PIGQ Antibody
Description
Overview of PIGQ Antibody
PIGQ antibodies are immunological reagents designed to detect and quantify the PIGQ protein, which is encoded by the PIGQ gene. This protein is part of the GPI-N-acetylglucosaminyltransferase (GPI-GnT) complex, essential for the first step of GPI-anchor synthesis—a glycolipid structure that anchors proteins to cell membranes . Deficiencies in GPI biosynthesis are linked to neurological disorders, congenital anomalies, and epilepsy .
Western Blot Analysis
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ABIN2782620: Validated using human cell lysate, detecting a band at ~58 kDa .
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H00009091-M02: Detects recombinant PIGQ at 84.1 kDa in transfected 293T cells .
Immunohistochemistry (IHC)
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HPA039828: Demonstrates strong staining in human placental tissue .
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H00009091-M02: Shows specific localization in formalin-fixed paraffin-embedded human placenta at 4 µg/mL .
ELISA and Immunofluorescence
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CAB17044: Suitable for sandwich ELISA with a detection limit of 0.1 ng/mL .
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ABIN2782620: Predicted reactivity of 100% with human samples and 79–93% with other mammals .
Biochemical and Functional Insights
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Role in GPI Biosynthesis: PIGQ catalyzes the transfer of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI), initiating GPI-anchor synthesis .
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Disease Associations: Mutations in PIGQ are linked to Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 4 and developmental epileptic encephalopathy .
Validation and Quality Control
Commercial PIGQ antibodies undergo rigorous validation:
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Specificity Testing: Antibodies-online uses peptide arrays and cross-reactivity panels .
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Functional Assays: Bio-Techne validates antibodies via transfected lysate comparisons and antigen-blocking experiments .
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Storage: Most antibodies are stable at -20°C, with avoidance of freeze-thaw cycles critical .
Limitations and Considerations
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Genome-wide association analyses identified four suggestive loci (PAX3, CCRN4L, PIGQ, and ADAM19) as potential determinants of disease progression in Alzheimer's disease. PMID: 25114068
- Genetic studies have identified two novel genes associated with Ohtahara Syndrome: KCNT1 and PIGQ. PMID: 24463883
Q&A
What experimental strategies validate PIGQ antibody specificity in GPI biosynthesis studies?
To confirm antibody specificity:
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Immunogen Alignment: Compare the antibody’s immunogen sequence (e.g., amino acids 1-200 of human PIGQ ) against homology regions in target species using tools like BLAST.
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Knockout Controls: Use CRISPR-edited PIGQ-null cell lines to verify loss of signal in Western blotting (WB) at expected molecular weights (84 kDa full-length vs. 32 kDa isoforms ).
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Competition Assays: Pre-incubate antibodies with recombinant PIGQ protein (e.g., residues 1-200) to test signal abolition .
Table 1: Validation Parameters for Common PIGQ Antibodies
How do PIGQ expression patterns inform experimental model selection?
PIGQ exhibits tissue-specific expression critical for GPI-anchored protein trafficking:
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Murine Models: Highest expression in brain (neuronal GPI-anchored proteins) and spleen (immune cell signaling), detectable at 1:500–1:2000 dilution in WB .
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Human Cell Lines: Lymphoblastoid cells show baseline mutant frequencies of 2.9–5.56 × 10⁻⁶, rising post-genotoxic exposure (e.g., radiotherapy ).
Methodological Note: For developmental studies, combine IHC (1:200–1:500 dilution ) with flow cytometry to track CD55/CD59 loss in PIGQ-mutant cells .
How can structural studies resolve contradictions in PIGQ antibody binding kinetics?
Conflicting reports on hapten-induced conformational changes require multi-angle scattering analysis:
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Neutron/X-ray Scattering: Measure radii of gyration (Rg) for free vs. hapten-bound antibodies. Non-precipitating antibodies show Rg reductions of 0.5–1.2 nm upon hapten binding, indicating Fab arm contraction .
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Dynamic Light Scattering: Confirm hydrodynamic radius (Rh) changes under physiological buffers.
Equation: For scattering intensity , the Guinier approximation determines Rg .
What protocols optimize PIGQ detection in mutagenesis assays?
The PIG-A/PIGQ somatic mutation assay requires:
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Flow Cytometry Setup:
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Stain with FITC-conjugated anti-CD55 (1:100) and PE-conjugated anti-CD59 (1:50).
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Gate GPI-negative populations using 0.1% fetal bovine serum to reduce background.
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Longitudinal Stability: Cryopreserve samples in 10% DMSO/90% FBS to maintain GPI anchors for ≤6 months .
Data Interpretation: Mutant frequencies >10⁻⁵ indicate genotoxic exposure (e.g., cigarette smoke increases mutants 2.3-fold ).
How do species-specific epitopes impact cross-reactivity in pig models?
Porcine PIGQ shares 89% homology with human PIGQ but requires tailored validation:
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Phage Display Libraries: Screen porcine antibody libraries (diversity ≤10¹⁰ clones ) against recombinant pig PIGQ.
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Epitope Binning: Compare binding of CAB17044 (mouse-reactive ) vs. 15658-1-AP (rat-reactive ) using SPR.
Table 2: Cross-Reactivity Profiles
Why do WB results vary between PIGQ isoforms, and how can this be resolved?
Discrepancies arise from alternative splicing (84 kDa vs. 65 kDa isoforms ):
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Gel Electrophoresis: Use 4–20% gradient gels to resolve isoforms.
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Peptide Competition: Pre-absorb antibodies with isoform-specific peptides (e.g., N-terminal 1-50 vs. 150-200).
Case Study: Proteintech’s 15658-1-AP detects all isoforms in human cells, while CAB17044 preferentially binds full-length PIGQ .
What controls are essential for immunofluorescence (IF) studies of PIGQ localization?
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Permeabilization Controls: Compare Triton X-100 (0.1% vs. 0.5%) to distinguish membrane vs. cytosolic pools.
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Co-Staining: Use GM130 (Golgi marker) to confirm PIGQ’s ER-Golgi localization .
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Mutation Rescue: Transfect PIGQ-null cells with wild-type vs. catalytic mutants (e.g., D129A).
How can PIGQ antibodies advance neurodegenerative disease research?
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GPI-AP Quantification: In Alzheimer’s models, correlate PIGQ levels (via ELISA ) with Aβ plaque density.
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CRISPR Interference: Knock down PIGQ in iPSC-derived neurons to assay synaptic GPI-anchored protein loss.
Recent Finding: PIGQ mutations account for 7% of early-onset epileptic encephalopathies (DEE77 ).
