NPGR2 Antibody

What is NPGR2 and why are antibodies against it valuable for research?

NPGR2 (sometimes referred to as nanodomain protein of Golgi and plasma membrane 2) is a protein involved in complex formation with PI4Kα1 and functions in cellular membrane organization. The antibody against NPGR2 is valuable for studying plasma membrane protein complexes, particularly those involved in phosphoinositide signaling. This antibody enables detection of NPGR2-containing complexes that play critical roles in membrane nanodomain formation and lipid kinase activity regulation .

What are the basic specifications of commonly used anti-NPGR2 antibodies?

Anti-NPGR2 antibodies are typically raised against residues 1-273 of NPGR2 protein. The antibody recognizes a band at approximately 80 kDa (the expected size of NPGR2 is 82 kDa) in immunoblotting applications. These antibodies demonstrate high specificity, as they do not cross-react with the related NPGR1 protein. The antibody can recognize both native NPGR2 and NPGR2 fusion proteins (such as NPGR2-mCITRINE, which appears at approximately 110 kDa on western blots) .

How can researchers validate the specificity of anti-NPGR2 antibodies?

Validation of anti-NPGR2 antibody specificity can be performed through multiple approaches:

• Western blot analysis comparing wild-type samples with npgr2 knockout mutants (e.g., npgr2-1, npgr2-3) and npgr1npgr2-1 double mutants

• Testing recognition of NPGR2-tagged proteins (e.g., NPGR2-mCITRINE) versus related proteins (e.g., NPGR1-mCITRINE)

• Peptide competition assays using the immunizing peptide

• Immunoprecipitation followed by mass spectrometry to confirm pulled-down proteins

What are the optimal conditions for using anti-NPGR2 antibodies in immunoprecipitation experiments?

For successful immunoprecipitation experiments with anti-NPGR2 antibodies:

• Use fresh tissue lysates prepared in a buffer containing:

  • PBS (pH 7.2)

  • Protease inhibitor cocktail

  • Mild detergent (0.5-1% NP-40 or Triton X-100)

  • Low salt concentration (150mM NaCl) to preserve protein-protein interactions

• For co-immunoprecipitation of NPGR2-containing complexes:

  • Pre-clear lysates with protein A/G beads

  • Incubate with anti-NPGR2 antibody (typically 2-5 μg per 1mg of total protein)

  • Use appropriate controls (e.g., IgG from the same species)

  • Wash stringently but avoid disrupting native complexes

  • Analyze by western blot using antibodies against suspected interaction partners (e.g., PI4Kα1, HYC2)

How can NPGR2 antibodies be used to study subcellular localization in plant cells?

NPGR2 antibodies can be effectively used for immunofluorescence microscopy to study subcellular localization:

• Sample preparation:

  • Fix plant tissues with 4% paraformaldehyde

  • Optionally perform cell wall digestion for better antibody penetration

  • Permeabilize with 0.1-0.5% Triton X-100

• Immunostaining procedure:

  • Block with 3-5% BSA in PBS

  • Incubate with primary anti-NPGR2 antibody (1:200-1:500 dilution)

  • Detect with fluorophore-conjugated secondary antibodies

  • Counterstain membranes with appropriate markers

• Analysis:

  • NPGR2 signal should be enriched at the plasma membrane

  • No signal should be detected in the cell wall

  • Co-localization studies can be performed with markers for membrane nanodomains

What is the recommended protocol for western blot detection of NPGR2 in plant samples?

For optimal western blot detection of NPGR2:

• Sample preparation:

  • Extract total proteins from plant tissues using a buffer containing:

    • 50 mM Tris-HCl (pH 7.5)

    • 150 mM NaCl

    • 1% Triton X-100

    • 0.5% sodium deoxycholate

    • Protease inhibitor cocktail

  • For membrane protein enrichment, perform subcellular fractionation

• SDS-PAGE and transfer:

  • Separate proteins on 8-10% SDS-PAGE gels

  • Transfer to PVDF membranes at 100V for 1 hour or 30V overnight

• Immunodetection:

  • Block with 5% non-fat milk in TBST

  • Incubate with anti-NPGR2 antibody (1:1000-1:5000 dilution)

  • Wash with TBST

  • Incubate with HRP-conjugated secondary antibody

  • Develop using ECL substrates

  • Expected NPGR2 band: ~80 kDa

  • Tagged NPGR2-mCITRINE: ~110 kDa

How can researchers overcome weak or absent signal when using anti-NPGR2 antibodies?

When facing challenges with weak or absent signals using anti-NPGR2 antibodies:

• Sample preparation improvements:

  • Ensure complete tissue disruption

  • Use fresh samples to minimize protein degradation

  • Add phosphatase inhibitors if phosphorylation status is important

  • Consider membrane enrichment through fractionation (NPGR2 is enriched in plasma membrane fractions)

• Western blot optimization:

  • Reduce antibody dilution (try 1:500 instead of 1:1000)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use more sensitive detection methods (fluorescent secondaries or enhanced chemiluminescence)

  • Adjust protein loading (try 50-100 μg total protein)

  • Optimize transfer conditions for high molecular weight proteins

• Immunoprecipitation optimization:

  • Adjust lysis buffer composition (try different detergents)

  • Increase antibody amount

  • Extend incubation time

  • Use protein A or protein G beads based on antibody isotype

What factors might cause non-specific binding with anti-NPGR2 antibodies and how can they be mitigated?

Non-specific binding issues with anti-NPGR2 antibodies may be addressed through:

• Identifying common causes:

  • Insufficient blocking

  • Excessive antibody concentration

  • Cross-reactivity with related proteins

  • Sample over-fixation (for immunohistochemistry)

• Optimization strategies:

  • Increase blocking time and concentration (try 5% BSA instead of 3%)

  • Use alternative blocking agents (fish gelatin, casein)

  • Include 0.1% Tween-20 in antibody dilution buffers

  • Pre-adsorb antibody with tissue/lysate from npgr2 knockout mutants

  • Perform more stringent washes (increase salt concentration to 250-300mM NaCl)

  • Validate results using npgr2 knockout controls

How can researchers distinguish between NPGR1 and NPGR2 when both proteins are expressed in the same tissue?

To distinguish between the related proteins NPGR1 and NPGR2:

• Antibody selection and verification:

  • Use the validated anti-NPGR2 antibody that does not recognize NPGR1-mCITRINE

  • Verify specificity using npgr1, npgr2, and npgr1npgr2 double mutants

• Experimental approaches:

  • Perform side-by-side western blot analysis with recombinant NPGR1 and NPGR2

  • Use size differences (if any) to distinguish between the proteins

  • Employ immunoprecipitation followed by mass spectrometry for definitive identification

  • Consider using epitope-tagged versions of the proteins for unambiguous detection

• Data analysis:

  • Compare band intensity patterns with known expression profiles of NPGR1 and NPGR2

  • Use densitometry to quantify relative expression levels

How can anti-NPGR2 antibodies be used to study protein complexes involving PI4Kα1 and HYC family members?

For advanced studies of NPGR2-containing protein complexes:

• Sequential co-immunoprecipitation approach:

  • First immunoprecipitate with anti-NPGR2 antibody

  • Elute under mild conditions

  • Perform second immunoprecipitation with anti-PI4Kα1 or anti-HYC2 antibody

  • Analyze resulting complexes by western blot or mass spectrometry

• Proximity labeling methods:

  • Fuse BioID or APEX2 to NPGR2

  • Allow proximity-dependent biotinylation to occur in vivo

  • Purify biotinylated proteins

  • Identify complex components by mass spectrometry

  • Confirm interactions using anti-NPGR2 antibody in western blots

• Quantitative analysis of complex formation:

  • Use anti-NPGR2 antibody to immunoprecipitate complexes from wild-type and mutant backgrounds

  • Quantify co-precipitating proteins by western blot

  • Calculate stoichiometry of complex components

What approaches can be used to study the dynamics of NPGR2-containing complexes in response to cellular signaling events?

To investigate dynamic changes in NPGR2 complexes:

• Stimulation experiments:

  • Treat samples with relevant stimuli (e.g., hormones, stress conditions)

  • Perform time-course analysis of complex formation

  • Immunoprecipitate with anti-NPGR2 antibody at different time points

  • Analyze changes in complex composition and post-translational modifications

• Phosphorylation analysis:

  • Immunoprecipitate NPGR2 complexes before and after stimulus

  • Perform phospho-specific western blots

  • Use phosphatase treatment to confirm phosphorylation events

  • Correlate phosphorylation status with complex assembly/disassembly

• Membrane fractionation studies:

  • Separate membrane microdomains (e.g., detergent-resistant membranes)

  • Analyze NPGR2 distribution using the antibody

  • Track dynamic redistribution upon cellular signaling events

How can researchers employ anti-NPGR2 antibodies in combination with super-resolution microscopy to study membrane nanodomains?

For nanoscale analysis of NPGR2 in membrane domains:

• Sample preparation for super-resolution microscopy:

  • Fix cells with PFA and mild permeabilization

  • Label with anti-NPGR2 antibody (1:100-1:200)

  • Use fluorophore-conjugated secondary antibodies optimized for super-resolution techniques

• Advanced imaging techniques:

  • STORM (Stochastic Optical Reconstruction Microscopy):

    • Use appropriate buffer systems containing oxygen scavengers

    • Collect 10,000-20,000 frames for reconstruction

    • Analyze NPGR2 cluster size and distribution

  • PALM (Photoactivated Localization Microscopy):

    • Combine with tagged interaction partners

    • Perform dual-color imaging to assess co-localization at nanoscale

• Data analysis:

  • Quantify NPGR2 nanodomain size, density, and distribution

  • Measure co-localization with PI4Kα1 and other complex members

  • Compare wild-type patterns with those in mutant backgrounds

What strategies can be employed to study the role of NPGR2 in regulating PI4Kα1 activity using anti-NPGR2 antibodies?

To investigate NPGR2's role in PI4Kα1 regulation:

• Activity assays following immunodepletion:

  • Deplete NPGR2 from lysates using anti-NPGR2 antibody

  • Measure remaining PI4Kα1 activity

  • Compare with control immunodepletions

• In vitro reconstitution:

  • Immunopurify NPGR2 complexes using the antibody

  • Add purified components to assess complex formation requirements

  • Measure PI4Kα1 activity in reconstituted systems

  • Evaluate effects of NPGR2 mutations on complex assembly and activity

• Membrane targeting analysis:

  • Use anti-NPGR2 antibody to track localization in wild-type and mutant backgrounds

  • Correlate NPGR2 localization with PI4Kα1 recruitment

  • Measure local phosphoinositide production

  • Analyze the impact of NPGR2 mutations on membrane targeting and lipid kinase activity

How should researchers interpret differences in NPGR2 detection patterns between immunoblotting and immunofluorescence experiments?

When analyzing discrepancies between different detection methods:

• Technical considerations:

  • Immunoblotting detects denatured proteins while immunofluorescence detects native conformation

  • Epitope accessibility may differ between techniques

  • Fixation methods may affect antibody binding

  • Protein complexes may mask epitopes in immunofluorescence

• Biological interpretations:

  • Different subcellular pools of NPGR2 may exist with distinct properties

  • Post-translational modifications may affect antibody recognition

  • Protein-protein interactions may compete with antibody binding in situ

• Methodological approaches to resolve discrepancies:

  • Use multiple antibodies recognizing different epitopes

  • Perform peptide competition in both techniques

  • Compare results with tagged NPGR2 constructs

  • Validate with knockout controls

What statistical approaches and controls are recommended when quantifying NPGR2 levels or interactions using anti-NPGR2 antibodies?

For robust quantification of NPGR2:

• Recommended controls:

  • Technical replicates (minimum 3)

  • Biological replicates (minimum 3 independent experiments)

  • Loading controls for western blots (housekeeping proteins)

  • npgr2 knockout negative controls

  • NPGR2 overexpression positive controls

• Quantification methods:

  • For western blots:

    • Use densitometry software with linear range validation

    • Normalize to appropriate loading controls

    • Generate standard curves with recombinant protein for absolute quantification

  • For immunofluorescence:

    • Measure mean fluorescence intensity in defined regions

    • Account for background signal

    • Use identical acquisition parameters across samples

• Statistical analysis:

  • Apply appropriate statistical tests (t-test, ANOVA with post-hoc tests)

  • Report exact p-values and confidence intervals

  • Consider using non-parametric tests for non-normally distributed data

  • Include power analysis to determine required sample size

How can researchers distinguish between direct and indirect interactions when studying NPGR2 complexes using immunoprecipitation with anti-NPGR2 antibodies?

To differentiate direct from indirect protein interactions:

• Experimental approaches:

  • Cross-linking prior to immunoprecipitation to capture direct interactions

  • Yeast two-hybrid assays to validate direct interactions observed in co-IP

  • In vitro binding assays with purified components

  • Sequential co-IP to identify core complex components

  • Varying salt concentrations to disrupt weak interactions

• Analytical methods:

  • Compare co-IP efficiency across different detergent and salt conditions

  • Analyze complex stability with varying buffer stringencies

  • Use deletion mutants to map interaction domains

  • Employ proximity-dependent labeling methods to identify spatial relationships

• Data integration:

  • Combine co-IP results with data from yeast two-hybrid screens

  • Correlate findings with structural predictions and domain analysis

  • Construct interaction maps with confidence scores for each interaction