pgk-1 Antibody

Basic Research Questions

• What are the primary experimental applications of PGK1 antibodies in cellular studies?
  PGK1 antibodies are used for:

  • Western blot (WB) detection in HeLa, HEK-293, and mouse/rat liver tissues at dilutions of 1:500–1:50,000 .

  • Immunofluorescence (IF) to localize PGK1 in cytoplasm/nucleus (e.g., fixed HeLa cells with 1:20 dilution) .

  • Immunoprecipitation (IP) for protein interaction studies (0.5–4.0 µg antibody per 1–3 mg lysate) .

  • Methodological tip: Optimize antigen retrieval for IHC using EDTA (pH 8.0) or citrate (pH 6.0) buffers .

• How do I validate PGK1 antibody specificity in Western blot?

  • Run parallel assays with negative controls (e.g., PGK1-knockout lysates) and confirm band size (~45 kDa) .

  • Use competitive inhibition assays: Pre-incubate antibody with recombinant PGK1 to block signal .

  • Cross-validate with mass spectrometry or siRNA-mediated PGK1 knockdown .

• Why do PGK1 antibody signals vary between cell types?

  • PGK1 expression levels differ by metabolic activity (e.g., higher in hypoxic HeLa cells; see 1% O₂ treatment in ).

  • Technical factors:

    • Electrophoresis conditions (10% SDS-PAGE at 70–90 V for 2–3 hours) .

    • Blocking agents (5% non-fat milk reduces background vs. BSA) .

Advanced Research Questions

• How to resolve contradictions in PGK1 subcellular localization across studies?

  • PGK1 distributes dynamically between cytoplasm/nucleus depending on stress (e.g., hypoxia, viral infection) .

  • Experimental design:

    • Compare normoxic vs. hypoxic cells using IF with α-Tubulin/Hoechst counterstains .

    • Validate with subcellular fractionation + WB .

• What mechanisms explain PGK1’s role in inflammatory signaling beyond glycolysis?

  • PGK1 regulates IL-1β/IL-6 via NRF2: Inhibitors (e.g., DC-PGKI) block LPS-induced cytokine production by stabilizing NRF2 .

  • Method: Use PGK1 inhibitors in macrophages + ChIP-seq to map NRF2 binding near Il-1β/Il-6 loci .

• How to assess PGK1’s ATP-generating function in viral replication?

  • Employ cell-free replicase assays:

    Step	Protocol	Outcome
    1	Purify TBSV replicase from PGK1-depleted yeast	3–10× reduced RNA replication
    2	Measure ATP levels in replication compartments using FRET-based ATeam sensors

Data Interpretation Challenges

• Why do PGK1 antibody cross-reactivity rates differ between species?

  • Commercial antibodies show variable reactivity:

    Antibody	Human	Mouse	Rat	Notes
    GTX107614	Yes	No	No	Validated in HeLa/293T
    PA2045	Yes	Yes	Yes	Detects 45 kDa band in rat brain

  • Solution: Pre-test species-specific lysates and use epitope alignment tools .

• How to distinguish PGK1’s metabolic vs. immune roles in disease models?

  • Use dual approaches:

    • Metabolic profiling: Measure lactate/ATP levels in PGK1-inhibited cells .

    • Immune profiling: Quantify anti-PGK1 IgG in patient sera (46% positivity in Kawasaki disease ).

Methodological Optimization Table