PGM1 Antibody

What is PGM1 and why is it significant in research?

PGM1 (Phosphoglucomutase-1) belongs to the phosphohexose mutase family and catalyzes the reversible transfer of phosphate between the 1 and 6 positions of glucose. This enzyme plays a critical role in carbohydrate metabolism, specifically in glycogen catabolism and synthesis . In most cell types, PGM1 isozymes predominate, representing approximately 90% of total PGM activity, with red blood cells being a notable exception where PGM2 is a major isozyme .

PGM1's significance in research stems from its central role in glucose trafficking between glycogen synthesis and glycolysis pathways. Recent studies have revealed its unexpected role in cancer biology, particularly as a potential tumor suppressor in hepatocellular carcinoma . Additionally, defects in PGM1 are associated with glycogen storage disease type 14 (GSD14), making it an important target for metabolic disorder research .

What applications are PGM1 antibodies validated for?

PGM1 antibodies have been validated for multiple research applications with specific recommended dilutions:

These antibodies have been successfully employed in multiple published studies focusing on hepatocellular carcinoma, metabolic disorders, and basic PGM1 biology .

How do I determine the optimal dilution for PGM1 antibody in my experiment?

For optimal results with PGM1 antibody, a titration experiment is recommended. Begin with the manufacturer's suggested dilution range (see table in question 1.2) and test at least 3-4 different concentrations. For Western blot, consider a dilution series (e.g., 1:2000, 1:5000, 1:10000, 1:16000) using positive control samples from tissues known to express PGM1, such as liver, skin, or heart tissues .

For immunohistochemistry applications, additional considerations include proper antigen retrieval methods. Published protocols suggest using TE buffer pH 9.0, although citrate buffer pH 6.0 has also been used successfully . Always include appropriate positive controls (e.g., human liver tissue) and negative controls (omission of primary antibody) to validate staining specificity.

Remember that optimal dilution can be sample-dependent. For example, PGM1 expression levels vary across tissues and disease states, as demonstrated in hepatocellular carcinoma studies where significant differences were observed between tumor and peritumoral tissues .

How should I validate the specificity of PGM1 antibody for my experiments?

Validating PGM1 antibody specificity requires a multi-approach strategy:

• Protein blocking assay: Perform pre-absorption with recombinant PGM1 protein to confirm specific binding. This approach was used by researchers studying PGM1 in hepatocellular carcinoma to validate their antibody .

• Genetic validation: Use cells with PGM1 knockdown (via shRNA) or knockout as negative controls. Compare these to wild-type cells or those with rescued PGM1 expression. Research has shown that PGM1 depletion using shRNA significantly reduced antibody signal, confirming specificity .

• Multiple antibody validation: When possible, compare results using antibodies from different sources or those targeting different epitopes of PGM1.

• Enzymatic activity correlation: Verify that PGM1 enzymatic activity correlates with protein levels detected by the antibody. Studies have demonstrated a positive correlation between PGM1 protein levels detected by antibodies and enzymatic activity measurements in primary HCC tumors and cell lines .

What controls should I include when using PGM1 antibody for immunohistochemistry?

For robust immunohistochemistry experiments with PGM1 antibody:

• Positive tissue controls: Include tissues known to express PGM1, such as normal liver tissue, heart tissue, or skin tissue .

• Negative controls:

  • Omission of primary antibody

  • Isotype-matched control antibody

  • Pre-absorption with recombinant PGM1 protein

• Internal controls: When studying tissues with variable PGM1 expression (e.g., tumor vs. adjacent normal tissue), use the non-altered tissue as an internal control. In HCC studies, peritumoral tissues served as excellent internal controls, showing consistently higher PGM1 expression compared to tumor tissues .

• Gradient of expression: Include samples with known differential expression of PGM1. For example, in HCC research, samples with varying degrees of differentiation showed corresponding differences in PGM1 expression (moderate to poor differentiation had lower expression compared to well-differentiated tumors) .

What are the key methodological considerations for PGM1 antibody in Western blot applications?

When performing Western blot with PGM1 antibody, consider these critical factors:

• Expected molecular weight: PGM1 has a calculated molecular weight of 61 kDa, which generally corresponds to its observed molecular weight in most tissues and cell lines .

• Sample preparation:

  • For tissue samples: Homogenization in RIPA buffer with protease inhibitors is recommended

  • For cell lines: Lysis in buffer containing phosphatase inhibitors may be important as PGM1 can be post-translationally modified

• Loading controls: Use appropriate housekeeping proteins (β-actin, GAPDH) and also consider metabolic enzymes in the same pathway for pathway-specific normalization.

• Detergent selection: PGM1 is predominantly cytoplasmic, so standard detergents like Triton X-100 or NP-40 are sufficient for extraction.

• Antibody incubation: Overnight incubation at 4°C with PGM1 antibody at 1:2000-1:16000 dilution typically yields optimal results for detecting endogenous protein levels .

Remember that PGM1 has two isoforms produced by alternative splicing, which may appear as closely migrating bands in some tissues .

How can PGM1 antibody be used to investigate metabolic reprogramming in cancer?

PGM1 antibody is a valuable tool for investigating metabolic reprogramming in cancer, particularly changes in glucose utilization pathways. Research has demonstrated that PGM1 regulates the balance between glycolysis and glycogen synthesis in cancer cells :

• Metabolic flux analysis: Use PGM1 antibody in combination with metabolic measurements to correlate protein expression with:

  • Glucose consumption rates

  • Lactate production

  • Glycogen content

  • G-1-P/G-6-P ratio

• Multi-parameter analysis: Combine PGM1 immunostaining with:

  • Other glycolytic enzyme markers (HK2, PKM2, LDHA)

  • Glycogen synthesis pathway components (GYS1, GBE1)

  • Proliferation markers (Ki-67, PCNA)

• Patient stratification: PGM1 expression levels can be used to stratify cancer patients for metabolic phenotyping. In HCC studies, patients with low PGM1 expression exhibited distinct metabolic characteristics and poorer clinical outcomes .

Research has shown that PGM1 overexpression significantly inhibits glucose consumption and lactate production while increasing glycogen content in cancer cells. Conversely, PGM1 depletion enhances glucose consumption and lactate production while decreasing glycogen content . These findings suggest PGM1 acts as a metabolic switch between glycolysis and glycogen synthesis.

How can I investigate the relationship between PGM1 enzymatic activity and its protein levels?

Investigating the correlation between PGM1 protein levels (detected by antibody) and enzymatic activity requires a multi-faceted approach:

• Enzymatic activity assay: Measure PGM1 activity using a coupled enzyme assay that tracks the conversion of G-1-P to G-6-P by monitoring NADPH production. This can be performed in cell or tissue lysates where PGM1 protein levels are simultaneously measured by Western blot.

• Structure-function analysis: Use PGM1 antibody to detect wild-type PGM1 and enzymatically inactive mutants (e.g., PGM1 G121R, which has significantly reduced activity). Research has demonstrated that while the antibody detects both proteins, only wild-type PGM1 demonstrates tumor-suppressive functions .

• Correlation studies: Analyze the relationship between PGM1 protein levels and enzymatic activity across multiple samples. Studies in HCC have shown positive correlation between PGM1 protein levels and enzymatic activity in both primary tumors and cell lines .

• Rescue experiments: Use PGM1 antibody to confirm expression in genetic rescue experiments comparing wild-type and enzymatically inactive mutants. Research has shown that only wild-type PGM1, not the inactive G121R mutant, could rescue the phenotypes caused by PGM1 depletion .

How does PGM1 expression correlate with clinical features in hepatocellular carcinoma?

Immunohistochemical analysis using PGM1 antibody has revealed significant correlations between PGM1 expression and clinical features in hepatocellular carcinoma:

What are common issues when using PGM1 antibody in immunohistochemistry and how can they be resolved?

Common challenges when using PGM1 antibody for IHC include:

• Weak or absent staining:

  • Solution: Optimize antigen retrieval. For PGM1, TE buffer pH 9.0 is recommended, although citrate buffer pH 6.0 is an alternative .

  • Approach: Increase antibody concentration or extend incubation time. For PGM1, dilutions between 1:400-1:1600 have been validated .

• High background:

  • Solution: Increase blocking time or use a different blocking reagent.

  • Approach: Dilute the antibody further or reduce incubation time.

• Non-specific staining:

  • Solution: Validate antibody specificity using protein blocking assays with recombinant PGM1, as demonstrated in HCC research .

  • Approach: Use tissue from PGM1-depleted models as negative controls.

• Variable staining intensity across samples:

  • Solution: Standardize fixation time and processing.

  • Approach: Include internal control tissues on each slide.

• Limited detection of PGM1 in certain tumor samples:

  • Solution: This may reflect genuine low expression, as seen in HCC tissues. Confirm with alternate detection methods like Western blot.

  • Approach: Use signal amplification methods if necessary, but validate that results correlate with other detection methods.

How can I optimize PGM1 antibody use for co-immunoprecipitation experiments?

For successful co-immunoprecipitation (co-IP) experiments with PGM1 antibody:

• Antibody selection: Use antibodies validated for IP applications. For example, the Proteintech antibody (15161-1-AP) has been validated for immunoprecipitating PGM1 from mouse skin tissue .

• Lysis conditions:

  • Use gentle non-ionic detergents (0.5% NP-40 or 1% Triton X-100)

  • Include phosphatase inhibitors, as PGM1 function may be regulated by phosphorylation

  • Maintain physiological salt concentration (150 mM NaCl)

• Antibody amount optimization:

  • Start with 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

  • Perform titration experiments to determine optimal antibody:protein ratio

• Pre-clearing lysates:

  • Pre-clear with appropriate control IgG and protein A/G beads to reduce non-specific binding

  • This is particularly important when studying PGM1 in tissues with high protein content

• Controls:

  • IgG control: Use matched isotype control antibody

  • Input control: Save a fraction of pre-IP lysate

  • Validation control: If possible, use lysates from PGM1-depleted cells

• Elution conditions:

  • For denaturing conditions: SDS sample buffer at 95°C

  • For native conditions: Consider competitive elution with PGM1 peptide

What approaches can I use to study PGM1 localization in different subcellular compartments?

While PGM1 is primarily cytoplasmic, studying its precise subcellular localization can provide insights into its regulation and function:

• Immunofluorescence optimization:

  • Use PGM1 antibody at 1:20-1:200 dilution for IF/ICC applications

  • Validated in cell lines such as HepG2

  • Consider paraformaldehyde fixation (4%, 10-15 minutes) followed by permeabilization with 0.1-0.5% Triton X-100

• Co-localization studies:

  • Combine PGM1 antibody with markers for:

    • Glycogen particles (glycogen synthase)

    • Glycolytic enzyme complexes (GAPDH, PKM2)

    • Cytoskeletal elements (potential regulatory interactions)

• Subcellular fractionation:

  • Separate cytosolic, membrane, nuclear, and cytoskeletal fractions

  • Use PGM1 antibody in Western blot to detect distribution across fractions

  • Include appropriate fraction markers (e.g., GAPDH for cytosol, histone H3 for nucleus)

• Activity-dependent localization:

  • Compare PGM1 localization under conditions that alter glucose metabolism:

    • Glucose deprivation/stimulation

    • Hypoxia (which alters glycolytic flux)

    • Glycogen depletion/accumulation conditions

• Super-resolution microscopy:

  • For detailed localization studies, consider:

    • STED microscopy

    • STORM/PALM approaches

    • Use highly specific PGM1 antibody conjugated to appropriate fluorophores

How can PGM1 antibody be used to investigate the role of metabolic enzymes in cancer progression?

PGM1 antibody offers powerful approaches to investigate metabolic enzymes in cancer:

• Tumor microenvironment studies:

  • Use multiplexed immunofluorescence combining PGM1 antibody with:

    • Markers of tumor zones (hypoxia, proliferation zones)

    • Immune cell infiltration markers

    • Other metabolic enzymes

• Patient-derived models:

  • Apply PGM1 antibody for stratification of patient-derived xenografts (PDX)

  • Research has demonstrated that PDX tumors with low PGM1 expression (n = 12) showed more rapid progression than those with high expression (n = 5)

  • Use this stratification to test metabolism-targeting therapeutics

• Therapy resistance mechanisms:

  • Investigate PGM1 expression changes in:

    • Chemotherapy-resistant tumors

    • Radiation-resistant populations

    • Immunotherapy non-responders

• Metabolic dependencies:

  • Combine PGM1 antibody staining with functional metabolic assays

  • Correlate PGM1 levels with sensitivity to glycolysis inhibitors or other metabolic interventions

• Cancer stem cell populations:

  • Examine PGM1 expression in cancer stem cell populations versus differentiated tumor cells

  • Investigate whether metabolic phenotypes correlate with stemness and treatment resistance

How does PGM1 enzymatic activity correlate with its tumor-suppressive function?

Research using PGM1 antibody has revealed critical insights into the relationship between PGM1's enzymatic activity and its tumor-suppressive function:

This evidence strongly suggests that PGM1's tumor-suppressive function is directly linked to its enzymatic activity in regulating glucose metabolism, specifically by directing glucose away from glycolysis and toward glycogen synthesis.

How can genetic and pharmacological modulation of PGM1 be validated using PGM1 antibody?

PGM1 antibody is essential for validating genetic and pharmacological modulations of PGM1:

• Genetic modulation validation:

  • Knockdown verification: Use PGM1 antibody to confirm protein reduction after shRNA treatment. Studies have used Western blot to verify >80% reduction in PGM1 protein levels following shRNA treatment .

  • Overexpression confirmation: Validate Flag-PGM1 expression in stable cell lines using both anti-Flag and anti-PGM1 antibodies to ensure proper protein expression and expected molecular weight (61 kDa) .

  • Rescue experiment validation: When performing rescue experiments with shRNA-resistant PGM1 constructs (wild-type or mutant), use PGM1 antibody to confirm comparable expression levels across experimental groups .

• Pharmacological modulation assessment:

  • Inhibitor specificity: When testing potential PGM1 inhibitors, use PGM1 antibody to:

    • Confirm target engagement via thermal shift assays

    • Assess whether inhibitors affect protein stability or expression levels

    • Determine potential conformational changes via limited proteolysis followed by Western blot

  • Indirect modulators: For compounds that affect PGM1 expression rather than activity directly, use PGM1 antibody to quantify dose-dependent and time-dependent changes in protein levels

• Combined approaches:

  • Correlate antibody-detected PGM1 protein levels with enzymatic activity measurements following various interventions

  • Use immunofluorescence to assess potential changes in subcellular localization upon modulation

  • Apply PGM1 antibody in xenograft models to confirm maintained genetic modulation in vivo

These validation approaches are critical for establishing the specificity of both genetic tools and potential pharmacological agents targeting PGM1 in research and therapeutic development.