At2g37240 Antibody

What applications is the At2g37240 Antibody validated for?

The At2g37240 antibody (e.g., product code CSB-PA692962XA01DOA) has been validated for specific applications including:

• Enzyme-linked immunosorbent assay (ELISA)

• Western blotting (WB)

These applications allow researchers to detect and quantify At2g37240 protein expression in plant samples . For Western blotting, the antibody enables identification of the target protein based on molecular weight separation, while ELISA provides quantitative measurement in complex biological samples.

What are the recommended storage and handling conditions for maintaining At2g37240 antibody activity?

For optimal preservation of antibody activity:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles that can degrade antibody performance

• The antibody is supplied in liquid form with preservative (0.03% Proclin 300)

• The storage buffer contains 50% Glycerol, 0.01M PBS, pH 7.4, which helps maintain stability

• For working solutions, store at 4°C for short-term use (1-2 weeks)

• Aliquoting the antibody before freezing is recommended to prevent repeated freeze-thaw cycles

How is the At2g37240 antibody produced and what are its general characteristics?

The At2g37240 antibody is:

• A polyclonal antibody raised in rabbits

• Immunogen: Recombinant Arabidopsis thaliana At2g37240 protein

• Purification method: Antigen affinity purified

• Isotype: IgG

• Form: Liquid, non-conjugated

• Species reactivity: Specific for Arabidopsis thaliana (Mouse-ear cress)

As a polyclonal antibody, it contains a heterogeneous mixture of antibodies recognizing multiple epitopes on the target protein, which can provide robust detection even if some epitopes are modified or masked.

What controls should be included when using At2g37240 antibody in immunological assays?

When designing experiments with the At2g37240 antibody, the following controls are crucial:

• Positive control: Include samples known to express At2g37240 (e.g., wild-type Arabidopsis leaf tissue)

• Negative control: Use tissues from At2g37240 knockout mutants if available, or tissues known not to express the protein

• Secondary antibody-only control: Omitting primary antibody to assess non-specific binding of the secondary antibody

• Blocking peptide control: Pre-incubation of the antibody with excess immunizing peptide should abolish specific binding

• Loading control: Include detection of a housekeeping protein (e.g., actin) to normalize protein loading

These controls help validate antibody specificity and experimental reliability, particularly important for new antibody lots or untested experimental conditions.

What sample preparation techniques are recommended for detecting At2g37240 in plant tissues?

For optimal detection of At2g37240 in plant tissues:

• Tissue extraction: Use fresh tissue when possible, or flash-freeze and store at -80°C

• Buffer selection: Extract with a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • Protease inhibitor cocktail

  • Consider including reducing agents like DTT (1 mM) since At2g37240 contains thioredoxin domains

• Membrane protein enrichment: Since At2g37240 may be associated with chloroplast membranes, consider fractionation methods to enrich for membrane proteins

• Denaturation: Heat samples at 70°C rather than boiling to prevent aggregation of membrane proteins

• Sample clarification: Centrifuge extracts at high speed (>10,000 × g) to remove insoluble materials

This careful preparation preserves protein integrity while maximizing extraction efficiency, especially important for potentially low-abundance proteins like At2g37240.

How should At2g37240 antibody be used for co-immunoprecipitation studies investigating protein-protein interactions?

For co-immunoprecipitation (co-IP) studies with At2g37240 antibody:

• Cross-linking (optional): Consider using membrane-permeable crosslinkers like DSP (dithiobis[succinimidyl propionate]) to stabilize transient protein interactions

• Extraction buffer: Use a gentle non-ionic detergent buffer:

  • 25 mM Tris-HCl (pH 7.4)

  • 150 mM NaCl

  • 1 mM EDTA

  • 1% NP-40 or 0.5% Triton X-100

  • Protease inhibitors

• Pre-clearing: Incubate lysate with protein A/G beads before adding antibody to reduce non-specific binding

• Antibody coupling: Consider covalently coupling At2g37240 antibody to beads using dimethyl pimelimidate to prevent antibody contamination in the eluate

• Elution strategy: Use competitive elution with excess immunizing peptide for gentle elution that preserves protein complexes

• Control IPs: Perform parallel IPs with non-immune rabbit IgG and with knockout/knockdown tissue

This approach is particularly valuable for investigating potential interactions between At2g37240 and STN7, as suggested by co-expression data in the literature .

What techniques can be used to verify At2g37240 subcellular localization in Arabidopsis?

To determine the subcellular localization of At2g37240:

• Immunogold electron microscopy:

  • Fix tissue with glutaraldehyde/paraformaldehyde

  • Embed in LR White resin

  • Prepare ultrathin sections

  • Incubate with At2g37240 antibody followed by gold-conjugated secondary antibody

  • Examine by transmission electron microscopy

• Subcellular fractionation:

  • Isolate intact chloroplasts using Percoll gradient centrifugation

  • Further fractionate chloroplasts into thylakoid membrane, stroma, and lumen

  • Analyze fractions by Western blot using At2g37240 antibody alongside marker proteins for each compartment

• Fluorescent protein fusion approach:

  • Create N- and C-terminal fusions of At2g37240 with GFP

  • Express in Arabidopsis using either transient expression or stable transformation

  • Analyze localization by confocal microscopy

  • Validate with immunofluorescence using the At2g37240 antibody

This multi-faceted approach helps resolve the current uncertainty about At2g37240's precise subcellular localization, which has been predicted but not conclusively demonstrated in the literature .

How can At2g37240 antibody be used to investigate potential thiol oxidoreductase activity in relation to STN7 kinase?

Recent research suggests a functional relationship between thiol oxidoreductases and the STN7 kinase in chloroplast signaling. To investigate this connection:

• Redox state analysis:

  • Extract proteins under conditions that preserve in vivo thiol redox states (protein extraction in TCA or with alkylating agents)

  • Perform non-reducing SDS-PAGE followed by Western blotting with At2g37240 antibody

  • Compare reduced and oxidized forms under different light conditions or in stn7 mutants

• Protein-protein interaction studies:

  • Conduct co-immunoprecipitation using At2g37240 antibody to pull down potential interacting partners

  • Analyze precipitates for presence of STN7 and other thylakoid proteins

  • Perform reciprocal IPs with STN7 antibodies

• Functional complementation assays:

  • Express At2g37240 in lto1 mutants (another thiol oxidoreductase)

  • Assess state transitions and STN7 phosphorylation activity

  • Use At2g37240 antibody to confirm expression and localization

This approach builds on observations from studies showing that LTO1 interacts with the lumenal domain of STN7, and At2g37240 has been identified as a protein with similar predicted domains .

What does co-expression analysis reveal about At2g37240 function and how can the antibody validate these predictions?

Bioinformatic co-expression analysis suggests functional relationships for At2g37240:

• Co-expression network:

  • At2g37240 appears in co-expression networks with STN7 according to the ATTED-II database

  • It belongs to a group of six proteins with predicted lumenal Trx domains

  • This suggests potential involvement in redox regulation pathways

• Validation experiments:

  • Use At2g37240 antibody for Western blot analysis across conditions where co-expressed genes are known to be regulated

  • Compare protein expression patterns in wild-type and mutant backgrounds of co-expressed genes

  • Perform chromatin immunoprecipitation (ChIP) with antibodies against transcription factors predicted to regulate both At2g37240 and co-expressed genes

• Quantitative proteomics:

  • Use At2g37240 antibody for immunoprecipitation followed by mass spectrometry

  • Analyze protein complex components under different physiological conditions

  • Compare with ProteomicsDB expression data that shows correlation between LTO1 and STN7 expression

This integrated approach connects bioinformatic predictions with experimental validation using the At2g37240 antibody as a key research tool.

How can researchers optimize immunolocalization protocols for At2g37240 in various plant tissue types?

For successful immunolocalization of At2g37240 across different plant tissues:

• Fixation optimization:

  • Test multiple fixatives: 4% paraformaldehyde, glutaraldehyde/paraformaldehyde mixtures, and methanol

  • Compare preservation of antigenicity through Western blot of fixed vs. unfixed samples

  • Optimize fixation time (2-24 hours) depending on tissue thickness

• Antigen retrieval methods:

  • Evaluate heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Test enzymatic antigen retrieval with proteases like proteinase K

  • Compare microwave, pressure cooker, and water bath heating methods

• Tissue-specific permeabilization:

  • For leaf tissue: 0.1-0.5% Triton X-100 (10-30 minutes)

  • For root tissue: 0.2-1.0% Tween-20 (15-45 minutes)

  • For thick sections: Longer permeabilization or higher detergent concentration

• Antibody dilution matrix:

  • Test serial dilutions (1:100 to 1:2000) to determine optimal concentration

  • Evaluate different incubation times (2 hours to overnight at 4°C)

  • Compare signal-to-noise ratio across conditions

This systematic optimization ensures specific detection of At2g37240 while minimizing background and preserving tissue morphology, essential for accurate localization studies.

What are common pitfalls when using At2g37240 antibody in Western blotting and how can they be addressed?

When using At2g37240 antibody for Western blotting, researchers may encounter several challenges:

• High background/non-specific binding:

  • Increase blocking time/concentration (5% BSA or milk)

  • Use more stringent washing (increase TBST concentration to 0.1-0.2% Tween-20)

  • Dilute antibody further (test 1:1000 to 1:5000 range)

  • Include 0.1-0.5% non-ionic detergent in antibody dilution buffer

• Weak or absent signal:

  • Ensure adequate protein loading (15-30 μg total protein)

  • Reduce transfer time for small proteins or increase for larger proteins

  • Try alternative membrane types (PVDF may retain more protein than nitrocellulose)

  • Decrease antibody dilution (1:500 or 1:250) if signal is weak

  • Extend primary antibody incubation time to overnight at 4°C

  • Use enhanced chemiluminescence (ECL) substrate with higher sensitivity

• Multiple unexpected bands:

  • Include reducing agent (5 mM DTT) to prevent disulfide-linked complexes

  • Use fresh tissue and protease inhibitors to prevent degradation

  • Compare with tissues from At2g37240 knockout plants to identify specific bands

  • Perform peptide competition assay to identify specific vs. non-specific bands

• Inconsistent results:

  • Standardize protein extraction and quantification methods

  • Use the same positive control across experiments

  • Prepare larger antibody working solutions to use across multiple experiments

  • Document detailed experimental conditions (exposure time, antibody lot, etc.)

These troubleshooting strategies address common technical issues while maintaining the scientific rigor necessary for published research.

How should researchers interpret multiple bands or unexpected molecular weights when using At2g37240 antibody?

When analyzing Western blot results with At2g37240 antibody:

• Expected vs. observed molecular weight:

  • The predicted molecular weight of At2g37240 should be compared to observed bands

  • Post-translational modifications may cause shifts in apparent molecular weight

  • Chloroplast transit peptide cleavage will reduce the observed size compared to predicted full-length protein

• Multiple band interpretation:

  • Higher molecular weight bands may represent:

    • Protein dimers or multimers (disappear under reducing conditions)

    • Protein-protein complexes that resist SDS denaturation

    • Post-translationally modified forms (glycosylation, phosphorylation)

  • Lower molecular weight bands may represent:

    • Proteolytic fragments (increase with sample age or inadequate protease inhibition)

    • Alternative splice variants

    • Cross-reactivity with related thioredoxin proteins

• Verification methods:

  • Immunoprecipitate with At2g37240 antibody followed by mass spectrometry

  • Compare band patterns in wild-type vs. At2g37240 knockout/knockdown plants

  • Use subcellular fractionation to determine compartment-specific forms

  • Treat samples with phosphatase or glycosidase to identify post-translational modifications

Understanding these patterns helps distinguish between artifact and biologically meaningful findings, critical for accurate data interpretation.

What approaches can help determine the specificity of At2g37240 antibody in diverse experimental contexts?

To rigorously validate At2g37240 antibody specificity:

• Genetic approaches:

  • Compare signal between wild-type and At2g37240 T-DNA insertion mutants

  • Use CRISPR/Cas9-generated knockout lines as negative controls

  • Analyze RNAi knockdown lines for corresponding reduction in signal intensity

  • Test overexpression lines for increased signal intensity

• Biochemical approaches:

  • Perform peptide competition assays by pre-incubating antibody with immunizing peptide

  • Use recombinant At2g37240 protein as positive control

  • Test cross-reactivity with recombinant related proteins (other Trx-domain proteins)

  • Compare results with commercially available antibodies against the same target

• Bioinformatic assessment:

  • Analyze epitope conservation across related proteins

  • Predict potential cross-reactive proteins based on epitope similarity

  • Assess potential post-translational modifications that might affect antibody recognition

• Sequential immunoprecipitation:

  • Deplete sample with At2g37240 antibody

  • Test depleted sample with the same antibody to confirm complete removal

  • Identify proteins in the immunoprecipitate by mass spectrometry

These approaches provide multiple lines of evidence for antibody specificity, essential for confidence in experimental results, especially for proteins like At2g37240 where detailed characterization is still ongoing.

How does At2g37240 relate to other chloroplast thiol oxidoreductases and what research questions remain open?

At2g37240 belongs to a family of chloroplast thiol oxidoreductases with varying degrees of characterization:

• Relationship to characterized thioredoxins:

  • At2g37240 is among six proteins with predicted lumenal Trx domains in Arabidopsis

  • Better-characterized members include HCF164, SOQ1, and LTO1

  • Unlike HCF164 and LTO1, At2g37240 function remains largely unknown

• Evolutionary context:

  • Comparative analysis across plant species can reveal conservation of At2g37240

  • Phylogenetic relationships between At2g37240 and other thioredoxins may suggest functional specialization

  • Use of At2g37240 antibody in multiple plant species can help determine evolutionary conservation

• Open research questions:

  • Does At2g37240 function redundantly with other thioredoxins?

  • What are its specific substrates and interaction partners?

  • How is its expression regulated under various stress conditions?

  • Does it participate in the same redox regulatory network as LTO1 and STN7?

• Experimental approaches using the antibody:

  • Comparative immunoprecipitation in various mutant backgrounds

  • Analysis of protein levels and redox state across developmental stages

  • Identification of redox-dependent protein-protein interactions

These investigations will help place At2g37240 within the complex network of chloroplast redox regulation, where several components have established roles in photosynthesis and stress responses.

What light and stress conditions should be tested when studying At2g37240 expression patterns?

Based on known functions of related thiol oxidoreductases and co-expression with STN7, researchers should consider:

• Light quality treatments:

  • Far-red light (activates PSI, oxidizes PQ pool)

  • Blue light (activates PSII, reduces PQ pool)

  • Red light (predominantly activates PSII)

  • Light intensity gradient (100-1000 μmol photons m⁻² s⁻¹)

  • Compare protein levels using At2g37240 antibody across these conditions

• State transition conditions:

  • State 1 to State 2 transition (PSII to PSI excitation pressure shift)

  • State 2 to State 1 transition (recovery phase)

  • Monitor At2g37240 protein levels alongside STN7 phosphorylation status

  • Compare with stn7 and lto1 mutants to understand functional relationships

• Oxidative stress conditions:

  • H₂O₂ treatment

  • Methyl viologen (paraquat) treatment

  • High light stress

  • UV-B exposure

  • Monitor both protein levels and redox state using non-reducing gels

• Developmental stages:

  • Seedling establishment

  • Mature leaves

  • Senescent leaves

  • Compare protein distribution using immunolocalization with At2g37240 antibody

This comprehensive analysis will help establish if At2g37240 functions in photosynthetic acclimation similar to STN7 and LTO1, or if it has distinct roles in stress responses.

How can At2g37240 antibody contribute to understanding redox regulation in chloroplasts?

The At2g37240 antibody provides several avenues for investigating chloroplast redox networks:

• Redox-state specific detection:

  • Modification of standard protocols to preserve in vivo redox states:

    • TCA precipitation to acid-trap thiols

    • Alkylation of free thiols during extraction

    • Non-reducing vs. reducing SDS-PAGE

  • Monitoring changes in oxidized vs. reduced forms of At2g37240

• Redox proteomics approaches:

  • Diagonal electrophoresis to separate proteins based on redox state

  • Redox-sensitive GFP fusions validated with At2g37240 antibody

  • OxICAT analysis with validation by Western blotting

• Integration with known redox networks:

  • Analysis of At2g37240 protein levels in:

    • STN7 kinase mutants

    • LTO1 thiol oxidoreductase mutants

    • Other photosynthetic mutants affecting redox balance

  • Correlation of protein levels with measurements of:

    • Plastoquinone redox state

    • ROS production

    • Photosynthetic electron transport rates

• Methodological innovations:

  • Development of redox-specific antibodies that recognize only oxidized or reduced forms

  • In situ proximity ligation assays to detect protein-protein interactions in intact tissues

  • Combination of fluorescent protein fusions with immunolocalization

These approaches can help establish whether At2g37240 functions in the same pathway as LTO1 in mediating redox regulation of STN7 and state transitions, or if it has distinct roles in chloroplast redox homeostasis .