GRIK2 Antibody, FITC conjugated

What is GRIK2 and what is its biological significance?

GRIK2 (Glutamate Ionotropic Receptor Kainate Type Subunit 2) functions as a cation-permeable ligand-gated ion channel that is gated by L-glutamate and the glutamatergic agonist kainic acid. It plays a critical role in excitatory neurotransmission in the central nervous system. When L-glutamate binds to this receptor, it induces a conformational change that leads to the opening of the cation channel, thereby converting a chemical signal into an electrical impulse. Following activation, the receptor rapidly desensitizes and enters a transient inactive state characterized by the presence of bound agonist .

Beyond its canonical role in neurotransmission, GRIK2 has additional functions that make it particularly interesting for researchers:

• It modulates cell surface expression of NETO2

• It participates in presynaptic facilitation of glutamate release at hippocampal mossy fiber synapses in association with GRIK3

• Independent of its ionotropic function, it acts as a thermoreceptor conferring sensitivity to cold temperatures

• Recent research indicates its involvement in cellular senescence pathways, suggesting potential roles in cancer biology

The protein has a calculated molecular weight of 103 kDa, though the observed molecular weight in experiments ranges from 103-115 kDa .

What are the available formats of GRIK2 antibodies and how do they differ in research applications?

GRIK2 antibodies are available in multiple formats to suit different experimental needs:

When selecting the appropriate format, researchers should consider:

• The required sensitivity for the target detection

• The experimental procedure (e.g., multiplexing needs)

• The available detection systems in the laboratory

• The sample type and preparation method

FITC-conjugated antibodies are particularly useful for flow cytometry applications and direct visualization of GRIK2 in immunofluorescence assays without requiring secondary antibody incubation steps .

What are the recommended protocols for using FITC-conjugated GRIK2 antibodies in flow cytometry?

When using FITC-conjugated GRIK2 antibodies for flow cytometry, the following protocol represents best practices based on published procedures:

Standard Protocol:

• Prepare single-cell suspension from your tissue of interest or cultured cells

• Fix cells with 2-4% paraformaldehyde for 10-15 minutes at room temperature (optional depending on experimental needs)

• If intracellular staining is required, permeabilize cells with 0.1% Triton X-100 or 0.5% saponin in PBS for 10 minutes

• Block non-specific binding sites with 2-5% BSA or serum from the same species as the secondary antibody (if used) for 30 minutes

• Incubate with FITC-conjugated GRIK2 antibody (typical dilution 1:50-1:200, but optimize for your specific antibody)

• Wash 3 times with PBS containing 0.5% BSA

• Resuspend cells in appropriate buffer for flow cytometric analysis

• Analyze using appropriate laser (488 nm excitation for FITC) and emission filter (typically 530/30 nm)

Validated Example:
Research by R&D Systems demonstrated successful detection of GRIK2 in CHO cell lines transfected with human GRIK2. Their protocol involved staining cells with Mouse Anti-Human GRIK2 Monoclonal Antibody followed by Phycoerythrin-conjugated Anti-Mouse IgG Secondary Antibody using their "Staining Membrane-associated Proteins protocol" .

For directly conjugated FITC-GRIK2 antibodies, appropriate controls should include:

• Isotype control antibody conjugated to FITC

• Unstained cells

• Blocking peptide competition (when available) to confirm specificity

What are the critical factors for successful immunostaining using GRIK2 antibodies?

Successful immunostaining with GRIK2 antibodies depends on several critical factors:

Optimal Dilution Determination:
Different applications require different antibody dilutions. Based on manufacturer recommendations:

• Western Blot (WB): 1:1000-1:6000 or 1:1000-1:4000

• Immunohistochemistry (IHC): 1:50-1:500

• Immunofluorescence (IF): 1:50-1:200

Sample-Specific Considerations:

• For brain tissue samples, particularly cerebellum, GRIK2 antibodies have been validated to work well in multiple species including human, mouse, rat, and pig samples

• For hippocampal samples, specific protocols have been validated for human tissue

Antigen Retrieval Methods:
For IHC applications with GRIK2 antibodies, the following antigen retrieval methods have been validated:

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

Buffer Compositions:
Many commercial GRIK2 antibodies are supplied in:

• PBS with 0.02% sodium azide and 50% glycerol pH 7.3

• Some smaller volume preparations (20μl) contain 0.1% BSA

• FITC-conjugated variants may contain preservatives like 0.03% Proclin 300

Researchers should be aware that improper sample preparation, antibody concentration, or incubation conditions can lead to false-negative results or nonspecific staining.

How can researchers validate the specificity of GRIK2 antibodies in their experimental systems?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For GRIK2 antibodies, consider these validation approaches:

Positive Controls:
Based on published data, these sample types show reliable GRIK2 detection:

• Mouse, rat, and pig cerebellum tissue

• Human hippocampus tissue

• A431 cells, PC-12 cells, SH-SY5Y cells

• CHO cells transfected with human GRIK2

Negative Controls:

• Isotype-matched control antibodies (e.g., Mouse IgG1 for monoclonal antibodies or normal rabbit IgG for rabbit polyclonals)

• Immunizing peptide competition assays where available

• Tissues known to have low or no expression of GRIK2

Molecular Validation:

• Western blot analysis should show bands at the expected molecular weight (103-115 kDa)

• RNA interference to knock down GRIK2 expression should reduce antibody signal

• Overexpression systems (as demonstrated with CHO cells transfected with human GRIK2)

Cross-Reactivity Assessment:
Some antibodies may cross-react with related glutamate receptors. For example, the EPR13726 antibody cross-reacts with both GRIK2/GluK2 and GRIK3/GluK3 . Researchers should carefully evaluate potential cross-reactivity with closely related proteins based on the immunogen sequence used to generate the antibody.

What are the known applications of GRIK2 antibodies in disease-related research?

GRIK2 antibodies have been applied in several disease-related research contexts:

Neurological Disorders:
Given GRIK2's role in glutamatergic neurotransmission, antibodies have been utilized to study various neurological conditions. GRIK2 mutations have been linked to autosomal recessive cognitive disability and neurodevelopmental disorders with impaired language and ataxia .

Cancer Research:
A significant application involves studying GRIK2's role in cellular senescence and cancer. A 2019 study demonstrated that:

• GRIK2 isoform expression in SKOV3 ovarian carcinoma cells induced senescence

• Transduced cells showed significantly reduced proliferation rates

• Complete cell-cycle arrest was achieved by 37 days post-transduction

• GRIK2-induced senescence was characterized by:

  • Activation of senescence-associated β-galactosidase

  • Decreased levels of active protein kinase B (AKT)

  • Increased abundance of inactive cyclin-dependent kinase 1 (CDK1)

These findings suggest GRIK2 as a potential target for therapeutic intervention in cancer cells, and GRIK2 antibodies serve as crucial tools for investigating these mechanisms.

Mechanistic Studies:
GRIK2 antibodies have been valuable in elucidating mechanistic details of the receptor's function, including:

• Its thermoreceptor activity conferring cold temperature sensitivity

• Its role in presynaptic facilitation of glutamate release

• Its functionality in dorsal root ganglion neurons

When designing disease-related research using GRIK2 antibodies, researchers should consider carefully selecting antibodies that recognize relevant functional domains or isoforms of interest.

How can researchers detect specific GRIK2 isoforms using antibodies?

GRIK2 has multiple isoforms due to alternative splicing and RNA editing, making isoform-specific detection an important consideration:

Isoform Awareness:
The human GRIK2 gene expresses up to 7 different isoforms, with varying functional properties . When selecting antibodies, consider:

• Epitope location: Verify that the immunogen sequence used to generate the antibody includes or excludes regions specific to your isoform of interest

• Validated reactivity: Check if the antibody has been validated against specific isoforms

For example, the CUSABIO GRIK2 antibody (CSB-PA618751LC01HU) uses a recombinant human GRIK2 protein fragment (amino acids 203-500) as its immunogen , which may recognize multiple isoforms depending on sequence conservation in this region.

Experimental Strategies:
To distinguish between isoforms:

• Use antibodies raised against isoform-specific regions where sequences diverge

• Combine with molecular techniques like RT-PCR to confirm isoform expression

• Consider using recombinant expression systems with defined isoforms as controls

• Use Western blotting to separate isoforms by molecular weight for differential detection

The study by Paarmann et al. (2019) successfully distinguished between GRIK2 isoforms by using EGFP-tagged GRIK2 isoform constructs and antibody detection, demonstrating that this approach can be useful for isoform-specific studies .

What common problems occur when using GRIK2 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with GRIK2 antibodies:

Problem: High Background in Immunofluorescence
Solutions:

• Increase blocking time/concentration (use 5-10% serum or BSA)

• Reduce primary antibody concentration (perform titration experiments)

• Include 0.1-0.3% Triton X-100 in blocking and antibody dilution buffers

• Include 0.1-0.3M NaCl in wash buffers to reduce non-specific ionic interactions

• For FITC-conjugated antibodies specifically, ensure samples are protected from light during all steps

Problem: Weak or No Signal in Western Blots
Solutions:

• Verify sample preparation - GRIK2 is a membrane protein requiring appropriate extraction methods

• For brain samples, use specialized extraction buffers containing mild detergents

• Increase protein loading (50-100μg recommended for brain tissue lysates)

• Verify transfer efficiency of high molecular weight proteins (103-115 kDa for GRIK2)

• Consider lower percentage gels (8%) for better resolution of larger proteins

Problem: Non-specific Bands in Western Blot
Solutions:

• Increase antibody dilution (1:4000-1:6000 for WB applications)

• Use freshly prepared samples to avoid degradation products

• Include protease inhibitors during sample preparation

• For analysis of GRIK2 in cerebellum tissue, validated dilutions of 1:1000-1:6000 have been shown to provide specific detection

Problem: Variable Results Between Experiments
Solutions:

• Standardize sample collection and preparation

• Prepare larger volumes of antibody dilutions to use across experiments

• Store antibodies according to manufacturer recommendations (-20°C for most GRIK2 antibodies)

• For long-term storage of aliquoted antibodies, -80°C may provide better stability

How can GRIK2 antibodies be used in multiparameter experiments?

Multiparameter experiments allow researchers to study GRIK2 in relation to other markers or cellular processes. FITC-conjugated GRIK2 antibodies are particularly useful in these contexts:

Flow Cytometry Multiplexing:
When using FITC-conjugated GRIK2 antibodies in multicolor flow cytometry:

• Pair with fluorophores that have minimal spectral overlap with FITC (e.g., PE, APC)

• Include proper compensation controls for each fluorophore

• Consider the brightness of FITC relative to other markers when determining antibody concentrations

• For cell surface GRIK2 detection combined with intracellular markers, perform surface staining before fixation/permeabilization

Co-localization Studies:
For immunofluorescence co-localization with other proteins:

• FITC-conjugated GRIK2 antibodies (green channel) can be combined with secondary antibodies conjugated to spectrally distinct fluorophores

• Consider using confocal microscopy for improved spatial resolution of co-localization

• Implement quantitative co-localization analyses using appropriate software

• Pre-absorb antibodies if cross-reactivity is suspected

Sequential Staining Protocols:
When performing sequential staining with multiple antibodies:

• Begin with the least sensitive target (often the most abundant protein)

• Apply FITC-conjugated GRIK2 antibody

• Block any remaining binding sites

• Apply subsequent antibodies

Practical Example:
A successful multiparameter experiment was demonstrated by R&D Systems, using anti-GRIK2 antibody detection in combination with flow cytometry. They validated their Mouse Anti-Human GRIK2 Monoclonal Antibody (Catalog # MAB9610) using CHO cell lines transfected with human GRIK2, using PE-conjugated secondary antibodies for detection .

How are GRIK2 antibodies being used to study its role in cellular senescence and cancer?

Recent research has revealed a novel role for GRIK2 in cellular senescence, particularly in cancer contexts:

Key Research Findings:
A 2019 study by Paarmann et al. provided compelling evidence for GRIK2's involvement in senescence induction:

• SKOV3 ovarian carcinoma cells transduced with GRIK2 showed significantly reduced proliferation

• Transduced cells demonstrated progressively increased doubling times

• Complete cell-cycle arrest was achieved by day 37

• The senescence phenotype was characterized by:

  • Activation of senescence-associated β-galactosidase

  • Cellular morphological changes (enlarged shape)

  • Absence of BrdU incorporation (confirming cessation of DNA replication)

  • Decreased levels of phosphorylated AKT (Ser473)

  • Increased levels of phosphorylated CDK1 (Tyr15)

Molecular Signaling Pathways:
GRIK2-induced senescence appears to involve alterations in two critical pathways:

• AKT signaling pathway: Reduced phosphorylation of AKT at Ser473 indicates inactivation of this pro-growth, pro-proliferation pathway

• Cell cycle regulation: Increased inhibitory phosphorylation of CDK1 at Tyr15 prevents progression through the G1 phase

Experimental Approaches:
Researchers investigating GRIK2's role in senescence typically employ these techniques:

• Retroviral transduction of GRIK2 expression constructs

• Proliferation assays (BrdU incorporation, MTT assays)

• Senescence-associated β-galactosidase staining

• Phospho-specific antibodies to detect changes in signaling molecules

• Long-term culture to monitor senescence progression

This research direction suggests GRIK2 as a potential therapeutic target for cancer intervention, highlighting the importance of GRIK2 antibodies as tools for further mechanistic investigations.

What considerations should researchers have when selecting GRIK2 antibodies for specialized applications?

When selecting GRIK2 antibodies for specialized applications, researchers should consider several factors:

Application-Specific Requirements:

Epitope Accessibility:
GRIK2's structure includes both extracellular and intracellular domains. For applications targeting the native receptor:

• Extracellular domain antibodies (N-terminal) are suitable for live cell applications

• Intracellular domain antibodies require permeabilization

• Transmembrane domain antibodies may have limited accessibility

The Cusabio FITC-conjugated GRIK2 antibody utilizes an immunogen comprising amino acids 203-500 of human GRIK2 , which includes portions of the extracellular domain.

Species Cross-Reactivity:
Many research applications benefit from antibodies that work across species:

• 66631-2-Ig antibody shows reactivity with human, mouse, rat, and pig samples

• 28550-1-AP antibody reacts with human, mouse, and rat samples

• Consider sequence homology when predicting cross-reactivity for untested species

Validation Requirements:
For specialized applications, additional validation may be necessary:

• Flow sorting: Validate antibodies for minimal impact on cell viability

• In vivo imaging: Test for absence of non-specific binding in relevant tissues

• Therapeutic applications: Evaluate immunogenicity and specificity profiles

What future developments can be expected in GRIK2 antibody technologies?

The field of GRIK2 antibody development is likely to advance in several directions:

Novel Conjugation Technologies:
While FITC conjugation remains common, newer fluorophores offer advantages:

• Quantum dots with higher brightness and photostability

• Near-infrared fluorophores for deeper tissue penetration

• Photoactivatable fluorophores for super-resolution microscopy

• Multi-modal probes combining fluorescence with MRI or PET detection capabilities

Isoform-Specific Reagents:
As understanding of GRIK2 isoform functions expands, more specific tools will emerge:

• Antibodies targeting splice-variant-specific epitopes

• RNA-edited isoform-specific detection reagents

• Editing site-specific antibodies that distinguish between edited and non-edited forms

Engineered Antibody Formats:
Beyond conventional antibodies, expect development of:

• Single-domain antibodies (nanobodies) for improved tissue penetration

• Bispecific formats targeting GRIK2 and interacting partners simultaneously

• Intrabodies designed for intracellular expression and binding

Therapeutic Applications:
If GRIK2's role in cellular senescence proves therapeutically relevant:

• Antibody-drug conjugates targeting GRIK2-expressing cancer cells

• Humanized therapeutic antibodies modulating GRIK2 function

• Antibody fragments optimized for tumor penetration

Integration with Systems Biology:
Future applications will likely leverage high-dimensional analyses:

• Antibody panels for comprehensive glutamate receptor profiling

• Integration with spatial transcriptomics data

• Computational modeling of GRIK2 signaling networks informed by antibody-based quantification