PGAM5 Antibody, HRP conjugated

What is PGAM5 and what are its primary cellular functions?

PGAM5 is a mitochondrial serine/threonine phosphatase with a molecular weight of approximately 32 kDa. Despite its name suggesting phosphoglycerate mutase activity, it primarily functions as a phosphatase that dephosphorylates various substrates involved in critical cellular processes. PGAM5 plays multifaceted roles in several biological pathways:

• Mitochondrial dynamics regulation through dephosphorylation of DNM1L/DRP1

• Protection of MFN2 from ubiquitination and degradation to promote mitochondrial network formation

• Regulation of mitophagy by dephosphorylating FUNDC1

• Modulation of anti-oxidative response through forming a tertiary complex with KEAP1 and NRF2

• Involvement in necroptosis by recruiting the RIPK1-RIPK3-MLKL complex to mitochondria

These diverse functions position PGAM5 at the intersection of multiple cellular pathways including apoptosis, necroptosis, and mitochondrial quality control.

What applications are commonly supported by PGAM5 antibodies?

PGAM5 antibodies support multiple experimental applications depending on their formulation and validation. Based on comprehensive analysis of commercially available options, the following applications are most commonly supported:

HRP-conjugated versions specifically eliminate the need for secondary antibody incubation in Western blotting and ELISA applications, streamlining experimental workflows .

How should researchers determine optimal sample preparation for PGAM5 detection?

Sample preparation is critical for successful PGAM5 detection due to its mitochondrial localization and relatively low abundance in some tissues. Consider these methodological approaches:

• For total cell lysates: Use RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors

• For enriched mitochondrial fractions: Employ differential centrifugation techniques to isolate intact mitochondria

• For fixed tissue samples in IHC:

  • Most products recommend antigen retrieval with TE buffer pH 9.0

  • Alternative antigen retrieval may be performed with citrate buffer pH 6.0

• For storage conditions: Keep samples at -20°C or -80°C in buffer containing 50% glycerol to prevent repeated freeze-thaw cycles

Validation studies indicate that PGAM5 detection is consistently successful in various human cell lines including A549, HeLa, HepG2, and MCF-7 cells, making these appropriate positive controls .

How does PGAM5 function in mitochondrial dynamics and related pathologies?

PGAM5 serves as a critical regulator of mitochondrial dynamics through its interaction with key fission and fusion proteins. Recent studies have demonstrated that PGAM5:

• Dephosphorylates DNM1L/DRP1 at Ser-637, which activates DRP1's GTPase activity and promotes mitochondrial fission

• Forms a complex with DRP1 that is resistant to ubiquitination, thus stabilizing DRP1 protein levels

• Dephosphorylates MFN2 in a stress-sensitive manner, protecting it from degradation and promoting mitochondrial network formation

These interactions place PGAM5 at the center of mitochondrial quality control mechanisms. Experimental approaches to study these interactions include:

• Co-immunoprecipitation assays using PGAM5 antibodies to pull down interacting partners

• Phosphorylation-specific Western blotting to monitor DRP1 Ser-637 phosphorylation status

• Confocal microscopy with PGAM5 antibodies to visualize mitochondrial morphology changes

Dysfunction in this pathway has been implicated in neurodegenerative conditions, particularly in Parkinson's disease models where PGAM5 stabilizes PINK1, a key protein in mitochondrial quality control .

How does PGAM5 contribute to tumor microenvironment regulation and immunotherapy response?

Recent findings have revealed a previously unrecognized role for PGAM5 in cancer immunology. Tumor-intrinsic PGAM5 significantly influences the tumor microenvironment, particularly through macrophage polarization:

• High PGAM5 expression correlates with poor prognosis in hepatocellular carcinoma (HCC) patients

• PGAM5 promotes tumor-associated macrophage (TAM) M2 polarization through CCL2 signaling

• Mechanistically, PGAM5 acts as a protein stabilizer of DRP1, which facilitates TAM M2 polarization

• Disruption of tumor-intrinsic PGAM5 enhances anti-PD-1 immunotherapy efficacy in mouse HCC models

These findings suggest that targeting PGAM5 could potentially improve immunotherapy outcomes. Experimental approaches to investigate this include:

• Flow cytometry with PGAM5 antibodies to analyze macrophage populations

• Multiplex cytokine assays to measure CCL2 and other inflammatory mediators

• Orthotopic and subcutaneous tumor models with PGAM5 knockdown combined with immune checkpoint inhibitors

What is the relationship between PGAM5 and PINK1 in neurodegenerative disease models?

PGAM5 has emerged as a potential link between mitochondrial homeostasis and Parkinson's disease pathogenesis through its interaction with PINK1:

• PGAM5 stabilizes wild-type PINK1 by increasing full-length PINK1 (~63kD) and decreasing the PARL-cleaved form (~54kD)

• This stabilization appears to be dependent on direct association between PGAM5 and PINK1

• The "di-RH" motif (amino acids 98-110) of PGAM5 is critical for this interaction

• Parkinson's disease-associated PINK1 mutants are generally resistant to PGAM5 stabilization

To effectively study this interaction, researchers can employ:

• Co-immunoprecipitation with PGAM5 antibodies to analyze PINK1 binding

• Western blotting to monitor PINK1 cleavage patterns in the presence/absence of PGAM5

• Site-directed mutagenesis of the di-RH motif to assess functional consequences

This PGAM5-PINK1 relationship provides a molecular link for studying mitochondrial homeostasis and movement disorders resembling Parkinson's disease.

What controls are essential for validating PGAM5 antibody specificity?

Establishing antibody specificity is crucial for meaningful PGAM5 research. Comprehensive validation should include:

• Positive controls:

  • Cell lines with confirmed PGAM5 expression (A549, HeLa, HepG2, MCF-7)

  • Tissue samples with known PGAM5 expression (human lung, liver, breast cancer tissues)

• Negative controls:

  • PGAM5 knockout cell lines (HEK293T PGAM5 KO cells have been used successfully)

  • siRNA or shRNA-mediated PGAM5 knockdown cells

  • Secondary antibody-only controls to assess non-specific binding

• Peptide competition assays:

  • Pre-incubation of antibody with immunizing peptide should abolish specific signal

A representative validation experiment demonstrated PGAM5 antibody specificity using wild-type and PGAM5 knockout 293T cell extracts (30 μg) separated by 12% SDS-PAGE. The membrane was probed with PGAM5 antibody diluted at 1:1000, followed by HRP-conjugated anti-rabbit IgG detection .

How should researchers optimize Western blot protocols for PGAM5 detection?

Optimal Western blot protocols for PGAM5 detection require attention to several key parameters:

• Sample preparation:

  • Include phosphatase inhibitors to preserve phosphorylation status

  • Use fresh samples when possible, or store properly at -80°C

  • Consider mitochondrial enrichment for low-expressing samples

• Gel electrophoresis:

  • 12% SDS-PAGE gels provide optimal resolution for the 32 kDa PGAM5 protein

  • Load 20-30 μg of total protein for most cell lines

• Transfer and blocking:

  • PVDF membranes generally provide better results than nitrocellulose

  • Block with 5% non-fat milk in TBST or PBST

• Antibody incubation:

  • Primary antibody dilutions range from 1:2000-1:14000 for Western blotting

  • For HRP-conjugated antibodies, eliminate secondary antibody step

  • Develop signal with enhanced chemiluminescence (ECL) substrate

When troubleshooting multiple bands, consider that PGAM5 can exist in different forms due to post-translational modifications and alternative splicing. The main band should be detected at approximately 32 kDa.

What methodological considerations are important for immunohistochemistry with PGAM5 antibodies?

Successful immunohistochemistry (IHC) with PGAM5 antibodies requires optimization of several experimental parameters:

• Tissue fixation and processing:

  • Formalin-fixed paraffin-embedded (FFPE) sections are commonly used

  • Section thickness of 4-6 μm is recommended

• Antigen retrieval:

  • Heat-induced epitope retrieval with TE buffer pH 9.0 is most effective

  • Alternative: citrate buffer pH 6.0 with slightly reduced sensitivity

• Antibody dilution and incubation:

  • Recommended dilutions range from 1:1000-1:4000

  • Overnight incubation at 4°C typically yields optimal results

• Signal detection:

  • For chromogenic detection, DAB substrate works well with HRP-conjugated antibodies

  • For fluorescent detection, appropriate fluorophore-conjugated secondary antibodies

PGAM5 expression has been successfully detected in human lung cancer tissue, human breast cancer tissue, and human liver cancer tissue, making these appropriate positive controls for protocol optimization.

How should researchers interpret PGAM5 expression patterns in pathological samples?

Interpretation of PGAM5 expression patterns in pathological samples requires consideration of both technical and biological factors:

• Normal expression patterns:

  • PGAM5 is primarily localized to mitochondria

  • In healthy tissues, moderate expression is observed in metabolically active tissues

• Pathological alterations:

  • Increased PGAM5 expression has been observed in hepatocellular carcinoma and correlates with poor prognosis

  • In neurodegenerative conditions, alterations in PGAM5 expression or localization may be observed

• Interpretative considerations:

  • Mitochondrial localization should be confirmed through co-staining with mitochondrial markers

  • Both intensity and pattern of staining should be considered

  • Quantification methods should be standardized across samples

Recent research demonstrates that high PGAM5 expression in HCC predicts poor prognosis and increased chemoresistance by inhibiting apoptosis. This suggests PGAM5 may serve as a prognostic marker in certain cancer types .

What strategies can address weak or absent PGAM5 signal in Western blotting?

When encountering weak or absent PGAM5 signal in Western blotting, consider these troubleshooting approaches:

• Sample-related issues:

  • Ensure adequate protein loading (30 μg recommended)

  • Verify sample integrity through detection of housekeeping proteins

  • Consider mitochondrial enrichment for low-expressing samples

• Technical optimization:

  • Adjust antibody concentration (try higher concentrations for weak signals)

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

  • Use high-sensitivity ECL reagents for detection

  • For HRP-conjugated antibodies, ensure the conjugate is active (avoid repeated freeze-thaw)

• Protocol modifications:

  • Try alternative blocking agents (BSA instead of milk)

  • Increase washing stringency to reduce background

  • Optimize transfer conditions for proteins in the 30-35 kDa range

If PGAM5 remains undetectable despite RNA expression confirmation, consider post-transcriptional regulation mechanisms or protein instability in your experimental system.

How can researchers optimize co-immunoprecipitation protocols for studying PGAM5 interactions?

Co-immunoprecipitation (Co-IP) is valuable for investigating PGAM5 protein interactions, particularly with partners like DRP1, PINK1, and components of the KEAP1-NRF2 complex:

• Lysis buffer selection:

  • Use mild non-denaturing buffers (e.g., NP-40 or Triton X-100 based)

  • Include protease and phosphatase inhibitors

  • Consider crosslinking for transient interactions

• Antibody considerations:

  • Confirm the antibody is validated for immunoprecipitation

  • Use 2-5 μg antibody per 500 μg protein lysate

  • Pre-clear lysates to reduce non-specific binding

• Washing conditions:

  • Optimize wash stringency to maintain specific interactions

  • Consider including low concentrations of detergent in wash buffers

• Controls:

  • Include IgG control immunoprecipitations

  • Compare results with and without treatment conditions (e.g., CCCP treatment can affect PGAM5-PINK1 interactions)

Published studies have successfully demonstrated PGAM5-DRP1 interactions through co-immunoprecipitation, confirming that these proteins mutually co-precipitate in HCC cells. This technique revealed that PGAM5 regulates DRP1 at the post-transcriptional level through modulation of ubiquitination .

How can PGAM5 antibodies be utilized to study mitochondrial dynamics in live cells?

While traditional fixed-cell immunofluorescence provides valuable information, studying mitochondrial dynamics in live cells offers unique insights into PGAM5 function:

• Combined approaches:

  • Use fluorescently tagged PGAM5 constructs for live imaging

  • Validate localization patterns with fixed-cell immunostaining using PGAM5 antibodies

  • Correlative live/fixed imaging to connect dynamic events with molecular markers

• Mitochondrial morphology analysis:

  • Quantify parameters such as mitochondrial length, branching, and connectivity

  • Measure fusion/fission events following manipulation of PGAM5 levels

  • Assess colocalization with other mitochondrial dynamic proteins (DRP1, MFN1/2)

• Functional assays:

  • Monitor mitochondrial membrane potential in conjunction with PGAM5 manipulation

  • Assess mitophagy rates in response to PGAM5 overexpression or knockdown

  • Evaluate mitochondrial ROS production in relation to PGAM5 activity

Recent studies have demonstrated that PGAM5-mediated dephosphorylation of DRP1 at Ser-637 is a key regulatory event in mitochondrial fission, highlighting the importance of studying these dynamics in live experimental systems.

What experimental approaches can investigate PGAM5's role in inflammation and macrophage polarization?

PGAM5's emerging role in inflammation and immune cell function can be investigated through several methodological approaches:

• Macrophage polarization assays:

  • Flow cytometry to quantify M1/M2 marker expression in macrophages

  • qRT-PCR to assess expression of polarization-associated genes

  • Cytokine profiling of macrophage secretome after PGAM5 manipulation

• Mechanistic studies:

  • Assessment of mitochondrial dynamics in macrophages using PGAM5 antibodies

  • Analysis of PGAM5-DRP1 interaction in different macrophage activation states

  • Investigation of mtROS production in relation to inflammatory signaling

• In vivo models:

  • Tumor models with PGAM5 manipulation to assess macrophage infiltration

  • Inflammatory disease models to evaluate PGAM5's role in pathogenesis

  • Combination with immunotherapy approaches to determine clinical relevance

Recent research has demonstrated that PGAM5 activity regulates the dephosphorylation of DRP1 in macrophages, leading to induction of proinflammatory responses. Upon LPS stimulation, PGAM5 interacts with DRP1 to form a complex that promotes mtROS production and polarization toward a proinflammatory phenotype .

How can PGAM5 antibodies contribute to drug discovery research for neurodegenerative diseases?

PGAM5 antibodies offer valuable tools for drug discovery research targeting neurodegenerative conditions, particularly Parkinson's disease:

• Target validation:

  • Confirm PGAM5 expression and localization in disease-relevant tissues

  • Evaluate PGAM5-PINK1 interactions in patient-derived samples

  • Assess correlation between PGAM5 function and disease progression

• High-throughput screening:

  • Develop ELISA-based assays using PGAM5 antibodies to screen compound libraries

  • Establish cell-based assays monitoring PGAM5 phosphatase activity

  • Create reporter systems for PGAM5-dependent signaling pathways

• Mechanism-of-action studies:

  • Determine how candidate compounds affect PGAM5-PINK1 stabilization

  • Assess impact on mitochondrial morphology and function

  • Evaluate effects on PGAM5-dependent phosphorylation of substrates

The genetic deficiency of PGAM5 causes a movement disorder similar to Parkinson's disease, suggesting that PGAM5 may provide a molecular link to study mitochondrial homeostasis and disease pathogenesis. This connection positions PGAM5 as a promising target for therapeutic intervention in certain neurodegenerative conditions .