GLR2.8 Antibody

What is GLR2.8 and why is it important in plant research?

GLR2.8 (Glutamate Receptor-like 2.8) is a member of the plant glutamate receptor-like family, which consists of 20 members in Arabidopsis thaliana divided into three clades: GLR1, GLR2, and GLR3. GLR2.8 belongs to the clade 2 of GLRs and forms a tandemly-arranged, closely-related cluster with GLR2.7 and GLR2.9 .

The importance of GLR2.8 lies in its role in plant immunity and calcium signaling. Studies have shown that GLR2.7, GLR2.8, and GLR2.9 were identified as "core immunity response" (CIR) genes, which are transcriptionally upregulated in response to pathogen-associated molecular patterns (PAMPs) but not to abiotic stresses . The triple mutants of these genes displayed defects in Ca²⁺ responses upon treatment with elicitors and reduced immunity against Pseudomonas syringae .

Methodologically, researchers interested in GLR2.8 should consider:

• Using GLR2.8-specific antibodies for protein detection and localization

• Employing Ca²⁺ imaging techniques to study its involvement in signaling

• Creating single, double, or triple mutants with related GLRs for functional characterization

How do I validate the specificity of a GLR2.8 antibody?

Validating antibody specificity is critical for reliable research outcomes. For GLR2.8 antibodies, consider the following methodological approach:

• Western blot analysis using positive and negative controls:

  • Positive control: Tissue samples known to express GLR2.8 (refer to expression databases)

  • Negative control: GLR2.8 knockout/knockdown plant lines

  • Additional control: GLR2.8 recombinant protein or overexpression lines

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide

  • Run parallel Western blots with blocked and unblocked antibody

  • Specific binding should be eliminated in the blocked sample

• Cross-reactivity assessment:

  • Test against closely related proteins (GLR2.7 and GLR2.9)

  • Use heterologous expression systems expressing single GLR variants

• Immunoprecipitation followed by mass spectrometry:

  • Verify that the immunoprecipitated protein is indeed GLR2.8

Example of successful antibody validation approaches can be found in studies of glutamate receptor antibodies where researchers used multiple techniques including rat brain immunohistochemistry, cell-based assays with standard secondary antibody, and cell-based assays with secondary and tertiary antibodies .

What are the optimal conditions for using GLR2.8 antibodies in Western blot analysis?

For optimal Western blot results with GLR2.8 antibodies, follow these methodological guidelines:

Sample preparation:

• Extract proteins using a buffer containing 10 mM Tris, 50 mM sodium chloride, with protease inhibitors at pH 7.4, similar to conditions used for other membrane proteins

• Include phosphatase inhibitors if studying phosphorylation status

• Maintain cold conditions throughout extraction

Gel electrophoresis and transfer:

• Use 8-10% SDS-PAGE gels for optimal separation of GLR2.8 (expected MW approximately 90-100 kDa based on other glutamate receptors)

• Transfer to PVDF membranes at 30V overnight at 4°C for better transfer of membrane proteins

Antibody incubation:

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

• Primary antibody: Use at 1:500 dilution (starting point based on similar antibodies)

• Incubation: Overnight at 4°C with gentle rocking

• Secondary antibody: Anti-rabbit HRP conjugate at 1:2000-1:5000 depending on detection system

Detection and controls:

• Use chemiluminescence detection for high sensitivity

• Include positive control: Arabidopsis tissue with known GLR2.8 expression

• Negative control: GLR2.8 knockout mutant tissue

• Loading control: Anti-Tubulin beta chain antibody (1:500 dilution)

How can GLR2.8 antibodies be used to study calcium signaling in plant immunity?

GLR2.8 has been implicated in calcium signaling during plant immune responses . To investigate this function using GLR2.8 antibodies, consider these methodological approaches:

Co-localization studies:

• Perform double immunostaining with GLR2.8 antibodies and calcium channel markers

• Use confocal microscopy to visualize subcellular localization during immune responses

• Track temporal changes in localization after pathogen challenge

Calcium flux measurement coupled with immunodetection:

• Use calcium reporters (e.g., Aequorin, GCaMP) in wild-type and GLR2.8 mutant plants

• Challenge with pathogens or pathogen-associated molecular patterns (PAMPs)

• Correlate calcium responses with GLR2.8 protein levels by Western blot

• Compare wild-type, single, and triple mutant plants (GLR2.7/2.8/2.9)

Immunoprecipitation and protein complex analysis:

• Use GLR2.8 antibodies to pull down protein complexes

• Identify interacting partners by mass spectrometry

• Focus on interactions that change during immune activation

• Validate interactions with other calcium signaling components

Example experimental design:

• Treatment groups: Mock, flg22 (bacterial PAMP), chitin (fungal PAMP)

• Time points: 0, 5, 15, 30, 60 minutes after treatment

• Measurements: Calcium flux (using fluorescent reporters), GLR2.8 protein levels and localization, interacting protein partners

This approach has been successful in studying other plant GLRs, where researchers found that GLR3.3 and GLR3.6 are required for calcium signals that propagate through the vasculature upon wounding .

What methodological considerations are important when using GLR2.8 antibodies in immunohistochemistry?

When performing immunohistochemistry with GLR2.8 antibodies, several methodological considerations are critical:

Tissue preparation:

• Fixation: Use 4% paraformaldehyde for 2-4 hours at room temperature

• Embedding: Paraffin embedding works well for plant tissues

• Sectioning: 5-10 μm sections are optimal for resolution

• Antigen retrieval: May be necessary due to fixation-induced epitope masking

  • Heat-mediated: Citrate buffer (pH 6.0) at 95°C for 10-20 minutes

  • Enzymatic: Proteinase K treatment (1-5 μg/ml) for 10 minutes

Antibody application:

• Blocking: 5% normal serum (matching secondary antibody host) with 1% BSA for 1 hour

• Primary antibody dilution: Start with 1:250 and optimize as needed

• Incubation time: Overnight at 4°C in a humid chamber

• Secondary antibody: Fluorescent-conjugated for co-localization studies

  • For plant tissues, Alexa Fluor 488 or 594 conjugates work well at 1:500-1:1000

Critical controls:

• No primary antibody control

• Peptide competition control

• GLR2.8 knockout/knockdown tissue sections

• Non-expressing tissue sections

Analysis considerations:

• Compare with known GLR2.8 expression patterns (RNA-seq data)

• Document any discrepancies between transcript and protein localization

• Consider developmental and stress-induced changes in expression

For studying GLR2.8 in plant immunity, focus on vascular tissues, as these have been implicated in systemic immune signaling involving related GLRs .

How can GLR2.8 antibodies be used to differentiate between closely related GLR family members?

Differentiating between the closely related GLR2.7, GLR2.8, and GLR2.9 proteins requires careful methodological consideration:

Epitope selection and antibody design:

• Analyze sequence alignments of the three proteins to identify unique regions

• Target antibody production against:

  • C-terminal regions (often most divergent)

  • Unique extracellular domain sequences

  • Specific post-translational modification sites

• Confirm epitope uniqueness using BLAST or other sequence comparison tools

Validation strategy:

• Express each recombinant protein (GLR2.7, GLR2.8, GLR2.9) in heterologous systems

• Perform Western blot analysis with each antibody against all three proteins

• Create a cross-reactivity matrix showing specificity profiles

Immunoprecipitation coupled with mass spectrometry:

• Perform IP with the GLR2.8 antibody

• Analyze peptides by LC-MS/MS

• Look for unique peptides that definitively identify GLR2.8

• Quantify any contamination from GLR2.7 or GLR2.9

Multiple antibody approach:

• Use a pan-GLR2 antibody to detect all three family members

• Use specific antibodies in parallel to distinguish individual proteins

• Combine with mutant lines lacking one or more of the proteins

This approach has been successful with other related receptor families, such as shown in studies of glutamate receptor delta 2 antibodies where researchers used multiple techniques to confirm specificity .

What are the considerations for using GLR2.8 antibodies in co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) with GLR2.8 antibodies requires specific methodological considerations for membrane proteins:

Buffer optimization:

• Membrane solubilization: Try different detergents

  • Start with 1% Triton X-100 or 0.5-1% NP-40

  • For stronger interactions, try milder detergents like digitonin (0.5-1%)

  • For challenging cases, test CHAPS, DDM, or OG

• Salt concentration: 100-150 mM NaCl is standard; adjust based on interaction strength

• pH: Typically 7.4, but consider the isoelectric point of GLR2.8

Protocol considerations:

• Pre-clearing: Critical to reduce non-specific binding

  • Use protein A/G beads with pre-immune serum

• Antibody binding: Two strategies

  • Direct: Antibody pre-bound to beads

  • Indirect: Antibody added to lysate, then captured with beads

• Controls:

  • Input control (5-10% of starting material)

  • IgG control (same species as GLR2.8 antibody)

  • No-antibody control

  • GLR2.8 knockout/knockdown tissue control

Analysis of interacting partners:

• Western blot: For known or suspected interactions

• Mass spectrometry: For unbiased identification of the interactome

  • Focus on proteins involved in calcium signaling

  • Look for interactions with other membrane receptors

  • Identify components of the plant immune signaling pathway

Crosslinking option for transient interactions:

• Use membrane-permeable crosslinkers (e.g., DSP, DTBP)

• Optimize crosslinking time (usually 15-30 minutes)

• Include a reducing agent to reverse crosslinking before SDS-PAGE

This approach has been effective in studying protein-protein interactions for other glutamate receptors, as demonstrated in studies identifying receptor complexes involved in immune signaling .

How can I optimize immunofluorescence protocols for GLR2.8 localization studies?

For optimal GLR2.8 localization using immunofluorescence, follow these methodological guidelines:

Sample preparation:

• Fixation options:

  • 4% paraformaldehyde in PBS, pH 7.4 for 30-60 minutes

  • For membrane proteins, add 0.1-0.5% glutaraldehyde to better preserve structure

• Permeabilization:

  • 0.1-0.5% Triton X-100 for 15-30 minutes

  • Alternative: 0.05-0.1% saponin (gentler for membrane proteins)

• For plant tissues:

  • Consider wall digestion with cellulase and macerozyme for better antibody penetration

  • Optimize digestion time to maintain tissue integrity

Staining protocol:

• Blocking: 5% normal serum with 1% BSA for 1 hour at room temperature

• Primary antibody:

  • Dilution: Start at 1:500 for immunofluorescence

  • Incubation: Overnight at 4°C

• Washing: 3-5 times with PBS + 0.1% Tween-20

• Secondary antibody:

  • Fluorophore selection: Consider Alexa Fluor 488 for green channel

  • Dilution: 1:500-1:1000

  • Incubation: 1-2 hours at room temperature

• Counter-staining:

  • Nucleus: DAPI (1 μg/ml)

  • Membrane: FM4-64 or similar membrane dye

  • Additional markers for co-localization studies

Controls and validation:

• Secondary antibody-only control

• Peptide competition control

• Non-expressing tissue control

• GLR2.8 knockout/mutant control

• Co-localization with known compartment markers:

  • Plasma membrane: PIP2A

  • ER: HDEL-tagged proteins

  • Golgi: ST-GFP

Example of successful localization:
This approach has been used effectively for visualizing tubulin in Arabidopsis thaliana epidermis cells, where specific localization to division plates was observed . A similar methodological approach could be applied to GLR2.8 localization studies.

What are common issues when working with GLR2.8 antibodies and how can they be resolved?

Several common issues may arise when working with GLR2.8 antibodies. Here are methodological solutions:

Problem: Weak or no signal in Western blot
Solutions:

• Increase antibody concentration (try 1:250 instead of 1:500)

• Optimize protein extraction:

  • Use stronger lysis buffers with 1% SDS or 8M urea

  • Include protease inhibitor cocktail

• Improve transfer efficiency:

  • Use PVDF membrane instead of nitrocellulose

  • Transfer at lower voltage overnight at 4°C

  • Add 0.05% SDS to transfer buffer for high MW proteins

• Enhance detection sensitivity:

  • Use highly sensitive chemiluminescent substrates

  • Consider signal enhancement systems

Problem: High background in immunofluorescence
Solutions:

• Optimize blocking:

  • Increase blocking time to 2 hours

  • Try different blocking agents (BSA, normal serum, casein)

• Improve washing:

  • Increase number of washes (5-6 times)

  • Use 0.2% Triton X-100 in wash buffer

• Dilute antibodies further:

  • Try 1:1000 instead of 1:500

• Filter antibody solution:

  • Use 0.22 μm filters to remove aggregates

Problem: Cross-reactivity with other GLRs
Solutions:

• Pre-absorb antibody:

  • Incubate with recombinant proteins of related GLRs

  • Use tissue lysates from plants overexpressing related GLRs

• Use peptide competition assays to confirm specificity

• Consider raising new antibodies against more unique epitopes

• Use GLR2.8 knockout plants as negative controls

Problem: Inconsistent immunoprecipitation results
Solutions:

• Optimize lysis conditions:

  • Test different detergents (Triton X-100, NP-40, CHAPS)

  • Vary salt concentration (100-500 mM)

• Improve antibody-bead coupling:

  • Use covalent coupling to protein A/G beads

  • Crosslink antibody to beads using DMP or BS3

• Modify wash stringency:

  • Increase or decrease salt concentration

  • Add low concentrations of detergent to wash buffer

These approaches have been effective in troubleshooting antibody applications in similar studies, such as those examining glutamate receptors in opsoclonus-myoclonus syndrome .

How can I determine the optimal antibody concentration for my specific GLR2.8 experiment?

Determining the optimal concentration for GLR2.8 antibodies requires systematic titration and validation:

Western blot titration:

• Prepare a dilution series of GLR2.8 antibody:

  • Standard range: 1:100, 1:250, 1:500, 1:1000, 1:2000

• Run identical Western blots with the same protein sample

• Process all blots identically

• Evaluate based on:

  • Signal-to-noise ratio

  • Background levels

  • Specific band intensity

  • Non-specific binding

Immunofluorescence titration:

• Prepare multiple tissue sections or cell preparations

• Test antibody dilutions: 1:100, 1:250, 1:500, 1:1000, 1:2000

• Keep all other variables constant:

  • Fixation conditions

  • Blocking reagents and time

  • Secondary antibody concentration

  • Exposure settings during imaging

• Evaluate based on:

  • Signal intensity at expected locations

  • Background fluorescence

  • Signal-to-noise ratio

Quantitative assessment:

• Measure signal and background intensities using image analysis software

• Calculate signal-to-noise ratios for each dilution

• Plot these values to determine the optimal range

• Confirm with biological replicates

Documentation table template:

This approach follows standard protocols for antibody optimization as used in studies of other receptor antibodies, where appropriate concentrations were determined through systematic testing .

How can I use GLR2.8 antibodies to study the role of this protein in plant-pathogen interactions?

To study GLR2.8's role in plant-pathogen interactions, consider these methodological approaches:

Time-course studies of GLR2.8 expression and localization:

• Challenge plants with pathogens or PAMPs (e.g., flg22, chitin)

• Collect samples at multiple time points (0, 15, 30, 60, 120 min, 24h)

• Perform:

  • Western blot analysis for expression levels

  • Immunofluorescence for subcellular localization changes

  • Co-IP for identifying dynamic interaction partners

Correlation with calcium signaling:

• Use GLR2.8 antibodies in parallel with calcium imaging:

  • Transform plants with calcium sensors (e.g., GCaMP6, Aequorin)

  • Challenge with pathogens

  • Correlate calcium flux patterns with GLR2.8 protein levels/localization

• Compare wild-type and GLR2.8 mutant/silenced plants

Integrated multi-omics approach:

• Combine antibody-based GLR2.8 protein detection with:

  • Transcriptomics (RNA-seq) to study gene expression changes

  • Phosphoproteomics to detect GLR2.8 phosphorylation status

  • Interactomics to identify protein-protein interactions

• Create a temporal map of GLR2.8 involvement in immune signaling

Experimental design example:

• Plant material: Wild-type, GLR2.8 knockout, GLR2.8 overexpressor

• Treatments:

  • Mock (control)

  • Bacterial pathogen (e.g., Pseudomonas syringae)

  • Fungal pathogen (e.g., Botrytis cinerea)

  • PAMPs (flg22, chitin)

• Measurements:

  • GLR2.8 protein levels and phosphorylation state

  • Calcium flux patterns

  • Defense gene expression

  • Pathogen growth/disease progression

This approach aligns with studies of related GLRs where researchers found that triple mutants displayed defects in Ca²⁺ responses upon treatment with elicitors and reduced immunity against P. syringae .

What are methodological considerations for using GLR2.8 antibodies in studies of protein-protein interactions?

For investigating protein-protein interactions involving GLR2.8, consider these methodological strategies:

Co-immunoprecipitation optimized for membrane proteins:

• Membrane solubilization buffer optimization:

  • Test different detergents: digitonin (0.5-1%), DDM (0.5-1%), CHAPS (0.5-1%)

  • Add cholesterol hemisuccinate (CHS, 0.1%) to stabilize membrane protein complexes

  • Include phosphatase inhibitors to preserve phosphorylation-dependent interactions

• Crosslinking options:

  • DSP (dithiobis(succinimidyl propionate)) at 0.5-2 mM for 30 min

  • Formaldehyde (0.5-1%) for 10 min

  • Both are reversible and can help capture transient interactions

Proximity-based approaches:

• BioID or TurboID fusion proteins:

  • Create GLR2.8-BioID fusion

  • Express in plants

  • Feed biotin and identify biotinylated proteins (proximity partners)

  • Validate with GLR2.8 antibodies

• APEX-based proximity labeling:

  • Similar approach with peroxidase-based labeling

Split reporter systems with antibody validation:

• Split-GFP, split-luciferase, or BRET approaches

• Express GLR2.8 fused to one half of the reporter

• Test candidate interactors fused to the complementary half

• Confirm interactions by Co-IP with GLR2.8 antibodies

Quantitative MS-based interactomics:

• SILAC or TMT labeling to compare interactomes:

  • Control vs. pathogen-challenged conditions

  • Wild-type vs. phosphorylation-site mutants

• Data analysis to identify:

  • Core GLR2.8 interactors (present in all conditions)

  • Dynamic interactors (changing with treatments)

  • Post-translationally modified interactors

Validation strategy:

• Reciprocal Co-IPs (pull down with partner antibody)

• In vitro binding assays with purified components

• Functional studies of identified interactions

This comprehensive approach has been effective for studying other receptor complexes, such as those involved in NMDA receptor clustering and autoimmune encephalitis .

How can I design experiments to resolve conflicting data about GLR2.8 function using antibody-based approaches?

When facing conflicting data about GLR2.8 function, systematic antibody-based approaches can help resolve discrepancies:

Validation of conflicting observations:

• Antibody re-validation:

  • Confirm specificity using multiple techniques:

    • Western blot against recombinant protein

    • Testing on GLR2.8 knockout tissue

    • Peptide competition assays

  • Test multiple GLR2.8 antibodies targeting different epitopes

• Cross-laboratory validation:

  • Exchange antibodies and protocols between labs

  • Perform identical experiments with standardized conditions

Systematic approach to resolve conflicts:

• Identify potential variables causing discrepancies:

  • Plant growth conditions

  • Developmental stages

  • Tissue-specific expression differences

  • Experimental conditions (buffers, temperatures, etc.)

• Design factorial experiments addressing these variables

• Use multiple detection methods in parallel:

  • Western blot

  • Immunofluorescence

  • Immunoprecipitation

  • Mass spectrometry

Recommendation table for resolving conflicting data:

Example resolution strategy:
If one study reports GLR2.8 involvement in calcium signaling while another does not, design experiments that:

• Use the same antibody in both experimental systems

• Include phosphorylation state analysis

• Test multiple stimulation conditions and timepoints

• Correlate protein detection with functional calcium imaging

• Examine both acute and long-term responses

This methodological approach aligns with studies that resolved conflicting data about glutamate receptor antibodies in opsoclonus-myoclonus syndrome , where systematic testing revealed that previously reported antibodies were not biomarkers of the disease.

How can I optimize GLR2.8 antibodies for plant tissue immunohistochemistry?

Optimizing GLR2.8 antibodies for plant tissue immunohistochemistry requires addressing several plant-specific challenges:

Plant tissue preparation optimization:

• Fixation options:

  • 4% paraformaldehyde (18-24 hours at 4°C)

  • FAA (formalin-acetic acid-alcohol) for better tissue preservation

  • Farmer's fixative for maintaining antigenicity

• Cell wall considerations:

  • Gentle cell wall digestion: 1% cellulase, 0.5% macerozyme for 10-30 minutes

  • Alternative: 1% driselase treatment

  • Monitor tissue integrity during digestion

• Sample embedding:

  • Paraffin embedding for thin sectioning

  • Cryosectioning for better antigen preservation

  • Vibratome sectioning for thicker sections with maintained 3D structure

Antigen retrieval methods:

• Heat-induced epitope retrieval:

  • Citrate buffer (pH 6.0) at 95°C for 10-20 minutes

  • Tris-EDTA buffer (pH 9.0) for different epitopes

• Enzymatic retrieval:

  • Proteinase K (1-5 μg/ml) for 5-15 minutes

  • Trypsin (0.05-0.1%) for 5-15 minutes

• Detergent-based permeabilization:

  • 0.1-0.5% Triton X-100 for 30-60 minutes

  • 0.05-0.1% saponin (gentler for membranes)

Plant tissue-specific blocking optimization:

• Test blocking agents:

  • 5% normal serum (matching secondary antibody host)

  • 2-5% BSA

  • 5% non-fat milk

  • 3% fish gelatin (low background for plant tissues)

• Include blockers of plant-specific binding:

  • 0.1-0.5% polyvinylpyrrolidone (PVP) to block phenolic compounds

  • 0.5% glycine to block aldehyde groups from fixation

Signal amplification options:

• Tyramide signal amplification (TSA):

  • 10-100× signal enhancement

  • Good for low abundance proteins like GLR2.8

• Biotin-streptavidin amplification:

  • Biotinylated secondary antibody

  • Streptavidin-fluorophore conjugate

• Tertiary antibody layer (as used in some GLR studies )

This approach incorporates techniques that have been successful in plant immunohistochemistry, adapting methodologies that were effective for tubulin visualization in Arabidopsis .

What are the best approaches for developing quantitative assays using GLR2.8 antibodies?

Developing quantitative assays with GLR2.8 antibodies requires attention to assay design, calibration, and validation:

ELISA development for GLR2.8 quantification:

• Assay format selection:

  • Direct ELISA: Simple but less sensitive

  • Sandwich ELISA: Higher sensitivity, requires two antibodies to different epitopes

  • Competitive ELISA: Good for small proteins or peptides

• Calibration curve preparation:

  • Use recombinant GLR2.8 protein or peptide

  • Prepare standard curves in the same matrix as samples

  • Include 7-8 concentration points with 2-3 fold dilutions

• Optimization steps:

  • Antibody concentration (capture and detection)

  • Blocking conditions (agent, time, temperature)

  • Sample dilution optimization

  • Incubation conditions (time, temperature)

Quantitative Western blot methodologies:

• Sample preparation standardization:

  • Consistent extraction method

  • Protein quantification before loading

  • Loading controls (tubulin or actin)

• Internal standards:

  • Recombinant GLR2.8 protein at known concentrations

  • Standard curve on each blot

• Detection optimization:

  • Use fluorescent secondary antibodies

  • Capture images within linear detection range

  • Analyze with appropriate software (ImageJ, etc.)

Multiplexed detection systems:

• Protein microarray approach:

  • Print samples on nitrocellulose-coated slides

  • Probe with GLR2.8 antibody

  • Compare signal to standard curve

• Automated capillary Western systems (e.g., Jess, Wes):

  • Higher reproducibility than traditional Western blots

  • Better quantification due to automated analysis

• Bead-based assays:

  • Couple GLR2.8 antibody to beads

  • Develop multiplex assay with other proteins of interest

  • Analyze using flow cytometry

Validation and quality control:

• Spike-and-recovery tests:

  • Add known amounts of recombinant GLR2.8 to samples

  • Calculate recovery percentage

• Linearity of dilution testing:

  • Dilute samples serially

  • Confirm proportional signal reduction

• Precision assessment:

  • Within-run precision (replicates in same assay)

  • Between-run precision (replicates across days)

  • Between-operator precision