NETO2 Antibody

What is NETO2 and why are antibodies against it important for neuroscience research?

NETO2 is a neurospecific type I transmembrane protein belonging to the NETO family. It contains two extracellular CUB domains followed by a low-density lipoprotein class A (LDLa) domain, and an intracellular FXNPXY-like motif . NETO2 functions as an accessory subunit of neuronal kainate-sensitive glutamate receptors, particularly GRIK2 and GRIK3, where it increases kainate-receptor channel activity, slows the decay kinetics of the receptors, and modulates their agonist sensitivity .

NETO2 antibodies are essential for neuroscience research because they enable:

• Detection and quantification of NETO2 protein in neural tissues

• Investigation of NETO2's role in regulating kainate receptor function and trafficking

• Study of NETO2's interaction with other proteins, including the K+-Cl- cotransporter KCC2

• Examination of NETO2's phosphorylation states and their functional implications

Human and mouse NETO2 share 98% amino acid sequence homology in their extracellular domains, making many antibodies cross-reactive across species .

What applications are NETO2 antibodies typically used for?

NETO2 antibodies have been validated for multiple experimental applications:

For phosphorylation-specific studies, custom phospho-specific antibodies like the Ser(P)-409 antibody have been developed, requiring specialized blocking conditions (5% PhosphoBLOCKER) and specific incubation parameters .

How should NETO2 antibodies be stored and handled for optimal results?

Proper storage and handling are critical for maintaining antibody performance:

Storage conditions:

• Store at -20°C for long-term preservation (12 months from receipt date)

• For reconstituted antibodies, store at 4°C for up to 1 week under sterile conditions

• For longer periods (up to 6 months), aliquot and store at -20°C to -70°C

• Avoid repeated freeze-thaw cycles by using a manual defrost freezer

Handling recommendations:

• Reconstitute lyophilized antibodies in specified buffer (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

• Centrifuge antibody vials before opening (10,000 x g for 5 min)

• For phospho-specific NETO2 antibodies, block membranes in 5% PhosphoBLOCKER rather than standard blocking agents

• For immunoprecipitation, incubate antibodies with lysates for 8 hours at 2-8°C

Specific reconstitution volumes vary by product format: 25 μL, 50 μL, or 0.2 mL double distilled water depending on sample size .

How can NETO2 antibodies be used to study kainate receptor function and trafficking?

NETO2 antibodies provide powerful tools for investigating kainate receptor (KAR) function and trafficking through several methodological approaches:

Co-immunoprecipitation studies:

• Prepare membrane fractions from brain tissue or cultured neurons

• Immunoprecipitate with NETO2 antibodies (1-2 μg/mL for 8 hours at 2-8°C)

• Detect co-precipitated KAR subunits by Western blotting

• Use protein G to absorb NETO2-antibody complexes

This approach has been instrumental in establishing NETO2 as an auxiliary subunit of KARs.

Surface expression analysis:

• Transfect neurons with HA-tagged GluK1 receptor alone or with Neto proteins

• Perform surface labeling using antibodies against extracellular epitopes

• Quantify surface expression through fluorescence microscopy

Research has demonstrated that both Neto1 and Neto2 can drive robust surface expression of GluK1, but Neto2 specifically affects receptor localization at synapses .

Electrophysiological studies combined with molecular approaches:

• Express KARs with or without NETO2 in heterologous systems

• Measure receptor kinetics through patch-clamp recordings

• Correlate with NETO2 expression confirmed by antibody-based techniques

Studies have shown that Neto2 specifically slows deactivation of GluK1-mediated currents and alters desensitization kinetics, with Neto1 and Neto2 having opposite effects on desensitization rate (Neto1 enhances while Neto2 slows desensitization) .

What considerations should be made when choosing a NETO2 antibody for detecting phosphorylated forms?

Detecting phosphorylated NETO2 requires specific antibody selection and experimental design:

Phosphorylation site specificity:

• NETO2 can be phosphorylated at Ser-409 by both CaMKII and PKA

• Choose antibodies specifically generated against phosphorylated epitopes (e.g., Ser(P)-409 antibody)

• Validate using phospho-deficient mutants (S409A) and in vitro kinase assays

Production of phospho-specific antibodies:

• Immunize with synthetic phosphopeptides (e.g., Ac-REKEIpSADLA-amide)

• Collect serum and affinity-purify using phosphopeptide-immobilized columns

• Test specificity using phosphorylated vs. non-phosphorylated proteins

Experimental validation protocol:

• Generate phospho-deficient mutants (e.g., S409A)

• Perform in vitro kinase assays using purified GST-Neto2 (WT or S409A)

• Confirm specific signal only with wild-type NETO2 when incubated with kinases

Optimized detection conditions:

• Block membranes in 5% PhosphoBLOCKER at room temperature for 1 hour

• Incubate with phospho-specific antibody at 4°C overnight (1 μg/μl)

• Use secondary antibody diluted in 1% PhosphoBLOCKER

This approach has successfully demonstrated that Neto2 is phosphorylated at Ser-409 by both CaMKII and PKA, both in vitro and in vivo, with phosphorylation being detected in brain tissue from young wild-type animals .

How can immunoprecipitation with NETO2 antibodies help identify novel protein interactions?

Immunoprecipitation (IP) using NETO2 antibodies has proven valuable for discovering protein interactions:

Sample preparation approach:

• Prepare membrane fractions from brain tissue to enrich for transmembrane proteins

• Use non-denaturing detergents (e.g., Triton X-100, NP-40) in lysis buffers

• Include protease inhibitors and phosphatase inhibitors (if studying phosphorylation-dependent interactions)

Immunoprecipitation protocol:

• Incubate brain membrane preparations with anti-Neto2 antibodies

• Absorb NETO2-antibody complexes using Protein G

• Analyze immunoprecipitated material by Western blotting or mass spectrometry

Demonstrated success:
Using this approach, researchers discovered that anti-Neto2 antibodies co-immunoprecipitated KCC2 protein from wild-type but not from Neto2-null mice . This finding was first identified through a GST pull-down experiment using a GST-Neto2 cytoplasmic domain fusion protein, which enriched a 145-kDa band later identified as KCC2 through peptide mapping .

Validation of interactions:

• Perform reciprocal co-IP (anti-KCC2 antibodies successfully co-immunoprecipitated Neto2)

• Test specificity with knockout controls (no co-IP of KCC2 from Neto2-null samples)

• Verify against related proteins (NETO2 did not co-immunoprecipitate with NKCC1 or KCC3)

This systematic approach revealed NETO2's previously unknown role in regulating neuronal chloride homeostasis through interaction with KCC2, demonstrating how IP can uncover novel functions beyond NETO2's established role with kainate receptors.

What are the differences in NETO2 antibody performance between immunofluorescence and Western blotting applications?

NETO2 antibodies demonstrate different performance characteristics across applications:

Western Blotting Characteristics:

• Typical band size: 59 kDa (predicted molecular weight)

• Optimal antibody dilutions: 1:2000-1:10000 for most recombinant antibodies

• Sample types: Brain tissue (particularly cerebellum and brain lysates) shows strong signal

• Buffer recommendations: PBS with 0.02% sodium azide and 50% glycerol for storage

• Blocking conditions: 5% non-fat milk or BSA (5% PhosphoBLOCKER for phospho-specific antibodies)

Immunofluorescence Considerations:

• Signal pattern: NETO2 shows distinct distribution patterns in neurons, with enrichment at the somatic periphery

• Fixation impact: Paraformaldehyde fixation (4%) is generally suitable

• Quantification approach: Measure both peak fluorescence intensity (at somatic periphery) and throughout soma

• Comparative analysis: Neto2-null neurons showed 27% decrease in somatic fluorescence intensity and 53% decrease in peak intensity

Key Differences Between Applications:

• Sensitivity: Immunofluorescence often requires higher antibody concentrations than Western blotting

• Background: Western blotting typically provides better signal-to-noise ratio

• Spatial information: Immunofluorescence preserves subcellular localization data

• Signal validation: Western blotting provides molecular weight confirmation

• Co-localization studies: Immunofluorescence allows assessment of NETO2 distribution relative to other proteins

For studying NETO2 distribution in neurons, immunofluorescence has revealed that NETO2 knockout significantly alters KCC2 distribution, with decreased fluorescence intensity both at the somatic periphery and throughout the soma .

How should proper controls be designed for NETO2 antibody experiments?

Rigorous control experiments are essential for reliable results with NETO2 antibodies:

Antibody Specificity Controls:

• Genetic Controls:

  • Compare wild-type vs. Neto2-null samples

  • Example: Anti-Neto2 antibodies co-immunoprecipitated KCC2 from wild-type but not from Neto2-null mice

• Peptide Competition:

  • Pre-incubate antibody with immunizing peptide

  • For phospho-specific antibodies, use both phosphorylated and non-phosphorylated peptides

• Mutant Controls:

  • For phospho-specific antibodies, compare wild-type vs. phospho-deficient mutants (S409A)

  • Example: Ser(P)-409 antibody signal was observed only with GST-Neto2 WT when incubated with kinases, not with the S409A mutant

Application-Specific Controls:

• Western Blotting:

  • Positive tissue control: Human cerebellum, SH-SY5Y cell lysates, human brain lysates

  • Loading control: Housekeeping protein (β-actin, GAPDH)

  • Expected band size: 59 kDa

• Immunoprecipitation:

  • Input sample: Run a portion of pre-IP lysate

  • IgG control: Non-specific IgG from same species

  • Reciprocal IP: If studying interactions, perform pull-down with antibodies against partner protein

• Phosphorylation Studies:

  • Activation controls: Treat with forskolin (PKA activator) or co-express constitutively active CaMKII (T286D)

  • Demonstration of endogenous phosphorylation: Immunoprecipitate from brain lysates and blot with phospho-specific antibody

Implementing these controls ensures experimental validity and facilitates troubleshooting when unexpected results occur.

What factors affect the cross-reactivity of NETO2 antibodies between species?

Cross-reactivity of NETO2 antibodies between species is influenced by several key factors:

Sequence homology:

• Human and mouse NETO2 share 98% amino acid sequence homology in their extracellular domains

• This high conservation enables many antibodies to recognize NETO2 across multiple species

Epitope selection considerations:

• Antibodies targeting highly conserved regions show broader cross-reactivity

• The extracellular domain (particularly Ile23-Lys345) is highly conserved and often used as an immunogen

• C-terminal regions may show more species variation

Validated cross-reactivity patterns:

• Many commercial antibodies are validated for human, mouse, and rat NETO2

• Some antibodies show broader reactivity with rabbit, bovine, dog, guinea pig, and horse NETO2

• Always verify species reactivity through experimental validation even if theoretically cross-reactive

Application-dependent cross-reactivity:

• An antibody may cross-react in Western blot but not in immunoprecipitation or immunohistochemistry

• Different applications expose different epitopes and may require different binding affinities

Recombinant vs. conventional antibodies:

• Recombinant monoclonal antibodies often show more consistent cross-reactivity

• Example: Recombinant rabbit monoclonal NETO2 antibody [EPR3497] is validated for human, mouse, and rat in Western blotting

When selecting NETO2 antibodies for cross-species studies, prioritize those with validation data in your target species and application, and conduct preliminary experiments to confirm reactivity before proceeding with full studies.

How can NETO2 antibodies be used to investigate phosphorylation-dependent interactions?

NETO2 undergoes phosphorylation that may regulate its interactions with other proteins. Here's how to investigate these phosphorylation-dependent interactions:

Identifying phosphorylation sites:

• Use mass spectrometry to identify phosphorylation sites

  • Two phosphorylation sites have been identified, with Ser-409 being detected in both HeLa cells and brain tissue

• Generate phospho-specific antibodies against identified sites

  • Example: Custom Ser(P)-409 antibody generated using synthetic phosphopeptide Ac-REKEIpSADLA-amide

• Validate antibody specificity using phospho-deficient mutants (S409A)

Investigating kinase regulation:

• Perform in vitro kinase assays with purified GST-Neto2

  • Both CaMKII and PKA phosphorylate NETO2 at Ser-409

• Co-transfect cells with Neto2 and constitutively active kinases

  • Constitutively active CaMKII (T286D) increases Neto2 Ser-409 phosphorylation

• Activate endogenous kinases and assess NETO2 phosphorylation

  • Forskolin treatment (PKA activator) increases NETO2 phosphorylation

Studying phosphorylation-dependent interactions:

• Prepare lysates under phosphorylation-preserving conditions

  • Include phosphatase inhibitors in all buffers

• Compare co-immunoprecipitation under different phosphorylation states

  • Phosphorylated vs. dephosphorylated (phosphatase-treated)

  • Kinase-activated vs. kinase-inhibited conditions

• Use phospho-mimetic (S409D/E) and phospho-deficient (S409A) mutants

  • Compare their interaction profiles with potential binding partners

Technical considerations:

• Use phospho-blocking reagents (PhosphoBLOCKER) during immunoblotting

• Consider proteomic approaches to identify phosphorylation-dependent interactors

• Validate findings in both heterologous systems and neuronal preparations

This systematic approach can reveal how phosphorylation of NETO2 at Ser-409 (and potentially other sites) regulates its interactions with kainate receptors, KCC2, or other neuronal proteins.

How can NETO2 antibodies be used to study developmental changes in expression and localization?

NETO2 expression and distribution change throughout development, making developmental studies a valuable application for NETO2 antibodies:

Developmental expression profiling:

• Prepare brain tissue samples from different developmental stages (embryonic, postnatal, adult)

• Perform Western blotting with NETO2 antibodies

• Quantify relative expression levels normalized to appropriate loading controls

• Note that young animals (P1-P2) show high expression of NETO2

Subcellular localization changes:

• Prepare brain sections or cultured neurons from different developmental stages

• Perform immunofluorescence with NETO2 antibodies

• Co-stain with developmental markers and synaptic proteins

• Quantify NETO2 distribution patterns and co-localization coefficients

Phosphorylation state during development:

• Immunoprecipitate NETO2 from brain tissue at different developmental stages

• Probe with phospho-specific antibodies (e.g., Ser(P)-409)

• Compare phosphorylation levels across development

• Correlate with expression or activity of relevant kinases (CaMKII, PKA)

Technical considerations:

• Age-dependent optimization of fixation protocols (younger tissue typically requires shorter fixation)

• Adjustment of antibody concentrations for developmental stages with lower expression

• Careful selection of normalization controls appropriate for developmental studies

Developmental studies using NETO2 antibodies can provide insights into the maturation of kainate receptor signaling and KCC2-mediated chloride homeostasis, both critical processes in neural circuit development.

What are the methodological approaches for using NETO2 antibodies in electrophysiological studies?

Combining antibody-based techniques with electrophysiology provides powerful insights into NETO2 function:

Correlation of protein expression with receptor function:

• Perform patch-clamp recordings to measure kainate receptor currents

• In the same preparation, conduct immunofluorescence with NETO2 antibodies

• Correlate NETO2 expression levels with:

  • Amplitude of kainate-evoked currents

  • Deactivation kinetics (Neto2 slows deactivation of GluK1)

  • Desensitization rates (Neto2 slows desensitization)

Molecular manipulation combined with antibody detection:

• Express wild-type or mutant NETO2 in neurons or heterologous cells

• Confirm expression and localization using NETO2 antibodies

• Measure functional parameters through electrophysiology

• For example, Neto1 and Neto2 have opposite effects on GluK1 desensitization (Neto1 enhances while Neto2 slows it)

Verification of knockout/knockdown efficiency:

• Generate or obtain Neto2-null models

• Confirm absence of NETO2 protein using antibodies

• Compare electrophysiological properties of wild-type vs. knockout preparations

• Example: Neto2-null neurons show altered KCC2 distribution and function

Technical implementation:

• For acute brain slices:

  • Perform electrophysiological recordings

  • Fix slice post-recording

  • Process for immunohistochemistry with NETO2 antibodies

• For cultured neurons:

  • Mark recorded neurons (e.g., with fluorescent dyes)

  • Fix and immunostain with NETO2 antibodies

  • Relocate recorded neurons for antibody signal quantification

This integrated approach has revealed that NETO2 modifies how kainate receptors respond to prolonged exposure to glutamate, with significant effects on receptor kinetics and synaptic function .

What are common problems and solutions when using NETO2 antibodies in Western blotting?

Researchers may encounter several challenges when using NETO2 antibodies for Western blotting:

Optimization tips:

• For brain samples, prepare membrane fractions to enrich for NETO2

• For phospho-NETO2 detection, block with 5% PhosphoBLOCKER instead of milk/BSA

• Include positive controls: human cerebellum, SH-SY5Y cells, or brain lysates

• Expected MW: 59 kDa for canonical NETO2

• For troubleshooting, test antibody on recombinant NETO2 or overexpression systems first

These approaches can help overcome common problems and achieve reliable Western blot results with NETO2 antibodies.

How can researchers validate the specificity of NETO2 antibodies in their experimental system?

Thorough validation of NETO2 antibody specificity is crucial for reliable experimental results:

Genetic validation approaches:

• Knockout/knockdown controls:

  • Test antibodies on samples from NETO2 knockout animals or cells with NETO2 knockdown

  • Absence of signal confirms specificity

  • Example: Anti-Neto2 antibodies did not co-immunoprecipitate KCC2 from Neto2-null mice

• Overexpression systems:

  • Compare signal between NETO2-transfected cells and vector-only controls

  • Use tagged NETO2 constructs to confirm co-localization of antibody signal with tag

  • Surface labeling experiments showed strong signal only when Neto proteins were co-expressed with GluK1

Biochemical validation approaches:

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide

  • Specific signals should be eliminated or dramatically reduced

  • Particularly useful for custom antibodies like phospho-specific ones

• Multiple antibodies targeting different epitopes:

  • Use at least two antibodies recognizing different regions of NETO2

  • Concordant results increase confidence in specificity

  • Compare monoclonal and polyclonal antibodies targeting different domains

• Immunodepletion experiments:

  • Perform sequential immunoprecipitations to deplete NETO2

  • Signal should progressively decrease with each round

  • Final depleted sample serves as negative control

Application-specific validation:

• Western blotting:

  • Confirm band at expected molecular weight (59 kDa)

  • Look for absence of band in knockout/knockdown samples

  • For phospho-specific antibodies, show signal reduction with phosphatase treatment

• Immunohistochemistry:

  • Compare staining pattern with published NETO2 distribution

  • Confirm absence of signal in knockout tissue

  • Demonstrate reduced signal with peptide competition

• Immunoprecipitation:

  • Show enrichment of NETO2, specifically at expected MW

  • Demonstrate co-precipitation of known interactors (KCC2, KARs)

  • Confirm absence of unrelated proteins as negative controls

Implementing these validation approaches ensures that experimental results reflect authentic NETO2 biology rather than antibody artifacts.