PIGO Antibody

What is PIGO and what role does it play in cellular biology?

PIGO (Phosphatidylinositol glycan anchor biosynthesis, class O) is a protein involved in glycosylphosphatidylinositol (GPI)-anchor biosynthesis. It functions as an ethanolamine phosphate transferase that specifically transfers ethanolaminephosphate (EtNP) to the third mannose in GPI. The GPI-anchor is a glycolipid containing three mannose molecules in its core backbone and is found on many blood cells, serving to anchor proteins to the cell surface through an amide bond .

PIGO is localized to the endoplasmic reticulum membrane as a multi-pass membrane protein . At least two alternatively spliced transcripts encoding distinct isoforms have been identified for the PIGO gene . Mutations in PIGO have been associated with hyperphosphatasia with mental retardation, highlighting its importance in normal cellular function and development .

What are the common applications of PIGO antibodies in laboratory research?

PIGO antibodies have several validated applications in research settings:

These applications allow researchers to detect, localize, and quantify PIGO protein in various experimental settings . When performing Western blot, HRP conjugated secondary antibody should be diluted 1:50,000 - 100,000 for optimal results .

How should PIGO antibodies be handled and stored for maximum effectiveness?

For optimal preservation of antibody activity:

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

• For short-term storage (up to 2 weeks), refrigerate at 2-8°C

• Aliquot upon receipt to prevent repeated freeze-thaw cycles

• Some lyophilized PIGO antibodies are supplied in PBS buffer with 2% sucrose and require reconstitution with 50 μL of distilled water to achieve a final concentration of 1 mg/mL

• Liquid formulations are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Antibodies are generally stable for one year after shipment when properly stored

Note that aliquoting is unnecessary for -20°C storage in some formulations, and 20μL sizes may contain 0.1% BSA as a stabilizer .

What positive controls should be used when working with PIGO antibodies?

Based on the validation data available, the following positive controls are recommended:

For Western blot:

• HeLa cells (human cervical cancer cell line)

• A431 cells (human epidermoid carcinoma)

• COLO 320 cells (human colorectal adenocarcinoma)

• Mouse spleen tissue

For IHC applications:

• Human liver tissue

• Human kidney tissue

• Human skin tissue

• Human spleen tissue

• Human testis tissue

For IF/ICC applications:

• MCF-7 cells (human breast adenocarcinoma)

When evaluating positive signals, note that the calculated molecular weight of PIGO is approximately 119 kDa (1089aa) or 50 kDa (454aa), but the observed molecular weight in Western blots is typically around 74 kDa .

How can PIGO mutations be studied using antibodies in disease models?

Studying PIGO mutations in disease models requires careful experimental design:

• Functional Rescue Assays: Set up a system similar to that described in Krawitz et al. (2012) where wild-type and mutant PIGO plasmids are transfected into PIGO-deficient cell lines. Use flow cytometry to measure restoration of GPI-anchored proteins (GPI-APs) on the cell surface, such as CD59 and uPAR .

• Mutation-Specific Analysis: For known mutations like p.Thr788Hisfs∗5 and Leu957Phe, compare antibody reactivity between wild-type and mutant proteins. As demonstrated in research, Leu957Phe mutation showed reduced protein levels, while the truncating mutation (c.2361dup) resulted in increased levels of the truncated protein .

• Immunoblot Analysis: Use GAPDH as a loading control when analyzing PIGO expression levels. Band intensities can be quantified to determine relative expression levels between wild-type and mutant PIGO proteins .

• Cell Line Selection: Consider using human CD59-expressing PIGO-deficient CHO cells derived from aerolysin-resistant clones from chemically mutagenized Chinese hamster ovary (CHO) cells as a model system .

What technical considerations are important for optimizing immunohistochemistry with PIGO antibodies?

For optimal IHC results with PIGO antibodies:

• Antigen Retrieval: Use TE buffer pH 9.0 for suggested antigen retrieval. Alternatively, citrate buffer pH 6.0 can be used if needed .

• Dilution Optimization: Begin with the recommended dilution range (1:20-1:200) but perform a titration in your specific testing system to determine optimal antibody concentration .

• Tissue Processing: For paraffin-embedded tissues, proper dewaxing and hydration are essential. After these steps, perform antigen retrieval using high pressure in the appropriate buffer .

• Blocking Step: Include a blocking step with 10% normal serum to reduce background staining .

• Controls: Always include positive control tissues (human liver, kidney, skin, spleen, or testis) and negative controls (omitting primary antibody) to validate staining specificity .

• Detection System: Select an appropriate detection system based on your research needs, considering factors such as sensitivity requirements and available instrumentation.

How can epitope considerations affect experimental design when using PIGO antibodies?

Understanding epitope locations is crucial when designing experiments with PIGO antibodies:

• C-terminal vs. N-terminal Antibodies: Different commercially available PIGO antibodies target different regions of the protein. For example, some target the C-terminal region (amino acids 955-983) , while others may target different domains.

• Impact on Protein Detection: Drawing from research on similar antibodies like anti-pIgR antibodies, epitope location can significantly influence protein trafficking detection and experimental outcomes. As seen with pIgR antibodies, those binding to different domains exhibited distinct trafficking patterns and cellular retention .

• Mutant Protein Studies: When studying truncated or mutated PIGO proteins, choose antibodies whose epitopes are preserved in the mutant protein. For instance, a C-terminal antibody would not detect a protein with C-terminal truncation .

• Domain-Specific Interactions: Based on analogous studies with pIgR, antibodies recognizing different domains can have different patterns of interaction with the target protein, affecting experimental outcomes like transcytosis efficiency .

• Epitope Accessibility: Consider the accessibility of the epitope in different experimental conditions. Protein conformation, post-translational modifications, or protein-protein interactions may mask certain epitopes in specific contexts.

What approaches can be used to optimize Western blot detection of PIGO?

For optimal Western blot results:

• Antibody Concentration: Use PIGO antibody at the recommended concentration of 1 μg/mL or at dilutions between 1:500-1:2000 .

• Secondary Antibody Selection: With HRP-conjugated secondary antibodies, use high dilutions (1:50,000-100,000) to minimize background .

• Sample Preparation: Validated samples include HeLa cells, A431 cells, COLO 320 cells, and mouse spleen tissue .

• Molecular Weight Considerations: Be aware that while the calculated molecular weight of PIGO is 119 kDa (1089aa) or 50 kDa (454aa), the observed molecular weight is typically around 74 kDa .

• Loading Controls: Use GAPDH as a loading control, as demonstrated in published PIGO research .

• Membrane Selection: Choose appropriate membrane type and pore size based on the molecular weight of PIGO and potential splice variants.

• Detection System: Select a detection system with sensitivity appropriate for your specific application needs.

How can PIGO antibodies be used to investigate GPI-anchor biosynthesis pathways?

To study GPI-anchor biosynthesis pathways:

• Co-immunoprecipitation Studies: Use PIGO antibodies to pull down protein complexes involved in GPI-anchor biosynthesis. This approach can help identify interaction partners in the biosynthetic pathway.

• Functional Complementation Assays: Similar to the approach used by Krawitz et al., transfect PIGO-deficient cells with wild-type or mutant PIGO constructs and measure restoration of GPI-AP surface expression using flow cytometry .

• Immunofluorescence Analysis: Use PIGO antibodies in combination with markers for the endoplasmic reticulum and other organelles to study the subcellular localization of PIGO and its potential redistribution under different conditions.

• RNAi and CRISPR Approaches: Combine gene silencing or knockout approaches with PIGO antibody detection to study the effects of PIGO depletion on GPI-anchor biosynthesis and protein trafficking.

• GPI-AP Surface Expression Analysis: Use flow cytometry to measure levels of GPI-anchored proteins such as CD59 and uPAR as functional readouts of PIGO activity and GPI-anchor biosynthesis efficiency .

This experimental approach builds on established methodologies in the field and allows for comprehensive analysis of GPI-anchor biosynthesis pathways in various cellular contexts.

What are common issues when working with PIGO antibodies and how can they be resolved?

Based on general antibody research practices and the specific information about PIGO antibodies:

• Non-specific Binding: May appear as multiple bands in Western blot or diffuse staining in IHC/IF.

  • Solution: Optimize antibody dilution (start with manufacturer recommendations), increase blocking time/concentration, and include additional washing steps.

• Weak or No Signal: Can result from insufficient antigen, degraded antibody, or suboptimal detection conditions.

  • Solution: Check antibody viability with known positive controls (HeLa, A431, COLO 320 cells for WB; human liver, kidney tissues for IHC) , optimize antigen retrieval methods, and ensure compatible secondary antibody selection.

• Variable Results Between Experiments: May be due to inconsistent sample preparation or handling.

  • Solution: Standardize protocols, prepare master mixes where possible, and maintain consistent incubation times and temperatures.

• Background in Immunofluorescence: Common in IF/ICC applications.

  • Solution: Use the recommended dilution range (1:10-1:100) , optimize fixation protocols, and include proper controls.

• Antibody Cross-reactivity: Potential concern when studying closely related proteins.

  • Solution: Validate specificity using knockout or knockdown controls, and compare results with antibodies targeting different epitopes.

How can researchers validate the specificity of PIGO antibodies?

Multiple validation approaches can ensure antibody specificity:

• Positive Control Tissues/Cells: Verify signal in known PIGO-expressing samples:

  • Western blot: HeLa, A431, COLO 320 cells, mouse spleen tissue

  • IHC: Human liver, kidney, skin, spleen, testis tissues

  • IF/ICC: MCF-7 cells

• Molecular Weight Verification: Confirm that observed bands align with expected molecular weight (calculated: 119 kDa/50 kDa; observed: typically 74 kDa) .

• siRNA/CRISPR Knockdown Controls: Demonstrate reduced signal following PIGO knockdown or knockout.

• Peptide Competition Assay: Pre-incubate antibody with the immunizing peptide and observe signal reduction.

• Multiple Antibody Comparison: Compare results from antibodies targeting different PIGO epitopes.

• Recombinant Protein Controls: Test antibody against wild-type and mutant PIGO variants, as demonstrated in studies with PIGO mutations .

• Immunoprecipitation-Mass Spectrometry: Confirm antibody pulls down PIGO protein via mass spectrometry analysis.

How can PIGO antibodies be utilized in studying disease mechanisms beyond GPI-anchor disorders?

PIGO antibodies can facilitate research in several disease contexts:

• Cancer Research: Given that PIGO antibodies have been validated in several cancer cell lines (HeLa, A431, COLO 320, MCF-7) , they can be used to investigate potential alterations in GPI-anchor biosynthesis in cancer cells, which might affect cell surface protein presentation and signaling.

• Neurodevelopmental Disorders: Building on the link between PIGO mutations and hyperphosphatasia with mental retardation , researchers can use PIGO antibodies to study neurodevelopmental mechanisms in relevant model systems.

• Immunological Research: Since GPI-anchored proteins are abundant on blood cells , PIGO antibodies can help investigate immune cell function and signaling that depends on proper GPI-anchor biosynthesis.

• Cell Surface Protein Organization: Combinatorial approaches using PIGO antibodies with super-resolution microscopy can provide insights into the organization of GPI-anchored proteins in membrane microdomains.

• Developmental Biology: Given the importance of GPI-anchored proteins in development, PIGO antibodies can be used to study tissue-specific or temporal patterns of GPI-anchor biosynthesis during organism development.

What methodological approaches can combine PIGO antibodies with advanced imaging techniques?

Integrating PIGO antibodies with advanced imaging offers powerful research capabilities:

• Super-resolution Microscopy: Combine PIGO antibodies with techniques like STORM or PALM to visualize subcellular localization with nanometer precision.

  • Methodology: Use fluorophore-conjugated secondary antibodies optimized for super-resolution imaging.

• Live-cell Imaging: Adapt immunofluorescence protocols with PIGO antibodies for live-cell applications.

  • Methodology: Consider using antibody fragments (Fab) conjugated to bright, photostable fluorophores for improved cell penetration and reduced impact on protein function.

• Co-localization Studies: Investigate PIGO interactions with other proteins in the GPI-anchor biosynthesis pathway.

  • Methodology: Employ multi-color confocal microscopy with careful controls to account for spectral overlap.

• FRET Analysis: Examine proximity relationships between PIGO and potential interaction partners.

  • Methodology: Label PIGO antibodies and partner proteins with appropriate FRET donor-acceptor pairs.

• Expansion Microscopy: Combine PIGO immunolabeling with physical expansion of specimens.

  • Methodology: Adapt standard IF protocols (starting with 1:10-1:100 dilution) to accommodate the expansion process, adjusting antibody concentration accordingly.