H6PD Antibody, HRP conjugated

What is G6PD and why is it an important research target?

G6PD (Glucose-6-phosphate dehydrogenase) is the rate-limiting enzyme in the pentose phosphate pathway (PPP) in glycolysis. It catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconolactone while reducing NADP+ to NADPH . G6PD's primary functions include providing reducing power (NADPH) and pentose phosphates for fatty acid and nucleic acid synthesis . The enzyme contains a substrate binding site for G6P and a catalytic coenzyme binding site that binds to NADP+/NADPH using the Rossman fold . G6PD is particularly important as a research target because genetic deficiency predisposes individuals to non-immune hemolytic anemia, and the enzyme exhibits significant genetic diversity with numerous variants showing varying levels of activity and clinical manifestations . Recent research has also revealed a novel role of G6PD in regulating mitophagy by maintaining PINK1 stability, connecting glucose metabolism, redox homeostasis, and mitochondrial quality control .

What detection methods are compatible with G6PD antibodies, and which one should I choose for my experiment?

G6PD antibodies are versatile research tools compatible with multiple detection methodologies. Western blot analysis is highly effective for detecting G6PD at approximately 58-59 kDa in various cell types including A549 human lung carcinoma and MCF-7 human breast cancer cell lines . Simple Western™ provides an automated alternative to traditional Western blotting with comparable results . For tissue samples, immunohistochemistry using both paraffin-embedded (IHC-P) and frozen sections (IHC-F) can be performed with appropriate dilutions (1:200-400 and 1:100-500, respectively) .

The choice of method depends on your specific research question:

• For protein quantity assessment or molecular weight confirmation, Western blot is optimal

• For high-throughput or small sample analysis, Simple Western™ is preferable

• For visualizing spatial distribution of G6PD in tissues or subcellular localization, use immunohistochemistry

• For protein-protein interaction studies, immunoprecipitation techniques are most appropriate

How should I optimize dilution factors for G6PD antibodies in different applications?

Optimization of antibody dilution is critical for obtaining specific signals while minimizing background. The following dilution ranges have been experimentally validated for HRP-conjugated G6PD antibodies:

For Western blot applications, experimental data shows that 1 μg/ml of Mouse Anti-Human G6PD Monoclonal Antibody followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody produces clear detection of G6PD at approximately 59 kDa in A549 and MCF-7 cell lines . For Simple Western™ applications, 10 μg/mL followed by 1:50 dilution of HRP-conjugated secondary antibody has been validated to detect G6PD at approximately 58 kDa . Always perform a dilution series to determine optimal concentration for your specific experimental conditions.

How can I effectively use G6PD antibodies in protein-protein interaction studies?

G6PD antibodies are valuable tools for investigating protein-protein interactions, particularly when studying the role of G6PD in cellular processes like mitophagy. Based on recent research, the following immunoprecipitation protocol has been validated for G6PD interaction studies:

• Transfect cells with tagged proteins of interest (e.g., myc-PINK1 or myc-tagged G6PD truncation mutants)

• After treatment, lyse cells in IP buffer (25 mmol/L Tris HCl pH 7.4, 150 mmol/L NaCl, 1% NP-40, 1 mmol/L EDTA, and 5% glycerol) containing protease and phosphatase inhibitors

• Sonicate lysates and centrifuge at 12,000 g for 15 min at 4°C

• Pre-clear supernatants with Protein A/G Agarose beads

• Incubate pre-cleared supernatants with either:

  • 20 μL agarose-conjugated G6PD antibody

  • 20 μL agarose-conjugated PINK1 antibody

  • 20 μL ANTI-FLAG® M2 Affinity Gel (for FLAG-tagged proteins)

  • 5 μL anti-Myc-tag antibody (for myc-tagged proteins)

• Add Protein A/G beads for free antibody IP

• Wash beads and analyze by Western blotting

This methodology has successfully demonstrated that a portion of G6PD localizes to mitochondria where it interacts with PINK1, revealing G6PD's role in regulating mitophagy through PINK1 stabilization .

How can I use G6PD antibodies to investigate variant forms of the enzyme in clinical samples?

G6PD exhibits significant genetic diversity with numerous variants showing different enzymatic activities. To investigate these variants:

• Generate recombinant G6PD variants using site-directed mutagenesis:

  • Amplify the G6PD gene from human cDNA using high-fidelity DNA polymerase

  • Introduce restriction enzyme sites (e.g., EcoRI and XhoI) in primers

  • Create variants using overlap-extension PCR (examples include G6PD Mahidol, G6PD Viangchan, and G6PD Viangchan + Mahidol)

• Express and purify the variants

• Confirm expression using Western blot analysis:

  • Separate purified proteins on 12% SDS-PAGE

  • Transfer to PVDF membrane

  • Block with PBS containing 5% skimmed milk

  • Incubate with anti-human G6PD antibody (1:3000 dilution)

  • Wash with PBST

  • Incubate with HRP-conjugated secondary antibody (1:2500 dilution)

  • Develop and analyze

This approach enables characterization of enzymatic properties of clinical G6PD variants, including activity levels, thermostability, and structural differences, providing insights into genotype-phenotype relationships in G6PD deficiency.

What are the key considerations when using HRP-conjugated G6PD antibodies for subcellular localization studies?

Recent research has revealed that G6PD has multiple subcellular localizations including cytoplasm, nucleus, and cell membrane , with a portion also localizing to mitochondria . When using HRP-conjugated G6PD antibodies for subcellular localization studies, consider:

• Fixation method: Different fixation protocols can affect epitope accessibility. Paraformaldehyde fixation preserves morphology but may mask certain epitopes.

• Permeabilization: Ensure adequate permeabilization for accessing intracellular compartments while preserving membrane structures.

• Controls: Include appropriate controls:

  • Cells with G6PD knockdown/knockout as negative controls

  • Co-staining with established subcellular markers (e.g., mitochondrial markers when studying mitochondrial localization)

  • Isotype controls (e.g., Mouse IgG1 Isotype Control)

• Signal amplification: For detecting low-abundance G6PD in specific compartments, tyramide signal amplification may be employed with HRP-conjugated antibodies.

• Resolution limitations: HRP-based detection may not provide the resolution needed for precise subcellular localization. Consider fluorescent detection methods for colocalization studies.

How can I design experiments to investigate G6PD's role in PINK1-Parkin-mediated mitophagy?

Recent research has identified G6PD as a key regulator of PINK1-Parkin-mediated mitophagy . To investigate this role:

• Generate G6PD knockout cell lines:

  • Use CRISPR-Cas9 with specific sgRNAs targeting G6PD

  • Verify knockout by Western blot with G6PD antibodies

  • Create rescue cell lines by reconstituting wild-type G6PD

• Evaluate mitophagy by monitoring:

  • Parkin translocation to mitochondria (fluorescence microscopy)

  • Degradation of mitochondrial proteins (Western blot)

  • Mitochondrial morphology (electron microscopy)

• Investigate interaction with PINK1:

  • Perform co-immunoprecipitation using G6PD and PINK1 antibodies

  • Assess PINK1 stabilization after mitochondrial depolarization

  • Evaluate ubiquitin phosphorylation using phospho-specific antibodies

• Test mitochondrial stress response:

  • Induce mitochondrial depolarization (e.g., with O/A treatment or CCCP)

  • Assess cell viability in WT vs. G6PD KO cells

  • Measure mitochondrial function parameters

When designing these experiments, it's important to include appropriate controls and validate findings using multiple approaches. The research by Cho et al. demonstrated that G6PD deletion significantly inhibited mitophagy and resulted in an impairment in PINK1 stabilization, establishing G6PD as a positive regulator of mitophagy .

What approaches can I use to investigate discrepancies between G6PD protein levels and enzymatic activity?

G6PD deficiency can result from reduced protein levels, decreased activity, or both. To investigate discrepancies:

• Parallel assessment of protein and activity:

  • Quantify G6PD protein levels using HRP-conjugated antibodies in Western blot

  • Measure enzymatic activity using spectrophotometric assays

  • Calculate specific activity (activity/protein ratio)

• Analysis of post-translational modifications:

  • Immunoprecipitate G6PD using specific antibodies

  • Perform mass spectrometry to identify modifications

  • Use modification-specific antibodies if available

• Investigation of protein stability:

  • Perform pulse-chase experiments to assess protein half-life

  • Conduct thermal shift assays to evaluate structural stability

  • Use trypsin digestion assays (as employed for G6PD Viangchan and G6PD Viangchan + Mahidol variants)

• Domain-specific analysis:

  • Generate and characterize truncation mutants

  • Assess substrate binding capacity versus catalytic efficiency

  • Investigate G6PD dimerization/tetramerization status

For instance, studies on G6PD Viangchan and G6PD Viangchan + Mahidol variants revealed that these mutations resulted in severe enzyme deficiency with remaining activity <10%, despite detectable protein levels. The combined G6PD Viangchan + Mahidol mutant showed an approximately 10-fold reduction in enzyme activity compared to single mutation variants .

How can I adapt protocols for G6PD antibodies when studying its interactions with non-canonical pathways?

The discovery of G6PD's role in mitophagy illustrates its involvement in non-canonical pathways beyond the pentose phosphate pathway . When investigating such interactions:

• Modify immunoprecipitation buffers:

  • Adjust detergent type and concentration based on interaction strength

  • Consider crosslinking for transient interactions

  • Optimize salt concentration to maintain specific interactions

• Use proximity labeling approaches:

  • Fuse G6PD to BioID or APEX2

  • Identify proximal proteins in different cellular compartments

  • Validate interactions using co-immunoprecipitation with G6PD antibodies

• Employ functional reconstitution:

  • Use G6PD KO cells as a background

  • Reconstitute with wild-type or mutant G6PD (catalytically inactive)

  • Assess pathway functionality

• Develop compartment-specific analyses:

  • Isolate subcellular fractions (e.g., mitochondria, nucleus)

  • Perform Western blot with G6PD antibodies on each fraction

  • Conduct immunoprecipitation from specific compartments

This approach revealed that while G6PD's catalytic activity is required for mitophagy regulation, the known PPP functions per se are not involved, highlighting a novel role for G6PD independent of its classical metabolic function .

How do I select the most appropriate G6PD antibody for my specific research application?

Selection of an appropriate G6PD antibody depends on multiple factors:

• Target species and epitope:

  • Ensure antibody reactivity matches your experimental species (e.g., human)

  • Consider epitope location relative to functional domains or variant sites

  • For variants studies, choose antibodies recognizing conserved regions

• Antibody type:

  • Monoclonal antibodies (e.g., Clone #1067503) offer high specificity and reproducibility

  • Recombinant antibodies provide consistency between lots

  • Consider host species compatibility with your experimental system

• Conjugation and detection system:

  • HRP-conjugated antibodies eliminate secondary antibody steps

  • Validate that the conjugation doesn't affect binding capacity

  • Consider signal strength requirements

• Validation data:

  • Review existing validation for your application (WB, IHC, IP)

  • Check for validated dilutions and expected band sizes (58-59 kDa for G6PD)

  • Assess specificity documentation in relevant cell types

For instance, Mouse Anti-Human G6PD Monoclonal Antibody (Clone #1067503) has been validated for detection of human G6PD in A549 and MCF-7 cell lines by Western blot at approximately 59 kDa , while Rabbit Recombinant G6PD Antibody with HRP conjugation has been validated for WB, IHC-P, and IHC-F applications .

What quality control procedures should I implement when working with G6PD antibodies?

Implementing rigorous quality control ensures reliable research outcomes:

• Antibody validation:

  • Verify specificity using G6PD knockout/knockdown cells

  • Confirm expected molecular weight (58-59 kDa)

  • Test cross-reactivity with related proteins

• Performance controls:

  • Include positive controls (cells/tissues known to express G6PD)

  • Use isotype controls (e.g., Mouse IgG1 Isotype Control)

  • Implement loading controls for quantitative analyses

• Storage and handling:

  • Store at -20°C in multiple aliquots to avoid freeze-thaw cycles

  • Use appropriate storage buffer (e.g., aqueous buffered solution containing 0.01M TBS with 1% BSA, 0.02% Proclin300, and 50% Glycerol)

  • Monitor performance over time

• Lot-to-lot variation assessment:

  • Test new lots against previous lots

  • Maintain reference samples for comparison

  • Document optimal working dilutions for each lot

• Application-specific validations:

  • For Western blot: verify band size and specificity

  • For IHC: validate staining pattern against literature

  • For IP: confirm enrichment of target protein

These quality control measures are particularly important when studying G6PD variants or when investigating novel functions like its role in mitophagy regulation.

How should I interpret discrepancies in G6PD localization data across different detection methods?

G6PD has been reported to localize in the cytoplasm, nucleus, cell membrane , and partially in mitochondria . When encountering localization discrepancies:

• Consider method-specific limitations:

  • Subcellular fractionation may cause cross-contamination between compartments

  • Fixation protocols can affect antigen accessibility

  • HRP-based detection may lack spatial resolution for precise localization

• Implement orthogonal validation:

  • Combine biochemical fractionation with immunofluorescence

  • Use multiple antibodies targeting different G6PD epitopes

  • Employ tagged G6PD constructs (being mindful of tag interference)

• Account for physiological state and cell type:

  • G6PD localization may change under stress conditions

  • Cell type-specific differences in localization may exist

  • Treatment conditions (e.g., O/A or CCCP) may alter distribution

• Quantitative assessment:

  • Quantify relative distribution across compartments

  • Perform time-course analyses to capture dynamic changes

  • Consider single-cell analyses to account for heterogeneity

The discovery that a portion of G6PD localizes to mitochondria where it interacts with PINK1 represents an important finding that expands our understanding of G6PD function beyond its classical role in the cytoplasmic pentose phosphate pathway.

What are the critical controls needed when investigating G6PD-protein interactions using immunoprecipitation?

When studying G6PD interactions, particularly novel ones like with PINK1, include these critical controls:

• Input controls:

  • Analyze a portion of pre-immunoprecipitation lysate

  • Verify expression of both G6PD and potential interacting partners

  • Assess consistency across experimental conditions

• Negative controls:

  • Use isotype-matched control antibodies (e.g., Mouse IgG1 Isotype Control)

  • Perform IP in cells lacking G6PD or the interacting protein

  • Include non-specific proteins to rule out promiscuous binding

• Reciprocal immunoprecipitation:

  • Perform reverse IP (pull down partner and probe for G6PD)

  • Compare interaction efficiency in both directions

  • Assess whether different domains are captured

• Competition controls:

  • Use excess untagged protein to compete with interaction

  • Assess binding under different buffer conditions

  • Test effect of substrate/cofactor binding on interaction

• Specificity controls:

  • Include closely related proteins that should not interact

  • Use truncation or point mutants to map interaction domains

  • Test interaction under conditions that should disrupt binding

For example, when investigating G6PD interaction with PINK1, researchers used control IgG, performed reciprocal IPs with both proteins, and validated direct interaction using purified proteins .

How can G6PD antibodies be integrated into multi-omics approaches for comprehensive pathway analysis?

Integrating G6PD antibody-based techniques with multi-omics approaches provides a comprehensive understanding of G6PD's diverse functions:

• Proteomics integration:

  • Use G6PD antibodies for immunoprecipitation followed by mass spectrometry

  • Correlate G6PD levels with global proteome changes

  • Identify post-translational modifications affecting G6PD function

• Metabolomics correlation:

  • Measure G6PD protein levels and activity alongside metabolite profiling

  • Correlate NADPH/NADP+ ratios with G6PD expression

  • Assess impact of G6PD variants on metabolic pathways

• Transcriptomics complementation:

  • Compare G6PD protein levels with mRNA expression

  • Identify discrepancies suggesting post-transcriptional regulation

  • Correlate transcriptional changes with G6PD activity

• Functional genomics validation:

  • Use G6PD antibodies to validate screening hits (e.g., CRISPR screens)

  • Confirm protein-level changes for genetic modulators

  • Assess downstream pathway effects of G6PD modulation

This integrated approach was effectively demonstrated in research identifying G6PD as a regulator of mitophagy, where G6PD was first identified through a whole-genome CRISPR-Cas9 screening and then validated through protein-level analyses using G6PD antibodies, followed by functional studies examining mitochondrial protein degradation and cell viability under stress conditions .