PIGK Antibody

What is PIGK and what role does it play in cellular functions?

PIGK (Phosphatidylinositol glycan anchor biosynthesis, class K protein) is a critical component of the GPI transamidase complex that mediates GPI anchoring in the endoplasmic reticulum. It functions by replacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GPI .

During this transamidation reaction, PIGK acts as the catalytic subunit, forming a carbonyl intermediate with the substrate protein . PIGK cleaves the C-terminal GPI signal of the precursor protein and forms an enzyme-substrate complex via a thioester bond . This process is essential for the attachment of GPI anchors to proteins that ultimately become attached to the plasma membrane.

PIGK has been observed to have two isoforms in endogenous conditions, with molecular weights of approximately 45 kDa and 36 kDa . Studies show that PIGK stability and function depends on other components of the GPI transamidase complex, particularly PIGT, indicating an interdependent relationship between these proteins .

What experimental applications can PIGK antibodies be used for?

PIGK antibodies have been validated for multiple experimental techniques:

When transitioning between applications, researchers should optimize antibody concentrations and conditions as performance can vary significantly across different experimental platforms .

What are the recommended sample preparation methods when working with PIGK antibodies in Western blotting?

For optimal results in Western blotting with PIGK antibodies:

• Cell lysate preparation: Extract proteins from cells using standard lysis buffers containing protease inhibitors to prevent PIGK degradation .

• Protein denaturation: Heat samples with loading buffer containing SDS and reducing agents at 95-100°C for 5 minutes to fully denature PIGK.

• Gel selection: Use 10-12% polyacrylamide gels for optimal separation as PIGK has observed molecular weights of 40-45 kDa .

• Transfer conditions: Transfer proteins to PVDF or nitrocellulose membranes using standard protocols, with transfer time optimized for the PIGK molecular weight range.

• Blocking: Block membranes with 5% non-fat milk or BSA in TBST to reduce background.

• Antibody dilution: Dilute primary antibodies according to manufacturer recommendations, typically 1:500-1:2000 for polyclonal antibodies and 1:5000-1:50000 for recombinant antibodies .

• Detection system: Both chemiluminescence and fluorescence-based detection systems are compatible with PIGK antibodies .

When troubleshooting, remember that unassembled PIGK undergoes proteasomal degradation, so proteasome inhibitors like MG132 may increase detection levels in certain experimental conditions .

How can researchers study the relationship between PIGK stability and other components of the GPI transamidase complex?

Research into PIGK stability and its relationship with other GPI transamidase components requires sophisticated approaches:

• Knockout strategy: Generate knockout cell lines of individual GPI transamidase components (PIGT, GPAA1, PIGU, PIGS) using CRISPR/Cas9 technology to observe effects on PIGK expression and stability .

• Protein expression analysis: Use PIGK antibodies for Western blotting to quantify protein levels in these knockout lines. Research has shown that knockout of PIGT significantly reduces PIGK expression to levels similar to PIGK-KO cells, suggesting PIGT is required for PIGK stability .

• Rescue experiments: Perform complementation studies by reintroducing wild-type or mutant forms of GPI transamidase components to confirm specificity of observed effects .

• RNA expression analysis: Employ RT-qPCR to quantify PIGK mRNA levels to distinguish between transcriptional effects and post-translational stability issues. Previous studies have shown that PIGK mRNA levels remain comparable between parental, Hrd1-KO, and PIGT-Hrd1-DKO cells, indicating regulation at the protein level .

• Protein-protein interaction studies: Use co-immunoprecipitation with PIGK antibodies to identify direct interactions between PIGK and other components of the complex .

This multi-faceted approach can help researchers delineate the complex interdependencies within the GPI transamidase complex and their impact on PIGK stability .

What is the role of Hrd1 in PIGK degradation and how can researchers investigate this pathway?

Hrd1 (also known as SYVN1) is a key ubiquitin ligase in the endoplasmic reticulum-associated degradation (ERAD) pathway that regulates unassembled PIGK degradation. To investigate this regulatory mechanism:

• Generate knockout cell lines: Create Hrd1 knockout cells using CRISPR/Cas9 genome editing in backgrounds with or without PIGT expression .

• Comparative analysis: Use PIGK antibodies in Western blotting to compare PIGK levels between wild-type, PIGT-KO, Hrd1-KO, and PIGT-Hrd1 double knockout cells. Research has shown that knockout of Hrd1 restores PIGK expression in PIGT-KO cells, suggesting Hrd1 specifically targets unassembled PIGK for degradation .

• Rescue experiments: Reintroduce wild-type or inactive mutant Hrd1 (C329S) in PIGT-Hrd1-DKO cells to confirm the role of Hrd1's ubiquitin-ligase activity. Studies demonstrate that wild-type Hrd1, but not the C329S mutant, reduces PIGK expression in these cells .

• Proteasome inhibition: Treat cells with proteasome inhibitors (e.g., MG132) to confirm the proteasome-dependent nature of this degradation pathway. Flow cytometry of permeabilized cells stained with anti-PIGK antibodies can quantify changes in PIGK levels .

• Ubiquitination assays: Use immunoprecipitation with PIGK antibodies followed by ubiquitin blotting to detect direct ubiquitination of PIGK by Hrd1.

This systematic approach can elucidate the specific role of Hrd1 in regulating PIGK levels and provide insights into ERAD quality control of GPI transamidase complex assembly .

How can researchers optimize immunofluorescence protocols for subcellular localization studies of PIGK?

For high-quality subcellular localization studies of PIGK using immunofluorescence:

• Cell preparation: Culture cells on glass coverslips coated with poly-L-lysine or other appropriate substrates depending on cell type.

• Fixation optimization: Test multiple fixation methods as they can significantly impact epitope accessibility:

  • 4% paraformaldehyde (PFA) for 10-15 minutes at room temperature preserves cellular architecture while maintaining PIGK antigenicity

  • Methanol fixation (-20°C for 10 minutes) may provide better access to certain PIGK epitopes

• Permeabilization: Use 0.1% Triton X-100 in PBS for 5-10 minutes to allow antibody access to intracellular PIGK without excessive membrane disruption .

• Blocking: Incubate with 1-5% BSA or normal serum (matched to secondary antibody host) to minimize non-specific binding.

• Primary antibody incubation: Dilute PIGK antibodies according to manufacturer recommendations, typically 1:500 for immunofluorescence applications . Incubate overnight at 4°C or for 1-2 hours at room temperature.

• Co-staining strategies: For co-localization studies, combine PIGK antibodies with markers of:

  • Endoplasmic reticulum (e.g., calnexin, PDI) as PIGK functions in GPI anchoring in the ER

  • Other GPI transamidase complex components (PIGT, GPAA1, PIGU, PIGS)

• Signal amplification: Consider using tyramide signal amplification for low abundance detection of PIGK.

• Confocal imaging: Use confocal microscopy with appropriate resolution to precisely determine the subcellular distribution of PIGK.

These optimizations can help researchers accurately characterize PIGK's intracellular distribution and its co-localization with other proteins involved in GPI-anchor attachment .

What are the key considerations when selecting between monoclonal and polyclonal PIGK antibodies for specific applications?

The choice between monoclonal and polyclonal PIGK antibodies should be made based on the specific experimental requirements:

For critical quantitative applications requiring reproducibility across experiments, recombinant monoclonal antibodies like EPR17843 may be preferable. For applications requiring detection of PIGK across diverse experimental conditions or detection of degraded PIGK in ERAD studies, polyclonal antibodies may offer advantages due to their multiple epitope recognition .

Some researchers adopt a dual-antibody approach, using both types to validate findings and ensure robust detection across experimental conditions .

How can PIGK antibodies be used to investigate disorders related to GPI anchor attachment defects?

PIGK antibodies can be valuable tools for investigating GPI anchor attachment disorders through several methodological approaches:

• Patient sample analysis: Use PIGK antibodies in Western blotting and immunohistochemistry to analyze PIGK expression levels in patient-derived samples (biopsies, cultured cells) compared to healthy controls.

• Flow cytometry applications: Employ PIGK antibodies alongside GPI-anchored protein markers to assess correlation between PIGK expression and GPI-anchored protein surface levels. This can be particularly informative in diseases like paroxysmal nocturnal hemoglobinuria where GPI-anchored proteins are deficient .

• GPI transamidase complex assembly evaluation: Use co-immunoprecipitation with PIGK antibodies to assess whether complex formation with other components (PIGT, GPAA1, PIGU, PIGS) is altered in patient samples.

• Cellular models of disease:

  • Create cellular models using CRISPR/Cas9 to introduce patient-specific mutations in PIGK or other GPI transamidase components

  • Use PIGK antibodies to monitor protein expression, stability, and localization in these models

  • Employ FLAER (fluorescently labeled inactive toxin aerolysin), which binds specifically to GPI-anchored proteins, alongside PIGK detection to correlate PIGK function with GPI-AP levels

• Rescue experiments: Attempt to rescue GPI anchor attachment defects by overexpressing wild-type PIGK in patient-derived cells and monitor changes in GPI-anchored protein expression.

These approaches can help elucidate the molecular mechanisms underlying GPI anchor attachment disorders and potentially identify therapeutic targets .

What controls and validation steps are essential when using PIGK antibodies in research?

Rigorous controls and validation are critical for ensuring reliable results with PIGK antibodies:

• Positive controls:

  • Cell lines known to express PIGK (e.g., HEK293, HT-1080, SMMC-7721, HepG2)

  • Recombinant PIGK protein or overexpression systems

  • Tissue sections with known PIGK expression patterns

• Negative controls:

  • PIGK knockout cell lines created using CRISPR/Cas9

  • Secondary antibody-only controls to assess non-specific binding

  • Isotype controls (matched to primary antibody class and host)

• Antibody validation techniques:

  • Peptide competition assays using the immunizing peptide (e.g., KLH-conjugated synthetic peptide from PIGK N-terminal region for ABIN389064)

  • siRNA knockdown of PIGK to confirm specificity of detected bands/signals

  • Western blot validation showing the expected 40-45 kDa band that disappears in PIGK-KO cells

• Cross-application validation:

  • Confirm findings using multiple applications (e.g., verify WB results with ICC/IF)

  • Use multiple antibodies targeting different PIGK epitopes to confirm specificity

• Experimental design considerations:

  • Include loading controls appropriate for the experimental context

  • Optimize antibody concentrations through titration experiments

  • Document batch numbers for reproducibility purposes

These validation steps ensure that observed results are specific to PIGK and not artifacts of non-specific binding or technical variables .

How can researchers optimize PIGK antibody performance in co-immunoprecipitation experiments?

For successful co-immunoprecipitation (Co-IP) experiments investigating PIGK interactions:

• Lysis buffer optimization:

  • Use mild non-denaturing buffers (e.g., NP-40 or Triton X-100 based) to preserve protein-protein interactions

  • Include protease inhibitors to prevent degradation during processing

  • Consider detergent concentration optimization since PIGK is a membrane-associated protein

• Antibody selection considerations:

  • Choose antibodies validated for immunoprecipitation applications

  • Recombinant monoclonal antibodies like EPR17843 may provide more consistent IP results compared to polyclonal antibodies

  • Confirm the epitope location does not interfere with protein-protein interaction sites

• Pre-clearing strategy:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Use isotype control antibodies to identify non-specific interactions

• Immunoprecipitation workflow:

  • Optimize antibody-to-lysate ratios through titration experiments

  • Consider crosslinking the antibody to beads to prevent antibody co-elution

  • For membrane protein complexes like GPI transamidase, extend incubation times (overnight at 4°C) to ensure complete binding

• Elution and detection:

  • Use elution conditions that maintain the integrity of co-precipitated proteins

  • Analyze precipitates by Western blotting using antibodies against suspected interaction partners (PIGT, GPAA1, PIGU, PIGS)

  • Consider mass spectrometry for unbiased identification of all interacting partners

• Reciprocal Co-IP:

  • Perform reverse Co-IP (precipitate with antibodies against interaction partners and detect PIGK) to confirm interactions

These optimizations can help researchers successfully investigate complexes containing PIGK and identify novel interaction partners in the GPI transamidase pathway .

What are the methodological considerations when using PIGK antibodies to study ERAD (ER-associated degradation) pathways?

When investigating PIGK in the context of ERAD pathways:

• Experimental design for degradation kinetics:

  • Perform cycloheximide chase assays using PIGK antibodies to track protein degradation rates in wild-type versus cells with compromised ERAD components

  • Compare PIGK stability in cells with knockout or inhibition of various ERAD components (Hrd1, gp78, TRC8, Doa10, RHBDL4)

• Proteasome inhibition studies:

  • Treat cells with proteasome inhibitors (MG132) or lysosome inhibitors (Bafilomycin) to determine the primary degradation pathway

  • Use PIGK antibodies in Western blotting, flow cytometry, or immunofluorescence to quantify accumulation of PIGK under these conditions

  • Research has established that PIGK degradation is proteasome-dependent rather than lysosome-dependent

• Ubiquitination analysis:

  • Immunoprecipitate PIGK under denaturing conditions after proteasome inhibition

  • Probe with anti-ubiquitin antibodies to detect ubiquitinated PIGK species

  • Compare ubiquitination patterns in wild-type versus Hrd1 knockout cells

• Co-localization with ERAD machinery:

  • Use immunofluorescence with PIGK antibodies alongside markers for ERAD components

  • Perform proximity ligation assays to detect direct interactions between PIGK and Hrd1

• Genetic manipulation approach:

  • Create systems with varying levels of PIGT to modulate PIGK assembly status

  • Use PIGK antibodies to monitor how assembly status affects ERAD targeting

  • Studies have shown that Hrd1 specifically regulates unassembled PIGK in PIGT-KO cells but not PIGK incorporated into the GPI-TA in PIGT-expressing cells

These approaches can help researchers understand the specific mechanisms by which unassembled PIGK is recognized and targeted for degradation by the ERAD machinery .

How can flow cytometry protocols be optimized when using PIGK antibodies for intracellular detection?

Optimizing flow cytometry protocols for intracellular PIGK detection requires careful attention to several parameters:

• Cell preparation:

  • Harvest cells using gentle methods (e.g., enzymatic dissociation with trypsin/EDTA) to maintain cellular integrity

  • Fix cells with 2% paraformaldehyde in phosphate buffer to preserve cellular architecture while maintaining PIGK antigenicity

• Permeabilization optimization:

  • Use 0.05% Triton X-100 in PBS for efficient permeabilization of cell membranes while preserving PIGK epitopes

  • Alternative permeabilization reagents like saponin (0.1-0.5%) or methanol may be tested if Triton X-100 yields suboptimal results

• Blocking strategy:

  • Include 1% BSA in staining buffers to reduce non-specific binding

  • Consider including 5-10% normal serum matched to the host of the secondary antibody

• Antibody titration:

  • Perform careful titration experiments to determine optimal antibody concentration

  • For PIGK-myc detection, 1:2000 dilution has been reported as effective

  • For direct detection of PIGK, follow manufacturer recommendations with titration optimization

• Multi-parameter analysis design:

  • Combine PIGK staining with markers of GPI-anchored proteins (e.g., CD59) to correlate PIGK expression with functional outcomes

  • Include viability dyes to exclude dead cells that may show non-specific antibody binding

• Controls for accurate analysis:

  • Include unstained, secondary-only, and isotype controls

  • Use PIGK knockout cells as negative controls

  • Consider including a positive control with PIGK overexpression

• Signal amplification considerations:

  • For low abundance detection, consider using biotin-streptavidin systems or other signal amplification methods

  • Fluorophore selection should account for instrument capabilities and other markers in the panel

These optimizations can enhance the specificity and sensitivity of PIGK detection in flow cytometry applications, enabling quantitative analysis of PIGK expression in various experimental conditions .

What strategies can researchers employ to troubleshoot weak or inconsistent signals when using PIGK antibodies?

When encountering weak or inconsistent signals with PIGK antibodies, consider these systematic troubleshooting approaches:

• Sample preparation optimization:

  • Ensure complete protein extraction using appropriate lysis buffers for membrane-associated proteins

  • For Western blotting, increase protein loading amount (50-80 μg total protein may be necessary)

  • Consider that PIGK is degraded by the ERAD pathway; adding proteasome inhibitors (10 μM MG132 for 6-8 hours) before cell lysis may increase detectable PIGK levels

• Antibody-specific considerations:

  • Verify antibody storage conditions (most PIGK antibodies should be stored at -20°C)

  • Prepare fresh dilutions of antibody for each experiment

  • Consider that different PIGK antibodies target different epitopes; epitope masking or modification may affect detection

• Application-specific troubleshooting:

  • For Western blotting:

    • Optimize transfer conditions for the 40-45 kDa range where PIGK is detected

    • Increase primary antibody incubation time (overnight at 4°C)

    • Test different membrane types (PVDF vs. nitrocellulose)

    • Use more sensitive detection systems (enhanced chemiluminescence substrates)

  • For immunohistochemistry/immunofluorescence:

    • Test different antigen retrieval methods (pH 6.0 citrate buffer vs. pH 9.0 TE buffer)

    • Extend primary antibody incubation times

    • Use signal amplification systems (tyramide, polymer-based detection)

• Biological context considerations:

  • PIGK expression depends on PIGT; verify PIGT expression in your experimental system

  • PIGK has multiple isoforms (45 kDa and 36 kDa); verify which isoform your antibody detects

  • Consider cell type-specific expression levels; PIGK has been detected in HT-1080, SMMC-7721, HepG2, HEK293, and other cell lines

• Validation with alternative approaches:

  • Use multiple antibodies targeting different PIGK epitopes to confirm results

  • Consider alternative detection methods (e.g., mass spectrometry)

  • Create positive controls by overexpressing PIGK in your experimental system

These strategies can help identify and address factors limiting PIGK detection in various experimental applications .

How can researchers effectively use PIGK antibodies to investigate protein-protein interactions within the GPI transamidase complex?

To investigate protein-protein interactions within the GPI transamidase complex using PIGK antibodies:

• Co-immunoprecipitation strategies:

  • Use PIGK antibodies validated for immunoprecipitation to pull down the entire GPI transamidase complex

  • Analyze co-precipitated proteins (PIGT, GPAA1, PIGU, PIGS) by Western blotting

  • Optimize lysis conditions to preserve native interactions (mild detergents like digitonin or CHAPS may better preserve membrane protein complexes)

• Proximity-based interaction techniques:

  • Perform proximity ligation assays (PLA) using PIGK antibodies paired with antibodies against other GPI transamidase components

  • Consider FRET (Fluorescence Resonance Energy Transfer) using fluorophore-conjugated antibodies against PIGK and interaction partners

• Sequential immunoprecipitation approach:

  • Perform tandem immunoprecipitation (first with anti-PIGK, then with antibodies against potential interactors) to identify direct versus indirect interactions

  • Compare complex composition in wild-type versus cells with individual components knocked out

• Cross-linking strategies:

  • Apply protein cross-linking before immunoprecipitation to stabilize transient interactions

  • Use mass spectrometry to identify cross-linked peptides and define interaction interfaces

• Domain-specific interaction mapping:

  • Use antibodies targeting different epitopes of PIGK (N-terminal , middle region, C-terminal) to determine which regions might be involved in protein-protein interactions

  • Compare immunoprecipitation efficiency with different antibodies to identify regions potentially occupied by interaction partners

• Comparative analysis approach:

  • Use PIGK antibodies to compare complex composition across different cell types, disease states, or experimental conditions

  • Investigate how complex formation correlates with GPI-anchored protein expression using flow cytometry

These approaches can provide valuable insights into the assembly, composition, and dynamics of the GPI transamidase complex and how PIGK contributes to its structure and function .

What methods can researchers use to validate PIGK antibody specificity in knockout and knockdown models?

Rigorous validation of PIGK antibody specificity using genetic models is essential for reliable research outcomes:

• CRISPR/Cas9 knockout validation:

  • Generate complete PIGK knockout cell lines using CRISPR/Cas9 gene editing

  • Verify knockout at the genomic level by sequencing the targeted region

  • Use PIGK antibodies in Western blotting to confirm absence of the specific band at 40-45 kDa in knockout cells compared to parental cells

  • Studies have shown that a band that migrates at approximately 45 kDa in parental cells is absent in PIGK-KO cells, confirming antibody specificity

• siRNA/shRNA knockdown approach:

  • Transfect cells with PIGK-targeting siRNA or transduce with shRNA

  • Include non-targeting siRNA/shRNA controls

  • Verify knockdown efficiency at the mRNA level using RT-qPCR

  • Use PIGK antibodies to demonstrate corresponding reduction in protein levels via Western blotting or other applications

  • Compare signal reduction between multiple PIGK antibodies targeting different epitopes

• Rescue experiment validation:

  • Reintroduce wild-type or epitope-tagged PIGK into knockout cells

  • Demonstrate restoration of antibody detection in rescued cells

  • Consider introducing PIGK with point mutations in the epitope region to confirm epitope specificity

• Functional validation approaches:

  • Verify that PIGK knockout results in expected functional outcomes (e.g., reduced GPI-anchored protein expression)

  • Use FLAER (fluorescently labeled inactive toxin aerolysin) staining to confirm reduced GPI-AP levels in PIGK knockout cells

  • Demonstrate that phenotypes correlate with loss of PIGK detection by antibodies

• Orthogonal detection methods:

  • Use mass spectrometry to confirm presence/absence of PIGK peptides in wild-type versus knockout samples

  • Employ RNA-scope or similar techniques to correlate protein detection with mRNA expression patterns