2-HG, α-KG).

  • Integrate protein expression, localization, and functional data.

• Biological replication:

  • Perform experiments with multiple biological replicates.

  • Report both technical and biological variability.

  • Use appropriate statistical tests for small sample sizes.

How does D2HGDH activity relate to epigenetic regulation, and what experimental designs can investigate this relationship?

The relationship between D2HGDH activity and epigenetic regulation can be investigated through:

• Integrated experimental design:

  • Overexpress wild-type D2HGDH and enzymatically inactive mutants .

  • Measure α-KG levels and correlate with histone and DNA methylation patterns .

  • Assess HIF1α hydroxylation as a readout of α-KG-dependent dioxygenase activity .

• Epigenomic profiling:

  • Perform ChIP-seq for histone modifications affected by α-KG-dependent demethylases (H3K9me3, H3K27me3, H3K4me3).

  • Use whole-genome bisulfite sequencing or reduced representation bisulfite sequencing to assess DNA methylation.

  • Compare epigenetic profiles between conditions with normal versus impaired D2HGDH function.

• Gene expression analysis:

  • Correlate epigenetic changes with transcriptomic alterations using RNA-seq.

  • Focus on genes regulated by HIF1α to connect metabolic changes to hypoxia response.

• Mechanistic investigation:

  • Use pharmacological inhibitors of epigenetic enzymes to determine causality.

  • Employ genetic approaches to manipulate levels of α-KG-dependent dioxygenases.

• Cancer context specificity:

  • Compare D2HGDH effects across different cancer types with varying baseline epigenetic profiles.

  • Focus on diffuse large B-cell lymphoma where D2HGDH mutants have been identified .

The complex nature of this relationship requires careful experimental design with appropriate controls and replication to establish meaningful connections between D2HGDH activity and epigenetic outcomes.

What emerging applications of D2HGDH antibodies show promise for advancing metabolic research?

Several emerging applications of D2HGDH antibodies hold potential for advancing metabolic research:

• Single-cell analysis:

  • Adapting D2HGDH antibodies for mass cytometry or imaging mass cytometry.

  • Combining with metabolic sensors to correlate D2HGDH expression with cellular metabolic states at single-cell resolution.

• Spatial metabolomics:

  • Using immunofluorescence with D2HGDH antibodies alongside metabolite imaging techniques.

  • Mapping D2HGDH distribution in tissues with metabolic heterogeneity, particularly in cancer.

• Protein interaction networks:

  • Employing D2HGDH antibodies for proximity labeling approaches (BioID, APEX).

  • Identifying novel D2HGDH interaction partners that may regulate its function or be regulated by it.

• Therapeutic targeting:

  • Developing screening assays using D2HGDH antibodies to identify compounds that modulate D2HGDH expression or activity.

  • Monitoring D2HGDH levels as pharmacodynamic biomarkers in metabolic intervention studies.

• Exosome research:

  • Investigating D2HGDH presence in extracellular vesicles using D2HGDH antibodies.

  • Exploring potential intercellular metabolic signaling involving D2HGDH or its metabolic products.

How might D2HGDH antibodies contribute to understanding the interplay between metabolism and cancer?

D2HGDH antibodies can significantly advance our understanding of metabolism-cancer connections:

• Metabolic reprogramming:

  • Profiling D2HGDH expression across cancer types and stages to identify patterns of metabolic adaptation .

  • Correlating D2HGDH levels with other metabolic enzymes to map cancer-specific metabolic networks.

• Tumor heterogeneity:

  • Using D2HGDH immunohistochemistry to assess metabolic heterogeneity within tumors .

  • Comparing D2HGDH expression between primary tumors and metastases to understand metabolic evolution.

• Therapeutic vulnerability:

  • Identifying cancer subtypes with altered D2HGDH expression or mutation as potentially vulnerable to metabolic intervention.

  • Developing D2HGDH expression signatures that predict response to therapies targeting related metabolic pathways.

• Epigenetic-metabolic axis:

  • Using D2HGDH antibodies alongside epigenetic markers to map the relationship between D2HGDH activity, α-KG levels, and epigenetic states in cancer .

  • Investigating how D2HGDH-mediated regulation of α-KG influences cancer cell differentiation and stemness properties.

• Immunometabolism:

  • Examining D2HGDH expression in tumor-infiltrating immune cells to understand metabolic crosstalk in the tumor microenvironment.

  • Investigating how tumor D2HGDH activity might influence immune cell function through metabolite-mediated signaling.