PGAM5 Antibody, FITC conjugated

What is PGAM5 and why is it significant in cellular research?

PGAM5 is a mitochondrial Serine/Threonine phosphatase primarily located in the inner mitochondrial membrane. It plays critical roles in mitochondrial homeostasis, particularly in contexts of cellular stress and disease conditions. The significance of PGAM5 stems from its involvement in key cellular processes including mitochondrial dynamics, mitophagy regulation, and cell death pathways. Recent studies have identified PGAM5 as an important regulator of mitochondrial function in pulmonary fibrosis and hepatocellular carcinoma, making it a valuable target for investigation in disease models . When studying PGAM5, researchers should consider its dual role as both a phosphatase and a structural component of mitochondrial complexes.

What are the key specifications of commercially available PGAM5 antibodies?

PGAM5 antibodies are available in various formulations including FITC-conjugated versions. The standard specifications for PGAM5 antibody, FITC conjugated include:

These antibodies are typically generated against recombinant human serine/threonine-protein phosphatase PGAM5 mitochondrial protein fragments (30-223AA) . For optimal results, researchers should consider the specific epitope recognized by their chosen antibody and ensure compatibility with their experimental system.

What cell and tissue types are most suitable for PGAM5 antibody applications?

PGAM5 antibodies have demonstrated successful applications across multiple cell lines and tissue types. According to validation studies, positive Western blot detection has been confirmed in A549 cells, HeLa cells, HepG2 cells, and MCF-7 cells . For tissue-based applications, immunohistochemistry has successfully detected PGAM5 in human lung cancer tissue, breast cancer tissue, and liver cancer tissue .

When designing experiments, researchers should consider that PGAM5 expression may vary across tissues, with particularly significant expression in metabolically active tissues and those with high mitochondrial content. Additionally, disease states may alter PGAM5 expression levels, as evidenced by studies in hepatocellular carcinoma where high PGAM5 expression correlates with poor prognosis . Preliminary optimization experiments are recommended when working with new tissue types or disease models.

How should researchers optimize PGAM5 antibody dilutions for different experimental applications?

Optimization of antibody dilutions is critical for obtaining specific signals while minimizing background noise. For PGAM5 antibodies, recommended dilution ranges vary by application:

For optimal IHC results with PGAM5 antibodies, antigen retrieval with TE buffer at pH 9.0 is typically recommended, although citrate buffer at pH 6.0 may serve as an alternative . When establishing optimal dilutions, researchers should implement a systematic titration approach, beginning with the manufacturer's recommended range and adjusting based on signal-to-noise ratio. Positive and negative controls should be included in optimization experiments, with A549 or MCF-7 cells serving as reliable positive controls for PGAM5 expression . For FITC-conjugated antibodies specifically, additional considerations regarding photobleaching and appropriate mounting media should be incorporated into experimental design.

What methodological approaches can distinguish between the two PGAM5 isoforms in experimental settings?

PGAM5 exists in two splice variants: PGAM5-L (long) and PGAM5-S (short), which have distinct subcellular localizations and functions. Distinguishing between these isoforms requires specific methodological considerations:

• Antibody selection: Verify whether the PGAM5 antibody recognizes epitopes common to both isoforms or is isoform-specific. Most commercial antibodies detect both variants but may show differential affinity.

• Western blot resolution: Use gradient gels (10-15%) to effectively separate the closely sized isoforms. Extended run times may be necessary for adequate separation.

• Subcellular fractionation: Employ differential centrifugation to separate mitochondrial and cytosolic fractions prior to immunoblotting, as PGAM5-L primarily localizes to mitochondria while PGAM5-S may redistribute under stress conditions.

• Immunofluorescence co-localization: Perform dual staining with mitochondrial markers (e.g., TOMM20) to assess co-localization patterns. Research has shown that PGAM5 predominantly co-localizes with TOMM20 during bleomycin-induced mitochondrial damage .

• Genetic approaches: For definitive isoform studies, researchers can employ isoform-specific siRNA knockdown or express tagged isoform-specific constructs.

When analyzing mitochondrial dynamics in relation to PGAM5 function, transmission electron microscopy (TEM) provides valuable structural insights that complement immunofluorescence approaches .

How does PGAM5 contribute to pulmonary fibrosis pathogenesis and what experimental models best demonstrate this relationship?

Research has identified PGAM5 as a key driver of mitochondrial dysfunction in experimental pulmonary fibrosis. In bleomycin-induced lung fibrosis models, PGAM5-deficient mice (Pgam5−/−) demonstrate significantly reduced fibrotic changes and architectural damage compared to wild-type controls . The experimental evidence suggests several mechanisms through which PGAM5 influences fibrosis progression:

• Mitochondrial dynamics regulation: PGAM5 affects mitochondrial morphology under stress conditions, with PGAM5 deficiency correlating with a more reticular organization of mitochondria during bleomycin treatment .

• Cell death modulation: A549 PGAM5-knockout cells show increased resilience to bleomycin-induced cytotoxicity, demonstrating the protein's role in cell death progression .

• Mitophagy pathway influence: PGAM5 facilitates PINK1 stabilization and subsequent initiation of mitophagic degradation, which becomes dysregulated during fibrotic processes .

For researchers studying PGAM5 in pulmonary fibrosis, recommended experimental approaches include:

• In vivo: Bleomycin-induced fibrosis in Pgam5−/− mice versus controls, with assessment via histopathology (Masson's trichrome staining), immunofluorescence for mitophagy markers (LC3B), and quantification of inflammatory mediators.

• In vitro: PGAM5-knockout epithelial cell lines (e.g., A549) treated with bleomycin, with assessment via real-time cell analysis, mitochondrial morphology assessment, and reconstitution experiments to confirm phenotype specificity .

These approaches provide complementary insights into PGAM5's multifaceted role in fibrotic disease progression.

What is the relationship between PGAM5 expression and cancer progression, particularly in hepatocellular carcinoma?

PGAM5 has emerged as a significant factor in cancer biology, with particularly well-documented roles in hepatocellular carcinoma (HCC). High PGAM5 expression in tumors correlates with poor clinical-pathological characteristics and prognosis in HCC patients . The relationship between PGAM5 and cancer progression involves several interconnected mechanisms:

• Immune microenvironment modulation: Tumor-intrinsic PGAM5 expression positively correlates with M2-phenotype tumor-associated macrophage (TAM) infiltration in both human HCC and mouse HCC tumor models .

• Mitochondrial dynamics regulation: In HCC cells, PGAM5 deficiency inhibits mitochondrial fission by promoting TRIM28 binding with DRP1, increasing its ubiquitination and degradation .

• Inflammatory signaling: PGAM5 deficiency reduces cytosolic mtDNA stress, attenuating TLR9 activation and downstream NF-κB-regulated CCL2 secretion, which impacts immune cell recruitment .

• Immunotherapy response: Disruption of tumor-intrinsic PGAM5 significantly facilitates CD8+ T cell activation and improves anti-PD-1 therapeutic efficacy .

For researchers investigating PGAM5 in cancer contexts, recommended experimental approaches include:

• Clinical correlation studies: Analyzing PGAM5 expression in tumor tissues in relation to immune cell infiltration patterns and patient outcomes.

• Mechanistic studies: Examining mitochondrial morphology, DRP1 regulation, and inflammatory signaling in PGAM5-manipulated cancer cell lines.

• Immunotherapy models: Testing combination approaches of PGAM5 targeting with immune checkpoint inhibitors in mouse models .

These findings suggest PGAM5 may represent a therapeutic target to enhance immunotherapy efficacy in HCC patients.

What controls and validation steps are essential when using PGAM5 antibodies in research applications?

Proper validation of PGAM5 antibodies is critical for experimental reliability. Essential controls and validation steps include:

• Positive controls: Use cell lines with confirmed PGAM5 expression such as A549, HeLa, HepG2, or MCF-7 cells . For tissue sections, human lung cancer, breast cancer, or liver cancer tissues provide reliable positive controls .

• Negative controls: Implement PGAM5 knockout cell lines (created via CRISPR/Cas9) as definitive negative controls. Alternatively, PGAM5 siRNA knockdown cells can serve as transient negative controls.

• Antibody specificity validation:

  • Western blot should detect a single band at approximately 32 kDa (observed molecular weight)

  • Peptide competition assays to confirm epitope specificity

  • Cross-validation with multiple antibodies targeting different PGAM5 epitopes

• Reconstitution experiments: To confirm phenotype specificity, perform reconstitution of PGAM5 in knockout systems. For example, transient expression of full-length PGAM5 in PGAM5-deficient cells should restore the wild-type phenotype, as demonstrated in bleomycin sensitivity experiments .

• Subcellular localization confirmation: Co-localization studies with established mitochondrial markers (e.g., TOMM20) to verify appropriate targeting .

For FITC-conjugated antibodies specifically, additional controls should include:

• Unstained samples to establish autofluorescence baselines

• Isotype controls conjugated to FITC to identify non-specific binding

• Photobleaching controls to determine signal stability over time

How can researchers effectively detect PGAM5-mediated mitochondrial dynamics changes using immunofluorescence techniques?

PGAM5 plays a significant role in regulating mitochondrial dynamics, particularly under stress conditions. Effective visualization and quantification of these changes require specialized immunofluorescence approaches:

• Co-staining protocols: Combine PGAM5 antibody (FITC-conjugated) with complementary mitochondrial markers:

  • TOMM20 (outer membrane)

  • Mitotracker dyes (membrane potential-dependent)

  • PINK1 (for mitophagy assessment)

  • LC3B (for autophagosome formation)

• Mitochondrial morphology assessment: Quantify parameters including:

  • Mitochondrial length and branching

  • Network connectivity

  • Perinuclear consolidation (a signature of stress-induced changes)

  • Reticular organization

• Advanced microscopy techniques:

  • Confocal microscopy with z-stack acquisition for three-dimensional analysis

  • Super-resolution microscopy for detailed mitochondrial substructure

  • Live-cell imaging for dynamic changes over time

• Quantitative analysis approaches:

  • Implement software-based morphometric analysis (e.g., MiNA plugin for ImageJ)

  • Assess polar perinuclear consolidation as a phenotypic marker of mitochondrial stress

  • Quantify co-localization coefficients between PGAM5 and other mitochondrial markers

Research has shown that bleomycin treatment induces a characteristic polar perinuclear consolidation of mitochondria (visualized via TOMM20) in PGAM5-proficient cells, which is significantly reduced in PGAM5-deficient cells . Additionally, PGAM5 deficiency correlates with a more pronounced reticular organization of mitochondria under stress conditions, which can be confirmed by both immunofluorescence and transmission electron microscopy .

How does PGAM5 integrate into mitochondrial quality control pathways, particularly in relation to PINK1-Parkin mediated mitophagy?

PGAM5 functions as a critical component in mitochondrial quality control pathways, with particular significance in the PINK1-Parkin mediated mitophagy system. Experimental evidence demonstrates several mechanistic interactions:

• PINK1 stabilization: PGAM5 influences PINK1 protein expression and localization patterns. Visualization of PINK1 in PGAM5-proficient cells after stress treatment (e.g., bleomycin) reveals focal clustering resulting in a granular staining pattern, which is markedly reduced in PGAM5-deficient cells .

• Mitophagy initiation: The spatial reorganization of PINK1 facilitated by PGAM5 reflects the initiation of mitophagic degradation. This has been observed both in vitro and in vivo, with LC3B clustering (indicating autophagy initiation) being reduced in bleomycin-challenged PGAM5-deficient systems compared to PGAM5-proficient counterparts .

• Mitochondrial dynamics interconnection: PGAM5 deficiency inhibits mitochondrial fission by promoting TRIM28 binding with DRP1, increasing DRP1 ubiquitination and degradation . This establishes a mechanistic link between PGAM5, mitochondrial morphology, and subsequent quality control pathways.

For researchers investigating these pathways, effective experimental approaches include:

• Dual immunofluorescence staining for PGAM5 and PINK1/Parkin

• Time-course analysis of mitochondrial depolarization using membrane potential-sensitive dyes

• Assessment of mitochondrial clearance using mt-Keima or similar pH-sensitive mitochondrial reporters

• Quantification of mitophagy-associated proteins in mitochondrial versus cytosolic fractions

These methodologies enable detailed dissection of PGAM5's role in coordinating mitochondrial quality control responses under various stress conditions.

What mechanisms explain the observed protective effects of PGAM5 deficiency in experimental disease models?

The protective effects of PGAM5 deficiency observed in multiple disease models can be attributed to several interconnected mechanisms:

• Altered mitochondrial dynamics: PGAM5 deficiency promotes mitochondrial fusion and prevents excessive fission under stress conditions. This maintains mitochondrial network integrity and function, as evidenced by the more reticular mitochondrial organization observed in PGAM5-knockout cells during bleomycin treatment .

• Reduced cell death susceptibility: A549 PGAM5-knockout cells display significantly reduced cytotoxicity when treated with bleomycin, as measured by real-time cell analysis. This protective phenotype can be reversed through reconstitution experiments expressing full-length PGAM5 .

• Dampened inflammatory signaling: In HCC models, tumor-intrinsic PGAM5 deficiency reduces cytosolic mtDNA stress, attenuating TLR9 activation and downstream NF-κB-regulated CCL2 secretion . This alters the recruitment and polarization of immune cells in the tumor microenvironment.

• Immune microenvironment remodeling: PGAM5 deficiency significantly reduces the infiltration of immunosuppressive M2-phenotype tumor-associated macrophages (TAMs) and promotes CD8+ T cell activation . This creates a more favorable immune landscape for anti-tumor responses.

• Enhanced therapeutic responses: Disruption of tumor-intrinsic PGAM5 improves the efficacy of immune checkpoint inhibitors, specifically anti-PD-1 therapy, suggesting potential combination strategies for cancer treatment .

For researchers investigating these protective mechanisms, comprehensive approaches should include:

• Mitochondrial function assessments (membrane potential, respiration, ROS production)

• Cell death pathway analysis (apoptosis, necroptosis, pyroptosis markers)

• Inflammatory cytokine profiling in cellular supernatants and tissue samples

• Immune cell phenotyping in tissue sections and tumor microenvironments

• Transcriptomic analysis of stress response pathways

Understanding these mechanisms provides opportunities for therapeutic targeting of PGAM5-dependent pathways in various disease contexts.

What are the current technical limitations when studying PGAM5 function and how might they be addressed?

Despite significant advances in PGAM5 research, several technical limitations persist:

• Antibody cross-reactivity concerns: Many commercial PGAM5 antibodies may have variable specificity across species or between isoforms. Researchers should:

  • Validate antibodies in knockout systems

  • Compare multiple antibodies targeting different epitopes

  • Use recombinant expression systems with epitope tags as complementary approaches

• Mitochondrial localization challenges: PGAM5's inner mitochondrial membrane localization can hinder accessibility in certain applications. Consider:

  • Optimized permeabilization protocols for immunofluorescence

  • Subcellular fractionation with high purity before biochemical assays

  • Electron microscopy for precise localization studies

• Dual function complexity: PGAM5 functions as both a phosphatase and a structural component, making it difficult to separate these roles. Approaches to address this include:

  • Phosphatase-dead mutants for functional studies

  • Domain-specific deletion constructs

  • Targeted inhibition of phosphatase activity versus protein-protein interactions

• Context-dependent effects: PGAM5's functions vary across cell types and stress conditions. Researchers should:

  • Perform comparative studies across multiple cell types

  • Test various stress inducers (oxidative, metabolic, inflammatory)

  • Consider microenvironmental factors in experimental design

Future methodological advances might include development of:

• Isoform-specific antibodies with improved validation

• Small molecule inhibitors with selectivity for PGAM5

• Improved live-cell imaging tools for mitochondrial dynamics

• Tissue-specific conditional knockout models for in vivo studies

How might targeting PGAM5 translate into therapeutic applications, and what experimental approaches would best validate these strategies?

Based on the roles of PGAM5 in disease processes, several therapeutic approaches show promise:

• Small molecule inhibitors of PGAM5 phosphatase activity: Could potentially mitigate damage in fibrotic disorders or enhance cancer immunotherapy. Validation approaches include:

  • In vitro phosphatase activity assays with candidate compounds

  • Cell-based phenotypic screens in disease models

  • Structure-based drug design targeting the catalytic domain

• Disruption of PGAM5-protein interactions: Targeting specific protein-protein interactions rather than catalytic activity may provide more selective effects. Experimental validation requires:

  • Identification of critical interaction interfaces through mutation studies

  • Development of peptide mimetics or small molecules targeting these interfaces

  • Confirmation of specificity using co-immunoprecipitation or proximity ligation assays

• Combination with existing therapies: PGAM5 targeting may enhance current treatments, particularly in cancer. Evidence suggests disruption of tumor-intrinsic PGAM5 significantly improves anti-PD-1 therapeutic efficacy . Validation approaches include:

  • Combination therapy testing in preclinical models

  • Analysis of treatment response biomarkers

  • Mechanistic studies of synergistic effects

• Gene therapy approaches: For genetic delivery of PGAM5 modulators in specific tissues. Validation would require:

  • Development of tissue-specific delivery systems

  • Optimization of expression constructs (knockdown, dominant negative)

  • Safety and efficacy testing in relevant disease models

The most promising near-term application appears to be in cancer immunotherapy, where PGAM5 inhibition could potentially overcome resistance to immune checkpoint inhibitors by reshaping the tumor microenvironment . Researchers should focus on developing specific inhibitors and validating their effects on immune cell recruitment and activation in cancer models.