GLR1.4 Antibody

What is the GluR4 antibody and what does it detect?

GluR4 antibodies detect the protein encoded by the gene GRIA4 (glutamate ionotropic receptor AMPA type subunit 4) in humans. This 902-amino acid protein belongs to the Glutamate-gated ion channel (TC 1.A.10.1) family, GRIA4 subfamily. GluR4 is primarily membrane-associated and contains multiple glycosylation sites. These antibodies are critical tools for studying AMPA receptor composition and distribution in neural tissues .

What species reactivity is available for GluR4 antibodies?

GluR4 antibodies demonstrate cross-reactivity across multiple species, with most commercial antibodies recognizing human, mouse, and rat orthologs. Some antibodies offer extended reactivity to chicken, monkey, and zebrafish GluR4. When selecting an antibody for your research, verify species reactivity in the product documentation, as epitope conservation varies across antibody clones .

What are the common applications for GluR4 antibodies?

GluR4 antibodies are validated for numerous research applications, with the most common being:

• Western Blot (WB): Detecting GluR4 protein expression levels

• Immunohistochemistry (IHC): Visualizing GluR4 distribution in tissue sections

• Immunocytochemistry (ICC): Examining cellular localization

• Immunofluorescence (IF): Determining subcellular localization

• Flow Cytometry (FCM): Quantifying cell populations expressing GluR4

• ELISA: Quantitative measurement of GluR4 levels

• Immunoprecipitation (IP): Isolating GluR4-containing protein complexes

What is the difference between polyclonal and monoclonal GluR4 antibodies?

Polyclonal GluR4 antibodies recognize multiple epitopes on the GluR4 protein, offering high sensitivity but potential cross-reactivity. They're ideal for applications where signal amplification is necessary, such as detecting low-abundance GluR4 in certain brain regions.

Monoclonal GluR4 antibodies (e.g., Cell Signaling Technology's D41A11 XP® Rabbit mAb) recognize a single epitope, providing higher specificity but potentially lower sensitivity. They excel in applications requiring consistent lot-to-lot reproducibility and minimal background, such as quantitative Western blots or high-resolution immunofluorescence microscopy .

What sample preparation protocols optimize GluR4 detection in Western blots?

For optimal GluR4 detection in Western blots:

• Tissue preparation: Rapidly extract brain tissue and immediately freeze in liquid nitrogen to prevent protein degradation

• Lysis buffer composition: Use RIPA buffer supplemented with:

  • Protease inhibitors (Complete™ or equivalent)

  • Phosphatase inhibitors (PhosSTOP™ or equivalent) if detecting phosphorylated GluR4

  • 1% sodium deoxycholate to effectively solubilize membrane proteins

• Homogenization: Perform on ice using a Dounce homogenizer (15-20 strokes)

• Centrifugation: 20,000g for 20 minutes at 4°C

• Protein concentration: Determine using Bradford or BCA assay

• Sample preparation: Mix 20-50μg protein with Laemmli buffer containing 5% β-mercaptoethanol

• Denaturation: Heat at 70°C for 10 minutes (avoid boiling which can cause GluR4 aggregation)

• Gel selection: 8% SDS-PAGE gels for optimal resolution of the ~102kDa GluR4 protein

• Transfer: Semi-dry transfer at 25V for 45 minutes or wet transfer at 30V overnight at 4°C

• Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature

Following this protocol ensures consistent GluR4 detection while minimizing background and non-specific binding .

How should phospho-specific GluR4 antibodies be validated?

Validating phospho-specific GluR4 antibodies (such as anti-GluR4 pS862) requires a systematic approach:

• Phosphatase treatment control: Split your sample and treat half with lambda protein phosphatase; the phospho-specific signal should disappear in the treated sample

• Stimulation paradigm: Compare samples from tissues/cells with known phosphorylation status:

  • Baseline (unstimulated)

  • Stimulated (e.g., with glutamate, AMPA, or PKA activators for S862 phosphorylation)

  • Stimulated plus kinase inhibitor

• Peptide competition: Pre-incubate antibody with phosphorylated vs. non-phosphorylated peptide

• Knockout/knockdown validation: Test in GluR4-null tissues or following siRNA-mediated knockdown

• Mass spectrometry correlation: Verify phosphorylation status using orthogonal methods

Document each validation step with appropriate positive and negative controls to ensure the observed signal truly represents phosphorylated GluR4 .

What fixation methods are optimal for GluR4 immunohistochemistry?

Optimizing fixation for GluR4 immunohistochemistry depends on the antibody epitope location:

For antibodies targeting extracellular epitopes:

• Light fixation with 2% paraformaldehyde for 20-30 minutes preserves antigenicity

• Avoid methanol fixation which can denature extracellular domains

For antibodies targeting intracellular epitopes:

• 4% paraformaldehyde fixation for 24 hours (for tissue blocks) or 15 minutes (for cultured cells)

• Post-fixation permeabilization with 0.1-0.3% Triton X-100

For phospho-specific antibodies:

• Rapid fixation in ice-cold 4% paraformaldehyde containing phosphatase inhibitors

• Phosphate-free fixatives may improve results

Always validate the fixation protocol through comparison experiments as GluR4 epitope accessibility varies among antibody clones .

How can GluR4 antibodies distinguish between synaptic and extrasynaptic receptor pools?

Distinguishing between synaptic and extrasynaptic GluR4 requires sophisticated immunolabeling strategies:

• Dual-immunofluorescence labeling:

  • Co-label with GluR4 antibody and established synaptic markers (PSD-95, Synapsin, Bassoon)

  • Quantify colocalization using Manders' overlap coefficient or Pearson's correlation coefficient

  • Non-overlapping GluR4 signal represents extrasynaptic pools

• Proximity ligation assay (PLA):

  • Use GluR4 antibody with antibodies against synaptic scaffold proteins

  • PLA signal indicates <40nm proximity, identifying synaptic GluR4

• Biochemical fractionation:

  • Prepare PSD (postsynaptic density), non-PSD synaptic, and extrasynaptic membrane fractions

  • Analyze GluR4 distribution across fractions via Western blot

  • Normalize to fraction-specific markers (PSD-95 for PSD, Na+/K+-ATPase for membrane)

• Super-resolution microscopy:

  • Employ STED or STORM imaging with GluR4 antibodies

  • Identify receptor nanoclusters relative to synaptic boundaries

  • This approach offers 20-50nm resolution of receptor organization

This multifaceted approach provides complementary data on GluR4 distribution across subcellular compartments, crucial for understanding receptor trafficking dynamics .

How should researchers troubleshoot inconsistent GluR4 antibody performance across experimental platforms?

When facing inconsistent GluR4 antibody performance, implement this systematic troubleshooting approach:

• Epitope availability analysis:

  • Determine if your antibody targets extracellular, transmembrane, or intracellular domains

  • Extracellular epitopes may be masked by protein interactions or glycosylation

  • C-terminal epitopes may be obscured by scaffold protein binding

• Application-specific optimization matrix:

• Receptor state considerations:

  • Test if receptor activation affects epitope accessibility

  • Compare antibody performance in high vs. low activity states

  • Consider using glutamate receptor antagonists during sample preparation

• Antibody validation hierarchy:

  • Test multiple GluR4 antibodies targeting different epitopes

  • Include knockout/knockdown controls

  • Use recombinant GluR4 as a positive control

  • Verify with orthogonal detection methods (e.g., mass spectrometry)

Thorough documentation of optimization parameters enables reproducible protocols across experiments .

What strategies exist for multiplexed detection of AMPA receptor subunits including GluR4?

Advanced multiplexed detection of AMPA receptor subunits requires careful experimental design:

• Antibody selection criteria for multiplexing:

  • Host species diversity (rabbit anti-GluR1, mouse anti-GluR2, goat anti-GluR3, guinea pig anti-GluR4)

  • Isotype diversity within same host species (IgG1 vs. IgG2a mouse antibodies)

  • Directly conjugated primary antibodies with spectrally distinct fluorophores

• Sequential immunostaining protocol:

  • First round: Incubate with first primary antibody, detect with secondary antibody

  • Elution step: Strip antibodies using glycine-HCl (pH 2.5) or SDS buffer

  • Subsequent rounds: Repeat with additional antibody pairs

  • Control for incomplete stripping and cross-reactivity

• Mass cytometry (CyTOF) approach:

  • Label anti-GluR antibodies with distinct metal isotopes

  • Enables simultaneous detection of 40+ parameters

  • Ideal for comprehensive AMPA receptor composition analysis

• Spectral unmixing fluorescence microscopy:

  • Uses antibodies with overlapping fluorescence spectra

  • Software algorithms separate signals based on spectral signatures

  • Increases multiplexing capacity to 8-10 targets simultaneously

• Technical considerations for all methods:

  • Test for antibody cross-reactivity extensively

  • Include controls for epitope masking when multiple antibodies target the same protein

  • Validate quantification against known standards

These approaches enable comprehensive analysis of AMPA receptor subunit composition across diverse neural circuits and experimental conditions .

How should researchers select the appropriate GluR4 antibody for studying receptor trafficking dynamics?

Selecting the optimal GluR4 antibody for trafficking studies requires consideration of several key parameters:

• Epitope location strategy:

  • N-terminal/extracellular antibodies: Ideal for live-cell surface labeling and internalization assays

  • C-terminal/intracellular antibodies: Better for total receptor pool quantification

  • Phospho-specific antibodies: Essential for activity-dependent trafficking (e.g., pS862 for PKA-mediated surface expression)

• Application-specific selection criteria:

  • Live imaging: Non-permeabilized conditions require extracellular epitope antibodies

  • Pulse-chase experiments: Primary antibodies that remain bound during endocytosis

  • Surface biotinylation: Antibodies validated to work in streptavidin pull-down conditions

• Experimental design considerations:

  • Time-course resolution requirements (seconds, minutes, hours)

  • Need for quantitative vs. qualitative assessment

  • Compatibility with pharmacological manipulations

• Validation experiments before trafficking studies:

  • Surface vs. intracellular staining patterns in control conditions

  • Antibody behavior following established trafficking stimuli (e.g., NMDA-induced internalization)

  • Correlation with tagged GluR4 constructs in heterologous systems

Proper antibody selection ensures accurate interpretation of GluR4 trafficking data in both physiological and pathological contexts .

What are the critical controls needed when comparing GluR4 levels across different brain regions?

Robust comparison of GluR4 levels across brain regions requires implementation of these critical controls:

• Normalization strategy matrix:

• Sample processing controls:

  • Process all regions simultaneously with identical protocols

  • Randomize sample order during processing and analysis

  • Include mixed-region standards on each gel/slide for inter-assay normalization

• Regional composition considerations:

  • Account for different white/gray matter ratios

  • Normalize for synaptic density using synaptophysin

  • Consider cell-type specific markers for regions with diverse neuronal populations

• Methodological validation:

  • Verify antibody specificity in each brain region separately

  • Confirm linear detection range encompasses expected GluR4 expression levels

  • Validate quantification with orthogonal methods (e.g., mass spectrometry, qPCR)

These rigorous controls enable reliable comparison of GluR4 expression across diverse brain regions with different cellular compositions and protein content .

How can researchers differentiate between GluR4-containing AMPA receptors and other AMPA receptor subtypes?

Differentiating GluR4-containing AMPA receptors from other subtypes requires sophisticated analytical approaches:

• Immunoprecipitation strategies:

  • Use GluR4-specific antibodies for IP followed by Western blotting for other subunits

  • Reverse approach: IP with other subunit antibodies, then blot for GluR4

  • Quantify relative abundance of heteromeric assemblies

• Pharmacological dissection:

  • GluR4-containing receptors show distinct sensitivity to:

    • Faster desensitization kinetics compared to GluR2-containing receptors

    • Higher calcium permeability in GluR2-lacking assemblies

    • Differential sensitivity to polyamine modulation

• Electrophysiological fingerprinting:

  • GluR4-dominant responses show:

    • Faster rise and decay kinetics

    • Distinct current-voltage relationships (particularly in GluR2-lacking receptors)

    • Characteristic single-channel conductance properties

• Advanced imaging approaches:

  • Single-molecule tracking of differentially labeled subunits

  • FRET analysis between GluR4 and other subunits

  • Multi-color STORM imaging for nanoscale colocalization

• Genetic manipulation controls:

  • Selective knockdown of GluR4 vs. other subunits

  • Use of subunit-specific dominant negative constructs

  • Rescue experiments with modified GluR4 constructs

These complementary approaches provide a comprehensive profile of GluR4-containing AMPA receptor populations and their functional significance .

How should researchers address conflicting results from different GluR4 antibodies?

When facing conflicting results from different GluR4 antibodies, implement this systematic resolution framework:

• Epitope mapping comparison:

  • Determine precise epitope locations for each antibody

  • Assess potential post-translational modifications at each epitope

  • Evaluate epitope conservation across species if using different model systems

• Validation hierarchy implementation:

  • Genetic validation: Test all antibodies in GluR4 knockout/knockdown tissues

  • Biochemical validation: Pre-absorb antibodies with immunizing peptides

  • Orthogonal method validation: Compare with mass spectrometry or in situ hybridization

• Technical parameter assessment:

  • Evaluate fixation/extraction effects on each epitope

  • Test different sample preparation methods systematically

  • Determine if conflicting results are application-specific

• Decision framework for resolving conflicts:

• Reporting recommendations:

  • Transparently document discrepancies in methods section

  • Include supplementary data with all antibody results

  • Discuss biological implications of different antibody behaviors

This structured approach transforms conflicting results into deeper insights about GluR4 biology and antibody performance .

What are the appropriate statistical approaches for analyzing GluR4 expression changes across experimental conditions?

Selecting appropriate statistical methods for GluR4 expression analysis depends on experimental design and data characteristics:

• Preliminary data assessment:

  • Test for normality using Shapiro-Wilk or Kolmogorov-Smirnov tests

  • Evaluate data spread and potential outliers

  • Assess homogeneity of variance with Levene's test

• Statistical approach decision tree:

Proper statistical analysis ensures robust interpretation of GluR4 expression changes while minimizing both false positives and negatives .

How can researchers distinguish between changes in GluR4 expression versus altered subcellular localization?

Differentiating between expression changes and redistribution of GluR4 requires parallel analytical approaches:

This multifaceted approach provides definitive evidence distinguishing between true expression changes and subcellular redistribution of GluR4 receptors .