At2g26030 Antibody

What is At2g26030 and why is it studied in Arabidopsis research?

At2g26030 is a protein-coding gene in Arabidopsis thaliana (Mouse-ear cress), a model organism widely used in plant molecular biology. The protein has the UniProt accession number Q8H1M0 and is studied for its role in plant cellular processes. Antibodies against this protein are essential tools for researchers investigating plant molecular pathways, protein interactions, and gene expression patterns. Understanding At2g26030's function contributes to our broader knowledge of plant biology, development, and stress responses .

What are the key specifications of commercially available At2g26030 antibodies?

At2g26030 antibodies are typically polyclonal IgG antibodies raised in rabbits using recombinant Arabidopsis thaliana At2g26030 protein as the immunogen. These antibodies are usually supplied in liquid form containing preservatives (such as 0.03% Proclin 300) and stabilizers (approximately 50% Glycerol in 0.01M PBS, pH 7.4). They are purified using antigen affinity methods to ensure specificity. Commercial antibodies are designed to react specifically with Arabidopsis thaliana samples and are validated for applications including ELISA and Western Blotting .

How should At2g26030 antibodies be stored to maintain optimal activity?

At2g26030 antibodies require proper storage conditions to maintain their binding efficacy and specificity. Upon receipt, these antibodies should be stored at either -20°C or -80°C for long-term preservation. It is critical to avoid repeated freeze-thaw cycles, as this can lead to protein denaturation and loss of antibody function. When working with the antibody, aliquoting into single-use volumes is recommended to minimize freeze-thaw damage. The storage buffer containing 50% glycerol helps maintain antibody stability during freezing, while the presence of 0.03% Proclin 300 prevents microbial growth during handling and short-term storage .

What is the recommended protocol for using At2g26030 antibody in Western blot applications?

For Western blot applications using At2g26030 antibody, researchers should follow this methodological approach:

• Sample Preparation: Extract proteins from Arabidopsis tissues using appropriate lysis buffer containing protease inhibitors.

• Protein Separation: Separate proteins (20-40 μg per lane) via SDS-PAGE using 10-12% polyacrylamide gels.

• Transfer: Transfer proteins to PVDF or nitrocellulose membranes using standard protocols.

• Blocking: Block the membrane with 5% non-fat milk or BSA in TBST for 1-2 hours at room temperature.

• Primary Antibody Incubation: Dilute the At2g26030 antibody (recommended starting dilution 1:500-1:2000) in blocking solution and incubate overnight at 4°C.

• Washing: Wash the membrane 3-5 times with TBST, 5 minutes each.

• Secondary Antibody: Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000-1:10000) for 1-2 hours at room temperature.

• Signal Detection: After washing, visualize using chemiluminescence detection reagents.

This protocol ensures optimal detection of the At2g26030 protein while minimizing background signals .

How can At2g26030 antibody be validated for specificity in plant tissue samples?

Validating At2g26030 antibody specificity requires multiple complementary approaches:

• Positive Control: Include protein extracts from wild-type Arabidopsis thaliana expressing the At2g26030 protein.

• Negative Control: Use protein extracts from At2g26030 knockout or knockdown lines.

• Peptide Competition Assay: Pre-incubate the antibody with excess purified At2g26030 protein or immunogenic peptide before application to the sample. Signal disappearance confirms specificity.

• Multiple Detection Methods: Verify detection using different techniques (Western blot, immunofluorescence, ELISA) to ensure consistent results.

• Cross-Reactivity Testing: Test against protein extracts from related plant species to assess potential cross-reactivity.

• Immunoprecipitation followed by Mass Spectrometry: Confirm the identity of the precipitated protein.

Thorough validation ensures experimental results can be interpreted with confidence and prevents misattribution of signals to non-specific binding .

What considerations should be made when designing experiments that combine At2g26030 antibody with recombinant expression systems?

When designing experiments that combine At2g26030 antibody with recombinant expression systems, researchers should consider:

• Tag Interference: Ensure N-terminal His-FLAG tags (as used in the pK7-HFN vector system) don't interfere with antibody epitope recognition. Conduct pilot experiments comparing tagged and untagged protein detection.

• Expression Level Optimization: The CaMV 35S promoter used in pK7HFN-AT2G26030 constructs provides strong constitutive expression, which may not reflect natural protein levels. Consider using inducible or native promoters for physiologically relevant studies.

• Background Control: Include empty vector controls to distinguish between recombinant At2g26030 detection and potential background signal.

• Antibody Dilution Optimization: Recombinant systems often produce higher protein levels than endogenous expression, requiring adjustment of antibody dilutions to prevent signal saturation.

• Subcellular Localization Verification: Compare localization of recombinant protein with endogenous protein to ensure physiological relevance.

• Functionality Assessment: Verify that the recombinant protein maintains biological activity through complementation studies in knockout lines .

How can At2g26030 antibody be utilized in decoy-based approaches to study protein-protein interactions?

At2g26030 antibody can be strategically employed in decoy-based approaches to investigate protein-protein interactions through the following methodology:

• Decoy System Design: Utilize the pK7HFN-AT2G26030decoy system, which employs N-terminal His-FLAG tags expressed under CaMV 35S promoter, to create protein decoys that mimic binding interfaces.

• Competition Experiments: The decoy proteins can compete with endogenous At2g26030 for binding partners, allowing researchers to identify interaction disruptions using the antibody as a detection tool.

• Co-Immunoprecipitation Validation: At2g26030 antibody can be used in co-IP experiments to verify disrupted or maintained interactions in the presence of decoy constructs.

• Quantitative Binding Analysis: Combine the antibody with techniques like microscale thermophoresis or surface plasmon resonance to quantify binding kinetics between At2g26030 and putative partners.

• Subcellular Localization Studies: Use immunofluorescence with the At2g26030 antibody to track changes in protein localization when decoy constructs are expressed.

This approach allows researchers to dissect complex protein interaction networks by selectively disrupting specific binding interfaces while monitoring consequences with the antibody .

What strategies can be employed to optimize At2g26030 antibody for energy-based preference studies in protein interaction analysis?

Optimizing At2g26030 antibody for energy-based preference studies requires sophisticated approaches to enhance binding specificity and affinity:

• Residue-Level Energy Decomposition: Apply computational methods to analyze the energy contribution of individual amino acid residues in the antibody-antigen interface. This allows identification of critical binding determinants through techniques similar to those described in energy-based preference optimization.

• Binding Affinity Enhancement: Modify critical residues in the antibody based on energy calculations to improve binding characteristics. This can be guided by direct energy-based preference optimization methods similar to those used in antibody design algorithms.

• Conflict Mitigation Between Energy Components: Apply "gradient surgery" techniques to optimize multiple energy parameters simultaneously (attraction vs. repulsion) to enhance antibody performance without sacrificing specificity or creating unfavorable interactions.

• Synthetic Data Training: Generate computational models using self-synthesized energy-based antibody preference data to overcome limitations of scarce experimental data, as demonstrated in advanced antibody optimization frameworks.

• Fine-Grained Residue-Level Analysis: Implement analysis at individual residue level to maximize efficiency of the optimization process rather than treating the antibody as a single unit .

How can At2g26030 antibody be integrated into multi-dimensional experimental designs addressing complex plant signaling networks?

Integrating At2g26030 antibody into multi-dimensional experimental designs requires sophisticated methodological approaches:

• Temporal Expression Profiling: Use the antibody in time-course Western blot analyses to track At2g26030 protein levels across developmental stages or following various stimuli, creating a temporal map of expression patterns.

• Spatial Organization Studies: Combine the antibody with tissue-specific sampling and immunohistochemistry to determine cell-type specific expression and subcellular localization, building a comprehensive spatial distribution map.

• Interactome Mapping: Deploy the antibody in large-scale co-immunoprecipitation studies followed by mass spectrometry to identify protein-protein interaction networks under various environmental conditions.

• Post-Translational Modification Analysis: Use the antibody in conjunction with modification-specific detection methods to identify how phosphorylation, ubiquitination, or other modifications affect At2g26030 function in signaling cascades.

• Multi-Omics Data Integration: Correlate protein levels detected by the antibody with transcriptomic, metabolomic, and phenomic data to establish a comprehensive systems biology perspective.

• In Vivo Dynamics: Utilize the antibody for FRET-based approaches when combined with fluorescently-tagged potential interaction partners to study dynamic interactions in living cells .

What are common issues encountered with At2g26030 antibody in Western blot applications and how can they be resolved?

Researchers frequently encounter several challenges when using At2g26030 antibody in Western blot applications. These issues and their methodological solutions include:

Implementing these methodological adjustments systematically can significantly improve the quality and reliability of Western blot results with the At2g26030 antibody .

How can researchers address challenges related to epitope accessibility in immunoprecipitation experiments using At2g26030 antibody?

Addressing epitope accessibility challenges in immunoprecipitation experiments with At2g26030 antibody requires systematic methodological refinements:

• Optimized Lysis Conditions: Test multiple lysis buffers with varying detergent types (CHAPS, NP-40, Triton X-100) and concentrations (0.1-1%) to identify conditions that maintain protein conformation while exposing the epitope.

• Epitope Unmasking Strategies: Implement gentle denaturation methods such as low concentrations of SDS (0.1%) or urea (1-2M) that can expose hidden epitopes without completely denaturing the protein structure.

• Cross-linking Optimization: If using formaldehyde cross-linking, titrate concentrations (0.1-1%) and duration (5-20 minutes) to balance between preserving protein complexes and maintaining epitope accessibility.

• Antibody Immobilization Approaches: Compare direct antibody immobilization to beads versus indirect capture using Protein A/G to identify the approach that provides optimal epitope access.

• Sequential Epitope Exposure: Implement a step-wise epitope exposure protocol where initial mild conditions are followed by progressively stronger conditions if initial immunoprecipitation fails.

• Pre-clearing Optimization: Extend pre-clearing steps (2-4 hours) to reduce non-specific binding that might interfere with epitope recognition .

What strategies can be employed to minimize cross-reactivity when using At2g26030 antibody in plant species other than Arabidopsis thaliana?

Minimizing cross-reactivity when using At2g26030 antibody across plant species requires systematic validation and optimization approaches:

• Sequence Homology Analysis: Before experimental work, conduct bioinformatic analysis to identify homologous proteins in the target plant species and assess sequence conservation in epitope regions.

• Epitope-Specific Validation: Synthesize peptides representing the epitope regions from both Arabidopsis and the target species to test antibody binding affinity and specificity differences.

• Graduated Stringency Protocol: Employ a series of increasingly stringent washing conditions (salt concentration 150-500 mM, detergent 0.05-0.5%) to identify optimal conditions that retain specific binding while eliminating cross-reactivity.

• Competitive Blocking: Pre-incubate the antibody with recombinant proteins or peptides from potentially cross-reactive species to absorb antibodies that might cause cross-reactivity.

• Dilution Optimization Matrix: Test a matrix of antibody dilutions (1:500, 1:1000, 1:2000, 1:5000) against protein concentrations from the non-target species to identify conditions that maximize signal-to-noise ratio.

• Species-Specific Controls: Include positive controls (Arabidopsis samples), negative controls (samples from evolutionarily distant plants), and target species samples in parallel experiments to clearly differentiate specific from non-specific signals .

How should quantitative Western blot data using At2g26030 antibody be normalized and statistically analyzed?

Proper normalization and statistical analysis of quantitative Western blot data using At2g26030 antibody should follow these methodological steps:

• Loading Control Selection: Use housekeeping proteins (such as actin, tubulin, or GAPDH) that maintain stable expression across experimental conditions as internal references. For plant samples, UBQ10 or EF1α are often appropriate.

• Signal Quantification Protocol:

  • Capture images within the linear dynamic range of the detection system

  • Measure integrated density of each band using image analysis software

  • Subtract background from an adjacent area for each band

  • Calculate the ratio of At2g26030 signal to loading control signal

• Technical Replication: Perform at least three technical replicates of each Western blot to account for technical variation.

• Biological Replication: Include a minimum of three biological replicates for each experimental condition.

• Statistical Analysis Framework:

  • Test for normal distribution using Shapiro-Wilk test

  • For normally distributed data, apply parametric tests (t-test for two conditions, ANOVA for multiple conditions)

  • For non-normally distributed data, use non-parametric alternatives (Mann-Whitney U, Kruskal-Wallis)

  • Apply appropriate post-hoc tests (Tukey's HSD, Bonferroni) for multiple comparisons

  • Set significance threshold at p < 0.05

This approach ensures robust quantification and appropriate statistical interpretation of At2g26030 protein expression data .

What considerations should researchers make when comparing results from different methodologies using At2g26030 antibody?

When comparing results from different methodologies using At2g26030 antibody, researchers should consider these critical analytical factors:

How can researchers effectively distinguish between specific signal and artifacts when using At2g26030 antibody in complex plant tissue samples?

Distinguishing between specific signals and artifacts when using At2g26030 antibody in complex plant tissues requires a systematic methodological approach:

• Multiple Control Integration Strategy:

  • Include wild-type plants as positive controls

  • Use knockout/knockdown lines (At2g26030 mutants) as negative controls

  • Perform secondary antibody-only controls to identify non-specific binding

  • Implement peptide competition assays using the immunizing peptide to confirm specificity

• Signal Validation Across Techniques: Verify the detected signal using multiple independent methods (Western blot, immunofluorescence, ELISA) to confirm consistency.

• Gradient Analysis: In tissues with expected expression gradients, verify that signal intensity correlates with expected biological patterns rather than showing random distribution indicative of artifacts.

• Extraction Method Comparison: Compare results using different protein extraction protocols to identify extraction-dependent artifacts.

• Molecular Weight Verification: For Western blots, verify that the detected band appears at the expected molecular weight of At2g26030 protein (with consideration for post-translational modifications).

• Biological Relevance Assessment: Correlate protein detection patterns with known biological responses or developmental stages where At2g26030 is expected to be regulated.

• Advanced Microscopy Techniques: For immunofluorescence, use super-resolution microscopy combined with co-localization studies to distinguish specific subcellular localization from artifactual signals .