GRIK2 Antibody, HRP conjugated

What is GRIK2 and why is it an important research target?

GRIK2 (Glutamate Receptor, Ionotropic, Kainate 2), also known as GluK2 or GluR6, is a kainate receptor subunit that functions as a glutamate-gated cation channel with diverse roles in the central nervous system. It has gained significant research interest due to its involvement in various neurological conditions and processes. Recent evidence suggests a unique role for GluR6 in controlling abnormalities related to behavioral symptoms of mania, making it a valuable target for understanding certain neuropsychiatric disorders . Additionally, bi-allelic loss of function of the KAR-encoding gene variants are likely pathogenic in certain neurological conditions, highlighting its importance in normal neuronal function .

What are the primary types of GRIK2 antibodies available for research?

Based on current literature and commercial offerings, GRIK2 antibodies are available in several configurations:

The selection of an appropriate antibody depends on the specific experimental application, target species, and epitope of interest .

How do GRIK2 antibody conjugations affect their applications?

Antibody conjugations, including HRP (horseradish peroxidase), significantly impact application methodologies and detection sensitivity. For GRIK2 research, while many primary antibodies are available unconjugated, HRP conjugation offers direct detection without secondary antibodies in applications like western blotting and ELISA.

The primary advantages of HRP-conjugated GRIK2 antibodies include:

• Elimination of secondary antibody incubation steps, reducing protocol time

• Minimization of background signal often associated with secondary antibodies

• Direct quantification of target proteins

• Compatible with multiple detection systems including enhanced chemiluminescence (ECL)

When working with unconjugated primary antibodies, researchers typically use HRP-linked secondary antibodies (anti-rabbit or anti-mouse IgG) as demonstrated in published protocols for GRIK2 detection .

What are the optimal applications for GRIK2 antibodies in neuroscience research?

GRIK2 antibodies have demonstrated utility across multiple experimental platforms in neuroscience research:

These applications provide complementary data for understanding GRIK2 expression, localization, and function in normal and pathological conditions .

How should GRIK2 antibodies be validated for specificity?

Rigorous validation of GRIK2 antibodies is essential for reliable experimental outcomes. Methodological approaches include:

• Positive and negative control samples:

  • Positive controls: Cell lines with known GRIK2 expression (e.g., Jurkat cells) or transfected cell lines overexpressing GRIK2

  • Negative controls: Knockout tissues/cells or cells with negligible GRIK2 expression

• Peptide competition assays:

  • Pre-incubation of antibody with immunizing peptide should abolish specific signal

  • Especially important for polyclonal antibodies targeting specific epitopes (e.g., regions between amino acids 156-205 of human GRIK2)

• Multiple antibody concordance:

  • Verification with alternative antibodies targeting different epitopes

  • Comparison of staining/binding patterns across techniques

• Isotype controls:

  • Use of appropriate isotype control antibodies (e.g., Mouse IgG2A for MAB9610)

  • Essential for flow cytometry applications to establish proper gating

These validation steps should be documented before undertaking extensive experimental work with any GRIK2 antibody .

What protocol adaptations are necessary when using HRP-conjugated GRIK2 antibodies for Western blotting?

When using HRP-conjugated GRIK2 antibodies for Western blotting, several methodological adaptations are needed compared to standard two-step detection protocols:

• Sample preparation:

  • Protein extraction should follow established protocols (e.g., TEEN-TX buffer: 50 mM Tris, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.1% Triton X-100 with protease inhibitor cocktail)

  • Load 5-10 μg of protein per lane as established in published protocols

• Blocking optimization:

  • Consider extended blocking periods (1-2 hours at room temperature)

  • Use 5% milk and 0.1% Tween 20 in TBS (TBS-T) as demonstrated effective for GRIK2 detection

• Antibody dilution and incubation:

  • Direct HRP-conjugated antibodies typically require higher concentrations than unconjugated primaries

  • Optimize dilution range based on signal-to-noise ratio (starting with 1:500-1:1000 dilution)

  • Incubate for 1 hour at room temperature or overnight at 4°C as established for other GRIK2 antibodies

• Washing procedure:

  • More stringent washing may be required (4-6 washes with TBS-T)

  • Increase washing duration to minimize background

• Detection considerations:

  • ECL substrate systems have proven effective for GRIK2 visualization

  • Exposure optimization may be needed based on expression levels

These adaptations help maintain sensitivity while reducing background when using directly conjugated antibodies .

How can GRIK2 antibodies be used effectively in brain tissue immunohistochemistry?

Effective immunohistochemical detection of GRIK2 in brain tissue requires specific methodological considerations:

• Tissue preparation:

  • Freshly prepared 4% paraformaldehyde fixation is preferable

  • Post-fixation time should be optimized to preserve epitope accessibility

  • For paraffin-embedded sections, antigen retrieval is critical (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Antibody selection:

  • For human tissue, multiple antibodies have demonstrated specificity

  • Cross-reactivity with other species should be verified (e.g., ab28697 is predicted to work with mouse, rat, cow, dog, and chimpanzee samples)

• Signal amplification:

  • For HRP-conjugated antibodies, tyramide signal amplification can enhance sensitivity

  • For unconjugated antibodies, biotin-streptavidin systems or polymer detection methods are effective

• Controls and quantification:

  • Include positive control tissues with known GRIK2 expression

  • Quantification should utilize standardized imaging parameters and analysis software (e.g., ImageJ)

  • For co-localization studies, spectral unmixing may be necessary to distinguish signals

• Protocol optimization:

  • Titrate antibody concentration to minimize background

  • Extended incubation times at 4°C may improve signal-to-noise ratio

  • Permeabilization conditions should be optimized for access to different cellular compartments

These methodological considerations enable reliable visualization of GRIK2 distribution in neuronal tissues .

How can researchers address non-specific binding issues with GRIK2 antibodies?

Non-specific binding is a common challenge with GRIK2 antibodies that can be addressed through systematic troubleshooting:

• Antibody validation:

  • Verify antibody specificity using knockout controls or peptide competition assays

  • For recombinant systems, compare transfected vs. non-transfected cells

• Blocking optimization:

  • Test alternative blocking agents (BSA, casein, commercial blocking buffers)

  • Increase blocking time or concentration (e.g., from 5% to 10% blocking agent)

  • Include additives like 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Antibody incubation conditions:

  • Reduce antibody concentration (perform titration series)

  • Add 0.1-0.2% BSA to antibody dilution buffer

  • For problematic samples, pre-adsorb antibody with tissue powder from the species being tested

• Washing optimization:

  • Increase wash buffer stringency (add 0.2-0.5M NaCl)

  • Extend washing times and number of washes

  • Use detergent combinations (Tween-20 plus Triton X-100)

• Cross-reactivity assessment:

  • Perform Western blot to identify potential cross-reactive proteins

  • Consider alternative antibodies targeting different epitopes

Systematic implementation of these approaches can significantly improve signal specificity .

What statistical approaches are recommended for quantifying GRIK2 expression data?

Quantitative analysis of GRIK2 expression requires appropriate statistical methodologies:

• Western blot quantification:

  • Signal intensities should be quantified using standardized systems (e.g., Kodak Imaging System, Li-Cor ImageStudio software)

  • Normalization to housekeeping proteins (e.g., β-actin) is essential

  • Generate standard curves of each protein for accurate quantification

• Statistical tests:

  • For normally distributed data: One-way ANOVA with Tukey's post hoc test

  • For non-parametric data: Kruskal-Wallis multiple comparison test

  • For two-group comparisons: Student's t-test

  • Software recommendations include STATISTICA, GraphPad Prism, or equivalent packages

• Experimental design considerations:

  • Minimum sample sizes should be determined through power analysis

  • Technical replicates (n=3 minimum) should be performed

  • Biological replicates across independent experiments are essential for reliability

• Data presentation:

  • Results should be presented as mean ± SEM from specified number of samples/cells

  • Significance thresholds should be clearly indicated (e.g., *p < 0.05, **p < 0.01, ***p < 0.001)

• Multiple testing correction:

  • When performing multiple measurements from the same recordings, correction for familywise error rates is necessary

  • Apply Bonferroni or similar corrections to maintain appropriate Type I error rates

These statistical approaches ensure rigorous analysis and interpretation of GRIK2 expression data .

How can GRIK2 antibodies be used to investigate receptor trafficking and membrane localization?

Investigation of GRIK2 trafficking and membrane localization requires specialized approaches:

• Surface biotinylation assay:

  • HEK293-T/17 cells transfected with GluK2a cDNA provide a controlled system

  • Incubate cells with 0.5 mg/mL biotin in cold PBS for 30 min at 4°C with gentle agitation

  • Quench with 100 mM glycine in cold PBS for 5 min at 4°C

  • Extract proteins using TEEN-TX buffer (50 mM Tris, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.1% Triton X-100) with protease inhibitors

  • Pull down biotinylated proteins and analyze by Western blotting with GRIK2 antibodies

• Live-cell imaging approaches:

  • Combine GRIK2 antibodies recognizing extracellular epitopes with fluorescent secondary antibodies

  • Monitor trafficking in real-time using confocal microscopy

  • For pulse-chase experiments, use temperature blocks to synchronize receptor movement

• Co-localization studies:

  • Combine GRIK2 antibodies with markers for specific cellular compartments

  • Quantify co-localization using Pearson's or Mander's coefficients

  • Super-resolution microscopy can provide nanoscale resolution of receptor clustering

• Receptor internalization assays:

  • Label surface receptors at 4°C, then allow internalization at 37°C

  • Strip remaining surface antibodies using acid wash

  • Quantify internalized receptors by microscopy or flow cytometry

These methodologies enable detailed characterization of GRIK2 trafficking dynamics and membrane expression .

What approaches can be used to study GRIK2 interactions with other proteins in neural signaling complexes?

Studying GRIK2 interactions within neural signaling complexes requires sophisticated methodological approaches:

• Co-immunoprecipitation strategies:

  • Extract proteins under non-denaturing conditions to preserve native interactions

  • Use GRIK2 antibodies for pull-down experiments followed by Western blotting for potential interacting partners

  • Validate interactions using reciprocal co-immunoprecipitation

• Proximity ligation assay (PLA):

  • Utilize GRIK2 antibodies in combination with antibodies against potential interacting proteins

  • This technique allows visualization of protein-protein interactions with subcellular resolution

  • Quantification of PLA signals provides semi-quantitative assessment of interaction strength

• BiFC (Bimolecular Fluorescence Complementation):

  • Genetic approach complementing antibody-based methods

  • Fusion of split fluorescent proteins to GRIK2 and potential interactors

  • Reconstitution of fluorescence occurs upon protein-protein interaction

• Mass spectrometry approaches:

  • Following immunoprecipitation with GRIK2 antibodies, analyze pulled-down complexes by LC-MS/MS

  • Filter results against appropriate controls to identify specific interactors

  • Validation of novel interactions using orthogonal methods is essential

• Advanced imaging techniques:

  • FRET (Fluorescence Resonance Energy Transfer) using antibody-based fluorophore systems

  • STED or STORM super-resolution microscopy for nanoscale colocalization analysis

  • Single-particle tracking to analyze dynamic interactions

These approaches provide complementary data on GRIK2 interactions, enabling comprehensive characterization of its role in signaling complexes .

How can GRIK2 antibodies be applied to study pathological alterations in neurological disorders?

GRIK2 antibodies can be instrumental in characterizing pathological alterations in neurological disorders through several methodological approaches:

• Post-mortem tissue analysis:

  • Comparative immunohistochemistry and Western blotting across control and disease tissues

  • Quantification of expression level changes in specific brain regions

  • Assessment of subcellular distribution alterations in disease states

• Animal model characterization:

  • Analysis of GRIK2 expression in genetic or pharmacological models of neurological disorders

  • Correlation of expression changes with behavioral phenotypes

  • Longitudinal studies to track progression of alterations

• Patient-derived cell models:

  • iPSC-derived neurons from patients with GRIK2 mutations

  • Immunocytochemistry and biochemical analysis of receptor expression and trafficking

  • Correlation of cellular phenotypes with clinical manifestations

• Pharmacological manipulation:

  • Use of GRIK2 antibodies to monitor receptor responses to therapeutic compounds

  • Assessment of receptor internalization, phosphorylation, or degradation following drug treatment

  • Combination with functional assays (calcium imaging, electrophysiology) to correlate structure with function

• Genetic validation:

  • Analysis of GRIK2 expression in genetically defined cases (e.g., clustered mutations in the GRIK2 gene associated with neurodevelopmental disorders)

  • Correlation of genetic variants with protein expression patterns

  • Structure-function relationships in receptors containing pathogenic variants

These applications provide critical insights into the role of GRIK2 in neurological disease pathogenesis and potential therapeutic targeting .