PGK1 Antibody

What is PGK1 and what are its primary cellular functions?

Phosphoglycerate Kinase 1 (PGK1), also known as PGKA, is a glycolytic enzyme encoded by the PGK1 gene. It catalyzes the conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate during glycolysis, generating one molecule of ATP in the process . Beyond its canonical glycolytic role, PGK1 exhibits moonlighting functions: it may act as a cofactor for polymerase alpha and is secreted by tumor cells where it participates in angiogenesis by reducing disulfide bonds in plasmin, leading to the release of angiostatin (a tumor blood vessel inhibitor) . PGK1 deficiency is associated with a spectrum of clinical phenotypes including hemolytic anemia and neurological impairment . The protein has a calculated molecular weight of 45 kDa and is typically observed at 40-45 kDa on Western blots .

What is the reactivity spectrum of commercially available PGK1 antibodies?

PGK1 antibodies demonstrate cross-reactivity with multiple species. According to the search results, most commercially available PGK1 antibodies show confirmed reactivity with:

When selecting a PGK1 antibody for your research, verify that it has been validated for your species of interest. Some antibodies may have broader cross-reactivity than others due to the high conservation of PGK1 across species .

What are the validated applications for PGK1 antibodies?

PGK1 antibodies have been validated for multiple research applications. The table below summarizes the applications supported by different commercial antibodies:

Most antibodies have been extensively tested for Western blotting, while applications like flow cytometry and PLA have more limited validation .

What is the recommended storage protocol for PGK1 antibodies?

Proper storage is critical for maintaining antibody activity. For PGK1 antibodies, the following storage conditions are recommended:

• Store at -20°C for long-term storage

• After reconstitution, can be stored at 4°C for up to one month

• For longer storage periods, aliquot and store at -20°C to avoid repeated freeze-thaw cycles

• Most PGK1 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Some smaller volume preparations (20μl) may contain 0.1% BSA

Aliquoting the antibody upon receipt is strongly recommended to prevent degradation from multiple freeze-thaw cycles, which can significantly reduce antibody activity and specificity .

What are the optimal dilution ratios for different PGK1 antibody applications?

Optimal dilution varies significantly by application type, antibody clone, and sample type. Based on validation data from manufacturers, the following dilution ranges are recommended:

These dilutions should be considered starting points for optimization. Researchers should conduct titration experiments to determine the optimal dilution for their specific experimental conditions and samples .

How can one validate PGK1 antibody specificity in experimental systems?

Validation of antibody specificity is critical for reliable experimental results. For PGK1 antibodies, consider these methodological approaches:

• Positive and negative control samples: Use cells/tissues known to express PGK1 (e.g., HeLa, A431, HepG2 cells) as positive controls. For negative controls, consider using PGK1 knockdown/knockout samples .

• Transfection controls: Compare non-transfected and PGK1-transfected 293T cells to confirm specificity, as demonstrated in validation data from manufacturers .

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight of 40-45 kDa .

• Physiological validation: Test antibody response under conditions known to affect PGK1, such as hypoxia (1% O2 treatment has been shown to affect PGK1 levels) .

• Multiple antibody comparison: Use antibodies from different sources or those targeting different epitopes of PGK1 to confirm consistent detection patterns.

• Subcellular localization: Verify that immunofluorescence staining shows the expected cytoplasmic and nuclear pattern consistent with PGK1's known localization .

These approaches collectively provide strong evidence for antibody specificity when consistent results are observed across multiple validation strategies.

What are the key considerations for using PGK1 antibodies in co-immunoprecipitation (Co-IP) experiments?

Co-immunoprecipitation with PGK1 antibodies requires careful optimization:

• Antibody selection: Choose antibodies validated specifically for IP/Co-IP applications. Not all PGK1 antibodies work effectively for precipitation, with polyclonal antibodies generally performing better than monoclonals for this application .

• Antibody amount: Use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate as a starting point . The optimal ratio may vary based on expression levels and experimental conditions.

• Cell/tissue selection: Several cell lines have been validated for successful PGK1 IP, including SW 1990 and HeLa cells . Start with these if possible.

• Lysis conditions: Use lysis buffers that preserve protein-protein interactions while effectively extracting PGK1. Avoid harsh detergents that may disrupt interactions of interest.

• Controls: Include appropriate controls:

  • IgG isotype control to identify non-specific binding

  • Input sample (pre-IP lysate) to confirm target protein presence

  • Reverse Co-IP (precipitate with antibody against suspected interacting protein, blot for PGK1)

• Washing stringency: Optimize wash conditions to remove non-specific binding while preserving genuine interactions.

Success in Co-IP experiments is highly dependent on the specific antibody clone and experimental conditions, so preliminary optimization is essential.

How can PGK1 antibodies be used to study hypoxia responses in cancer cells?

PGK1 is known to be upregulated under hypoxic conditions due to its role in glycolysis. Methodological approaches for studying this relationship include:

• Hypoxia treatment protocols: Exposing cells to 1% O2 treatment for 24 hours has been shown to effectively modulate PGK1 expression . Western blot analysis using PGK1 antibodies can then detect changes in expression levels.

• Comparative analysis: Western blot samples should be run with both normoxic (control) and hypoxic treatment conditions, using appropriate loading controls such as beta-actin .

• Subcellular localization changes: Immunofluorescence staining using PGK1 antibodies can detect potential changes in PGK1 localization (cytoplasmic vs. nuclear) under hypoxic conditions .

• Correlation with HIF-1α: Co-staining or parallel blotting for hypoxia-inducible factor 1-alpha (HIF-1α) can provide confirmation of hypoxic response activation.

• Functional assays: Combine antibody detection of PGK1 with functional assays of glycolytic activity to correlate expression levels with metabolic changes.

This approach allows researchers to investigate the role of PGK1 in metabolic adaptation to hypoxia, which is particularly relevant in cancer research where the glycolytic pathway is often dysregulated.

What is the significance of PGK1 autoantibodies in immuno-related pancytopenia?

Recent research has identified PGK1 as a potential autoantigen in immuno-related pancytopenia (IRP). Methodological insights from this research include:

• Detection method: Anti-PGK1 antibodies can be screened in patient serum using ELISA techniques. This approach was used to compare untreated IRP patients with severe aplastic anemia (SAA) patients .

• Diagnostic value: Serum levels of anti-PGK1 antibodies were significantly higher in untreated IRP patients compared to SAA patients, suggesting potential diagnostic value in differentiating these conditions .

• Clinical correlations: In recovered IRP patients, PGK1-Ab levels showed:

  • Positive correlation with white blood cell (WBC) counts

  • Positive correlation with circulating immune complex (CIC) levels

  • Negative correlation with platelet (PLT) levels

  These correlations suggest PGK1 autoantibodies may have mechanistic involvement in the disease process .

• Treatment monitoring: Monitoring of anti-PGK1 antibody levels, along with CD34+ IgM positivity, CD5+ B cells, and complement components C3 and C4, may have value in assessing treatment efficacy in IRP patients .

This research demonstrates how PGK1 antibodies can be used not only as research tools but also potentially as diagnostic markers and for monitoring treatment response in certain hematological disorders.

How can PGK1 antibodies be utilized in cancer research?

PGK1 plays significant roles in cancer metabolism and progression. Methodological approaches using PGK1 antibodies in cancer research include:

• Expression profiling: PGK1 antibodies can be used for immunohistochemistry (IHC) to evaluate PGK1 expression levels in cancer tissues. Multiple antibodies have been validated for detection in human liver cancer tissue .

• Metabolic reprogramming studies: Western blot analysis with PGK1 antibodies can quantify changes in glycolytic enzyme expression in cancer cells under various conditions or treatments, providing insights into metabolic adaptations.

• Co-localization studies: Immunofluorescence with PGK1 antibodies, combined with markers for specific organelles or other proteins, can reveal cancer-specific changes in subcellular localization or protein interactions.

• Secreted PGK1 investigation: Some research suggests PGK1 can be secreted by tumor cells to participate in angiogenesis . Antibodies can be used to detect extracellular PGK1 in conditioned media or patient samples.

• Therapy response monitoring: Changes in PGK1 expression following anticancer treatments may serve as a biomarker for metabolic adaptation or resistance mechanisms.

These approaches can provide valuable insights into how cancer cells adapt their metabolism to support proliferation and survival, potentially identifying new therapeutic targets or biomarkers.

What are the critical parameters for optimizing Western blot protocols with PGK1 antibodies?

Western blot optimization for PGK1 detection requires attention to several key parameters:

• Sample preparation: PGK1 antibodies have been validated with multiple cell types including A431, HepG2, HEK-293, HeLa, PC-12, PC-3, NIH/3T3 cells, and various tissue samples including liver and testis tissue . Extract proteins using buffers that effectively solubilize cytoplasmic proteins.

• Loading amount: While standard protein amounts (20-30 μg) are typically sufficient for PGK1 detection due to its abundant expression in most cells, optimization may be needed for tissues or cells with lower expression levels.

• Gel percentage: 10% SDS-PAGE has been successfully used for PGK1 separation and detection . This percentage provides good resolution in the 40-45 kDa range where PGK1 is observed.

• Transfer conditions: Standard transfer protocols are generally effective for PGK1, but optimization of transfer time and buffer composition may improve results with specific antibody clones.

• Antibody dilution: While recommended ranges are 1:5000-1:50000 for Western blotting , the optimal dilution should be determined empirically for each experimental system.

• Detection method: HRP-conjugated secondary antibodies (such as anti-rabbit IgG or anti-mouse IgG depending on the primary antibody host) have been validated for effective visualization of PGK1 .

• Positive controls: Include positive control samples such as HeLa or A431 cell lysates that consistently express PGK1 at detectable levels .

These parameters should be systematically optimized to achieve reliable and reproducible PGK1 detection with minimal background.

How should researchers optimize immunofluorescence protocols for PGK1 antibodies?

Successful immunofluorescence staining with PGK1 antibodies can be achieved by optimizing these key parameters:

• Fixation method: 4% paraformaldehyde fixation for 15 minutes at room temperature has been validated for PGK1 detection in immunofluorescence applications .

• Antibody dilution: Start with a dilution range of 1:400-1:1600 for immunofluorescence applications . Some protocols have used more dilute solutions (1:20) , highlighting the importance of empirical optimization.

• Cell types: A431 and HepG2 cells have been successfully used for PGK1 immunofluorescence detection , making them good candidates for initial protocol optimization.

• Co-staining markers:

  • Cytoskeletal markers like alpha-tubulin can help visualize cell structure (validated with alpha Tubulin antibody [B-5-1-2])

  • Nuclear counterstains like Hoechst 33342 help define nuclear boundaries and assess potential nuclear localization of PGK1

• Subcellular localization: PGK1 has been detected in both cytoplasm and nucleus in immunofluorescence studies . Proper optimization should allow visualization of this dual localization pattern.

• Controls: Include negative controls (primary antibody omission) and positive controls (cell types known to express PGK1) to validate staining specificity.

• Antigen retrieval: While not always necessary for cultured cells, antigen retrieval may improve staining in tissue sections, similar to the recommendations for IHC applications .

Careful optimization of these parameters will help achieve specific and reproducible PGK1 staining in immunofluorescence applications.

What are common issues encountered when using PGK1 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with PGK1 antibodies:

• Multiple bands in Western blot:

  • Issue: Detection of bands other than the expected 40-45 kDa.

  • Solution: Optimize antibody dilution (try more dilute), improve blocking conditions, or test alternative antibody clones. Verify if additional bands represent alternative isoforms, post-translational modifications, or degradation products.

• Weak or no signal:

  • Issue: Insufficient detection despite adequate protein loading.

  • Solution: Reduce antibody dilution, increase protein loading, optimize antigen retrieval (for IHC/IF), or try different antibody clones. Consider whether PGK1 expression might be genuinely low in your sample.

• High background:

  • Issue: Non-specific staining making specific signal difficult to interpret.

  • Solution: Increase antibody dilution, improve blocking (try different blocking agents or longer blocking times), add additional washing steps, or test alternative antibody clones.

• Inconsistent immunoprecipitation results:

  • Issue: Variable success in pulling down PGK1 or interacting partners.

  • Solution: Optimize antibody amount (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate is recommended) , try different lysis buffers, and consider cross-linking antibodies to beads for cleaner results.

• Unexpected localization in immunofluorescence:

  • Issue: Staining pattern doesn't match expected cytoplasmic and nuclear localization.

  • Solution: Validate with multiple antibodies, optimize fixation conditions, and consider that localization may genuinely differ in your experimental conditions (e.g., hypoxia, cell cycle stage).

• Species cross-reactivity issues:

  • Issue: Antibody doesn't work in species of interest despite claimed reactivity.

  • Solution: Verify the exact immunogen sequence used to generate the antibody and its homology across species. Consider antibodies raised against more conserved epitopes.

Systematic optimization and thorough controls can address most of these common issues encountered with PGK1 antibodies.

How can researchers determine if a PGK1 antibody is suitable for studying post-translational modifications?

Studying post-translational modifications (PTMs) of PGK1 requires specialized considerations:

• Epitope mapping: Determine if the antibody's epitope overlaps with or is adjacent to known PTM sites on PGK1. Contact manufacturers for detailed epitope information if not provided in product documentation.

• Modification-specific antibodies: For common PTMs (phosphorylation, acetylation, etc.), consider using modification-specific antibodies designed to recognize PGK1 only when modified at specific residues.

• Validation strategies:

  • Compare detection patterns in samples treated with modifying enzymes (kinases, acetylases) versus control samples

  • Use samples treated with demodifying enzymes (phosphatases, deacetylases) to confirm specificity

  • Employ modified and unmodified peptide competition assays to verify specificity

• Complementary techniques: Combine antibody-based detection with mass spectrometry to unambiguously identify and localize PTMs on PGK1.

• 2D gel analysis: Use two-dimensional gel electrophoresis followed by Western blotting with PGK1 antibodies to separate differently modified forms of PGK1 based on charge differences.

• Controls for specific modifications:

  • For phosphorylation: Treatment with phosphatase inhibitors or activators of pathways known to modify PGK1

  • For glycosylation: Treatment with deglycosylating enzymes

  • For ubiquitination: Proteasome inhibitors to accumulate ubiquitinated forms

These approaches can help determine whether a specific PGK1 antibody is suitable for studying PTMs and provide strategies for validating findings from such studies.