NPGR1 Antibody

What is NPGR1 and what cellular processes is it involved in?

NPGR1 (Nanodomain and phosphoinositide binding protein with repetitive motifs related 1) is a member of the NPG family that includes NPG1 and NPGR2. These proteins share high sequence identity and similar architecture. The C-terminal region of NPGR1 (amino acids 501-694) contains a high density of TPR (tetratricopeptide repeat) motifs that likely mediate interactions with PI4Kα1, a phosphatidylinositol 4-kinase . This interaction suggests NPGR1 plays a role in phosphoinositide signaling pathways, which are crucial for various cellular processes including membrane trafficking, cytoskeletal organization, and signal transduction. NPGR1 expression patterns are similar to those of NPGR2 but at considerably lower levels, which often makes NPGR2 a preferred subject for experimental studies .

What types of antibodies can be generated against NPGR1?

Researchers can generate several types of antibodies against NPGR1:

• Polyclonal antibodies: Developed by immunizing animals (typically rabbits) with synthetic peptides or recombinant NPGR1 proteins.

• Monoclonal antibodies: Created using hybridoma technology after immunizing mice with NPGR1 antigens.

• Recombinant antibodies: Engineered antibodies developed through phage display or similar technologies.

• Single-domain antibodies (VHHs): Smaller antibodies that can be computationally designed using approaches like RFdiffusion to target specific epitopes on NPGR1 .

For optimal specificity, antibodies are typically raised against unique regions of NPGR1 that differ from NPG1 and NPGR2, particularly outside the conserved TPR motifs, to prevent cross-reactivity within this protein family.

How should I validate the specificity of an NPGR1 antibody?

Proper validation of NPGR1 antibodies requires multiple complementary approaches:

• Western blot analysis using:

  • Recombinant NPGR1 protein as a positive control

  • Cell/tissue lysates from wildtype and NPGR1 knockout/knockdown samples

  • Comparison with lysates expressing related proteins (NPG1, NPGR2) to confirm specificity

• Immunoprecipitation followed by mass spectrometry to confirm the antibody pulls down NPGR1 specifically.

• Immunofluorescence comparing wildtype and NPGR1-deficient samples, with appropriate controls for secondary antibody background.

• Cross-adsorption tests with recombinant NPG1 and NPGR2 proteins to demonstrate lack of cross-reactivity with related family members .

A study examining NPGR family members found that antibodies raised against one member may not recognize others, as demonstrated when antibodies against NPGR2 did not recognize NPGR1-mCITRINE fusion proteins . This highlights the importance of thorough validation for antibody specificity within this protein family.

How can I optimize immunoprecipitation protocols for NPGR1 to investigate protein-protein interactions?

Optimizing immunoprecipitation (IP) of NPGR1 requires careful consideration of several technical factors:

• Lysis buffer composition:

  • Use buffers containing 1% NP-40 or 0.5% Triton X-100 with protease inhibitors

  • Include phosphatase inhibitors if phosphorylation-dependent interactions are of interest

  • Consider including specific detergents that preserve membrane-associated interactions

• Crosslinking strategies:

  • Implement reversible crosslinking (DSP or formaldehyde) to capture transient interactions

  • Optimize crosslinking time to maintain complex integrity without creating non-specific aggregates

• IP conditions:

  • Pre-clear lysates with appropriate control IgG and protein A/G beads

  • Compare different antibody concentrations (typically 2-5 μg per mg of protein lysate)

  • Optimize antibody incubation time (4-16 hours) and temperature (4°C)

• Washing stringency:

  • Implement a gradient of salt concentrations to eliminate non-specific binding

  • Consider using TAP-tagged (tandem affinity purification) NPGR1 constructs for improved specificity

Research on related proteins demonstrated successful co-immunoprecipitation of PI4Kα1 with NPGR2-mCITRINE using anti-GFP antibodies, suggesting similar approaches could be effective for NPGR1 studies . When designing these experiments, include appropriate controls, such as unrelated membrane proteins (like Lti6b-CITRINE), to confirm specificity of the interactions .

What are the key considerations when designing experiments to study NPGR1 and PI4Kα1 interactions?

Given the known interaction between NPGR family proteins and PI4Kα1, designing robust experiments to study NPGR1-PI4Kα1 interactions requires:

• Experimental systems selection:

  • Transient expression systems: HEK293T or COS-7 cells for initial validation

  • Stable expression: Create inducible expression systems to control expression levels

  • Endogenous systems: Primary cells or tissues where both proteins are naturally expressed

• Interaction domains mapping:

  • Create truncation constructs focusing on the C-terminal region (aa 501-694) of NPGR1 where TPR motifs are concentrated

  • Design point mutations in predicted interaction surfaces based on structural predictions

  • Use yeast two-hybrid assays with fragments to identify minimal interaction domains

• Functional studies:

  • Measure PI4K activity in the presence/absence of NPGR1

  • Assess changes in phosphoinositide levels using specific biosensors

  • Analyze subcellular localization of both proteins using confocal microscopy

• Competition assays:

  • Test whether NPGR1 competes with NPGR2 or NPG1 for binding to PI4Kα1

  • Use purified proteins or peptides corresponding to interaction domains

The research approach should be similar to that used in the study of NPGR2, which successfully demonstrated interaction with PI4Kα1 through both yeast two-hybrid assays and co-immunoprecipitation experiments .

How can computational approaches be used to design antibodies with improved specificity for NPGR1?

Advanced computational approaches offer promising strategies for designing highly specific NPGR1 antibodies:

• Structure-based design using RFdiffusion:

  • This approach enables the de novo design of antibodies targeting specific epitopes on NPGR1

  • The RFdiffusion method can be fine-tuned predominantly on antibody complex structures

  • The framework sequence and structure can be specified at inference time, allowing customization

  • The rigid body position (dock) between antibody and target epitope is designed along with the framework structure

• CDR loop design optimization:

  • ProteinMPNN can be used to design CDR loop sequences that specifically target unique regions of NPGR1

  • This approach creates antibodies that make diverse interactions with the target epitope while differing significantly from existing antibodies

• Validation using RoseTTAFold2:

  • Fine-tuned RoseTTAFold2 network can filter designs based on predicted structural similarity

  • This computational validation step has been shown to correlate well with experimental success

• Epitope targeting strategy:

  • Focus on regions that differ between NPGR1 and its family members (NPGR2, NPG1)

  • Target non-immunodominant epitopes to improve specificity and reduce cross-reactivity

These computational approaches offer significant advantages over traditional methods, potentially producing antibodies with higher specificity while reducing time and costs associated with immunization or library screening approaches .

What are the best expression systems for producing recombinant NPGR1 antigens for antibody development?

Selecting appropriate expression systems for NPGR1 antigen production depends on several factors:

• Bacterial expression systems:

  • E. coli BL21(DE3): Ideal for producing fragments of NPGR1, particularly domains without complex folding requirements

  • Modifications: Use fusion tags (MBP, SUMO, or TRX) to enhance solubility

  • Limitations: Full-length NPGR1 may not fold correctly or may form inclusion bodies

• Insect cell expression:

  • Sf9/High Five cells: Better for full-length NPGR1 expression with proper folding

  • Advantages: More likely to maintain tertiary structure and post-translational modifications

  • System: Baculovirus expression vector system (BEVS) with polyhistidine or GST tags for purification

• Mammalian expression:

  • HEK293F/Expi293: Optimal for production of NPGR1 with native folding and modifications

  • Methods: Transient transfection with high-density suspension cultures

  • Applications: Particularly valuable when native conformation is critical for antibody recognition

• Cell-free expression systems:

  • Wheat germ extract: Alternative for difficult-to-express protein segments

  • Advantages: Rapid production and avoidance of inclusion body formation

For experimental screening of antigen expression, parallel testing of different domains and expression systems is recommended. Based on patterns observed in related proteins, focusing on the C-terminal region (aa 501-694) of NPGR1 containing the TPR motifs may yield antigens that generate antibodies recognizing functionally relevant epitopes .

How can I distinguish between antibodies that recognize NPGR1 versus related family members like NPGR2?

Distinguishing antibody specificity between closely related NPGR family members requires rigorous validation strategies:

• Parallel testing with all family members:

  • Express tagged versions of NPGR1, NPGR2, and NPG1 (e.g., with mCITRINE or similar tags)

  • Perform western blot analysis with the antibody of interest against all three proteins

  • Compare band intensities to assess relative affinity and cross-reactivity

• Competitive binding assays:

  • Pre-incubate antibodies with recombinant NPGR2 or NPG1 before testing against NPGR1

  • Measure the decrease in signal to quantify cross-reactivity

• Epitope mapping:

  • Use peptide arrays covering unique and conserved regions of all family members

  • Identify specific epitope(s) recognized by the antibody to predict cross-reactivity

• CRISPR-Cas9 validation:

  • Test antibody in wildtype cells versus NPGR1 knockout cells

  • If signal persists in knockout cells, assess whether this is due to recognition of NPGR2/NPG1

Research has demonstrated that antibodies raised against specific NPGR family members may not cross-react with others. For example, antibodies specifically recognizing NPGR2 did not recognize NPGR1-mCITRINE, indicating distinct epitope recognition . This suggests careful epitope selection can yield antibodies that discriminate between these related proteins.

What controls should be included when using NPGR1 antibodies in immunoprecipitation-mass spectrometry experiments?

Robust immunoprecipitation-mass spectrometry (IP-MS) experiments for NPGR1 interactome analysis require comprehensive controls:

• Negative controls:

  • IgG control: Use matched isotype control antibodies from the same species

  • Knockout/knockdown control: Perform parallel IP from NPGR1-deficient samples

  • Competition control: Pre-incubate antibody with excess recombinant NPGR1 antigen

• Positive controls:

  • Spike-in control: Add known quantities of recombinant NPGR1 to samples

  • Known interactor control: Confirm detection of established binding partners (e.g., PI4Kα1)

• Technical validation:

  • Reciprocal IP: Confirm key interactions by IP with antibodies against identified partners

  • Two different NPGR1 antibodies: Compare interactomes obtained with antibodies targeting different epitopes

  • Tagged NPGR1: Compare native antibody results with anti-tag IP results

• Data analysis controls:

  • Implement stringent statistical filters (fold change >2, p-value <0.05)

  • Use CRAPome database to filter common contaminants

  • Apply quantitative approaches (SILAC, TMT, LFQ) with appropriate normalization

Research with related protein NPGR2 effectively used tagged versions (NPGR2-mCITRINE) and unrelated membrane proteins (Lti6b-CITRINE) as controls to demonstrate specific co-immunoprecipitation with PI4Kα1 . Similar control strategies should be applied to NPGR1 studies to ensure reliable identification of genuine interactors.

How can NPGR1 antibodies be used to study its role in phosphoinositide signaling pathways?

NPGR1 antibodies can be strategically employed to investigate phosphoinositide signaling through several approaches:

• Localization studies:

  • Use immunofluorescence to track NPGR1 localization during cellular responses

  • Combine with phosphoinositide biosensors to correlate NPGR1 positioning with PI4P production

  • Implement super-resolution microscopy to visualize nanodomain organization

• Interaction dynamics:

  • Employ proximity ligation assays (PLA) to visualize NPGR1-PI4Kα1 interactions in situ

  • Use FRET-based approaches with labeled antibodies to measure interaction kinetics

  • Implement BiFC (Bimolecular Fluorescence Complementation) with antibody fragments

• Functional manipulation:

  • Develop function-blocking antibodies that disrupt NPGR1-PI4Kα1 interactions

  • Use antibody microinjection to acutely inhibit NPGR1 function

  • Combine with phosphoinositide measurements to assess impact on PI4P production

• Pathway analysis:

  • Apply antibodies in ChIP-seq if NPGR1 has nuclear functions

  • Use for proximity biotinylation (BioID) to identify context-specific interactors

  • Implement for immunoprecipitation before and after pathway stimulation

Evidence from related family members suggests NPGR1 likely interacts with PI4Kα1 through its C-terminal TPR motifs (aa 501-694) . This interaction places NPGR1 within a molecular complex involved in phosphoinositide metabolism, making antibodies valuable tools for dissecting its precise role in these signaling networks.

What are the challenges in developing antibodies that recognize post-translationally modified forms of NPGR1?

Developing antibodies against post-translationally modified (PTM) forms of NPGR1 presents several unique challenges:

• Identification of relevant modifications:

  • Perform phosphoproteomics, ubiquitylomics, or other PTM-specific analyses to identify actual modification sites on NPGR1

  • Prioritize modifications that change upon cellular stimulation or in disease states

  • Focus on modifications within functional domains, particularly the TPR motifs

• Antigen design strategies:

  • Synthesize peptides containing the specific modification of interest

  • Ensure sufficient peptide length (15-20 amino acids) with the modification centrally positioned

  • Consider using branched peptides to increase immunogenicity of small modifications

• Specificity validation:

  • Test against both modified and unmodified recombinant proteins

  • Validate with cell lysates treated with phosphatases or other enzymes that remove specific PTMs

  • Employ CRISPR knock-in mutations at modification sites as negative controls

• Technical considerations:

  • Include modification-stabilizing phosphatase/protease inhibitors in all buffers

  • Consider using multiple modifications in a single antigen if they co-occur in vivo

  • Develop blocking peptides for both modified and unmodified forms for validation

Given that TPR motif-containing proteins like NPGR1 often undergo phosphorylation that regulates their protein-protein interactions, developing phospho-specific antibodies may be particularly valuable for understanding how NPGR1 function is dynamically regulated within phosphoinositide signaling pathways .

How do antibody-based detection methods compare with genetic tagging approaches for studying NPGR1?

Both antibody-based detection and genetic tagging offer complementary approaches for NPGR1 research, each with distinct advantages and limitations:

Research on related proteins has successfully employed both approaches, as seen with native antibodies against PI4Kα1 and tagged versions of NPGR2 (NPGR2-mCITRINE) . For optimal results, combining both methods provides validation through complementary approaches. For example, co-immunoprecipitation studies can be performed both with antibodies against native proteins and with tagged versions to confirm interactions .

Why might my NPGR1 antibody show inconsistent results across different experimental techniques?

Inconsistent performance of NPGR1 antibodies across different applications can stem from several technical factors:

• Epitope accessibility issues:

  • Protein conformation differences between applications (native in IP vs. denatured in WB)

  • Masking of epitopes by protein-protein interactions or post-translational modifications

  • Fixation-induced epitope alterations in immunofluorescence

• Protocol-specific considerations:

  • Western blot: Insufficient blocking, inappropriate transfer conditions for high MW proteins

  • Immunoprecipitation: Detergent choice affecting protein complex stability

  • Immunofluorescence: Fixation method incompatible with epitope recognition

• Sample preparation variables:

  • Lysis buffer composition affecting protein solubility and epitope exposure

  • Fixation protocols (PFA vs. methanol) altering protein conformation

  • Storage conditions leading to protein degradation or modification changes

• Technical remediation strategies:

  • Epitope retrieval methods for immunohistochemistry/immunofluorescence

  • Membrane stripping and re-probing with alternative antibodies

  • Testing multiple antibodies recognizing different epitopes

Research with antibodies against related proteins has shown that antibody performance can vary significantly between applications. For instance, an antibody might successfully detect a protein in western blot but fail in immunoprecipitation due to conformation-dependent epitope recognition . Thorough validation in each specific application is essential.

What strategies can address cross-reactivity issues with NPGR1 antibodies?

Addressing cross-reactivity with NPGR1 antibodies requires systematic troubleshooting and validation:

• Identifying cross-reactivity sources:

  • Test against recombinant NPGR1, NPGR2 and NPG1 to quantify family member cross-reactivity

  • Perform immunoprecipitation followed by mass spectrometry to identify all captured proteins

  • Utilize knockout/knockdown validation in combination with overexpression systems

• Antibody purification approaches:

  • Implement affinity purification against the specific NPGR1 peptide used as immunogen

  • Perform negative selection by passing antibody through columns containing related proteins

  • Use competitive elution to obtain highest-affinity antibody fractions

• Blocking strategies:

  • Pre-incubate antibodies with recombinant NPGR2/NPG1 to block cross-reactive antibodies

  • Use peptide competition with epitope-specific peptides to confirm specificity

  • Include recombinant competing proteins in immunoprecipitation buffers

• Analytical solutions:

  • Implement higher stringency washing conditions in western blot and IP protocols

  • Use gradient gels to better separate similarly-sized family members

  • Employ two-color detection systems to simultaneously visualize multiple proteins

The reported lack of cross-reactivity between antibodies against NPGR2 and NPGR1-mCITRINE suggests that despite high sequence similarity, properly designed antibodies can distinguish between these family members . Focus on unique regions outside the conserved TPR motifs when designing antigens for antibody development.

How can I optimize immunofluorescence protocols for detecting low-abundance NPGR1 in tissue samples?

Detecting low-abundance NPGR1 in tissue samples requires optimized immunofluorescence protocols:

• Signal amplification methods:

  • Tyramide signal amplification (TSA): Can increase signal 10-100 fold

  • Polymer-based detection systems: HRP-conjugated polymers with multiple secondary antibodies

  • Sequential application of primary and secondary antibodies (multiple rounds)

• Epitope retrieval optimization:

  • Test multiple retrieval buffers (citrate pH 6.0, EDTA pH 8.0, Tris-EDTA pH 9.0)

  • Optimize retrieval time and temperature (microwave, pressure cooker, water bath)

  • Combine heat-induced and enzymatic retrieval for difficult epitopes

• Background reduction strategies:

  • Extended blocking (overnight at 4°C) with 5-10% normal serum

  • Include detergents (0.3% Triton X-100) and carrier proteins (BSA, casein)

  • Implement avidin/biotin blocking if using biotinylated detection systems

• Technical enhancements:

  • Use thin sections (5 μm or less) to improve antibody penetration

  • Extend primary antibody incubation (24-48 hours at 4°C)

  • Employ fluorophores with distinct spectral properties from tissue autofluorescence

  • Implement spectral unmixing to separate signal from autofluorescence

• Controls and validation:

  • Include absorption controls with immunizing peptide

  • Compare staining patterns with in situ hybridization for NPGR1 mRNA

  • Use tissue from NPGR1 knockout animals as negative controls

Since NPGR1 expression is lower than NPGR2 , these signal optimization techniques are particularly important when studying this family member in native tissues where its abundance may be near detection limits with standard protocols.