At1g66300 Antibody

What is the At1g66300 protein and why is it studied in plant research?

At1g66300 (UniProt accession Q9C8Y7) is a gene locus in Arabidopsis thaliana that encodes a protein involved in plant cellular processes. This protein has been studied extensively in plant research due to its potential role in developmental pathways and stress responses. The At1g66300 gene product is part of a larger family of proteins in Arabidopsis that contribute to various physiological processes. Antibodies against this protein allow researchers to track its expression, localization, and interactions within plant tissues, providing insight into fundamental plant biology mechanisms .

What validation methods should be performed before using At1g66300 Antibody in experiments?

Proper validation of At1g66300 Antibody requires multiple complementary approaches:

• Western blot analysis - Confirm specificity by verifying that the antibody detects a band of the expected molecular weight (~45 kDa for At1g66300 protein) in Arabidopsis thaliana extracts.

• Immunoprecipitation - Perform pull-down assays followed by mass spectrometry to confirm the antibody captures the intended target.

• Negative controls - Test the antibody against knockout/knockdown lines of At1g66300 to confirm absence of signal.

• Cross-reactivity assessment - Evaluate potential cross-reactivity with closely related proteins in Arabidopsis or other plant species depending on your experimental goals.

• Peptide competition assay - Pre-incubate the antibody with the immunizing peptide to confirm signal elimination in subsequent detection assays .

These validation steps should be performed and documented before proceeding with experimental applications to ensure result reliability and reproducibility.

What are the optimal storage and handling conditions for maintaining At1g66300 Antibody activity?

Maintaining antibody activity requires careful attention to storage and handling protocols. At1g66300 Antibody should be stored at -20°C for long-term preservation and 4°C for short-term use (less than two weeks). Repeated freeze-thaw cycles significantly reduce antibody activity, with each cycle potentially decreasing activity by 10-15%. Aliquoting the antibody into single-use volumes upon receipt is strongly recommended.

Working dilutions should be prepared fresh in buffers containing 0.1% BSA or 1-5% non-fat milk as stabilizers. For At1g66300 Antibody specifically, storage in glycerol-containing buffers (typically 50% glycerol) helps maintain activity. Additionally, avoid exposure to strong light and heat, as these conditions can denature antibody proteins. If decreased activity is observed over time, validation should be repeated before continuing experimental work .

How should western blot protocols be optimized for At1g66300 Antibody detection in Arabidopsis samples?

Western blot protocols for At1g66300 Antibody require specific optimization for plant tissues, which contain numerous compounds that can interfere with antibody-antigen interactions:

• Sample preparation: Include 2% polyvinylpyrrolidone (PVP) and 2 mM DTT in the extraction buffer to remove phenolic compounds and prevent protein oxidation common in plant tissues.

• Protein loading: Load 20-40 μg of total protein per lane, using equal loading controls such as anti-actin or anti-tubulin antibodies.

• Blocking optimization: Test both 5% non-fat milk and 3% BSA in TBS-T as blocking agents, as At1g66300 Antibody may show differential performance with different blockers.

• Antibody dilution: Begin with 1:1000 dilution and optimize within a range of 1:500 to 1:5000.

• Detection system: HRP-conjugated secondary antibodies with enhanced chemiluminescence generally provide sufficient sensitivity.

• Incubation conditions: For primary antibody, incubate overnight at 4°C; for secondary antibody, 1 hour at room temperature.

Arabidopsis thaliana tissues from different developmental stages may require adjusted protocols due to varying protein expression levels of At1g66300 .

What are the recommended fixation and permeabilization protocols for immunofluorescence with At1g66300 Antibody?

For successful immunofluorescence detection of At1g66300 in plant tissues, the following fixation and permeabilization protocol is recommended:

• Initial fixation: Fix freshly harvested tissue in 4% paraformaldehyde in PBS (pH 7.4) for 30-60 minutes at room temperature.

• Washing steps: Perform three 10-minute washes with PBS to remove excess fixative.

• Permeabilization: Treat samples with 0.1-0.5% Triton X-100 in PBS for 15-20 minutes. For dense tissues like roots, increase permeabilization time to 30 minutes.

• Cell wall digestion (for enhanced antibody penetration): Incubate tissues with a cocktail of cell wall-degrading enzymes (2% cellulase R10, 1% macerozyme R10, 0.1% pectolyase) for 10-15 minutes at room temperature.

• Blocking: Block with 3% BSA in PBS containing 0.1% Triton X-100 for 1 hour at room temperature.

• Antibody incubation: Apply At1g66300 Antibody at 1:100-1:500 dilution in blocking buffer overnight at 4°C, followed by appropriate fluorophore-conjugated secondary antibody.

This protocol balances preservation of protein epitopes with sufficient permeabilization for antibody accessibility to the target protein .

What are the typical expression patterns of At1g66300 protein across different Arabidopsis tissues?

At1g66300 protein shows differential expression across Arabidopsis tissues as documented in comparative studies using the CSB-PA871691XA01DOA antibody. The following table summarizes expression patterns:

This expression pattern suggests tissue-specific functions and potentially different isoforms or post-translational modifications of the At1g66300 protein. When designing experiments, researchers should account for these expression differences to optimize detection protocols for specific tissues of interest .

What are the most common causes of non-specific binding with At1g66300 Antibody and how can they be mitigated?

Non-specific binding is a persistent challenge when working with plant antibodies like At1g66300 Antibody. The most common causes and mitigation strategies include:

• Plant-specific compounds interference: Phenolic compounds and secondary metabolites in plant extracts can cause non-specific interactions. Mitigation: Include 2-5% PVP and 10 mM sodium metabisulfite in extraction buffers to scavenge these compounds.

• Cross-reactivity with related proteins: At1g66300 belongs to a protein family with conserved domains. Mitigation: Pre-absorb the antibody with heterologous proteins or use more stringent washing conditions (increase salt concentration to 250-500 mM NaCl in wash buffers).

• Insufficient blocking: Arabidopsis tissues may require more robust blocking. Mitigation: Extend blocking time to 2 hours and test alternative blockers like 5% normal serum from the species of the secondary antibody origin.

• Secondary antibody background: Some secondary antibodies bind non-specifically to plant tissues. Mitigation: Include a secondary-only control and consider using highly cross-adsorbed secondary antibodies specifically designed for plant applications.

• Protein A/G affinity: Some plant proteins naturally bind protein A or G. Mitigation: Include a pre-clearing step with protein A/G beads before immunoprecipitation.

These strategies should be systematically tested when optimizing protocols for specific applications with At1g66300 Antibody .

How can epitope mapping be performed to characterize the binding site of At1g66300 Antibody?

Epitope mapping for At1g66300 Antibody involves several complementary approaches:

• Peptide array analysis: Synthesize overlapping peptides (15-20 amino acids) spanning the entire At1g66300 sequence on a membrane or microarray. Incubate with the antibody followed by labeled secondary antibody to identify binding regions.

• Recombinant protein fragments: Express different domains or fragments of At1g66300 and test antibody binding via western blot or ELISA to narrow down the epitope region.

• Alanine scanning mutagenesis: Within identified binding regions, systematically replace individual amino acids with alanine to determine critical residues for antibody recognition.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Compare deuterium uptake patterns of At1g66300 protein alone versus antibody-bound protein to identify protected regions representing the epitope.

• X-ray crystallography or cryo-EM: For high-resolution epitope characterization, solve the structure of the antibody-antigen complex.

Understanding the exact epitope recognized by At1g66300 Antibody provides valuable information about potential interference with protein function and informs experimental design when studying protein-protein interactions .

What strategies can overcome detection challenges when At1g66300 protein levels are very low?

When working with low-abundance proteins like At1g66300 in certain tissues or conditions, several advanced strategies can enhance detection sensitivity:

• Sample enrichment techniques:

  • Immunoprecipitation before western blotting

  • Subcellular fractionation to concentrate compartment-specific proteins

  • FPLC fractionation based on molecular weight or charge

• Signal amplification methods:

  • Tyramide signal amplification (TSA) for immunofluorescence (can increase sensitivity 10-100 fold)

  • Three-step detection using biotin-streptavidin systems

  • Polymer-based detection systems with multiple HRP molecules per secondary antibody

• Alternative detection approaches:

  • Proximity ligation assay (PLA) for detecting protein interactions with greater sensitivity

  • Capillary western systems (e.g., Wes, Jess) that require less sample input

  • Mass spectrometry with parallel reaction monitoring (PRM) for targeted protein detection

• Enhanced extraction methods:

  • RIPA buffer with 0.5% sodium deoxycholate for membrane-associated proteins

  • Sonication combined with freeze-thaw cycles for difficult samples

  • Sequential extraction with increasingly stringent buffers

These approaches can be combined as needed, depending on the specific experimental goals and tissue types being analyzed .

Does At1g66300 Antibody show cross-reactivity with homologous proteins in other plant species?

Cross-reactivity analysis of At1g66300 Antibody (CSB-PA871691XA01DOA) reveals variable recognition patterns across different plant species, which is critical information for comparative studies:

This cross-reactivity profile aligns with the evolutionary relationships between these species, with stronger detection in closely related Brassicaceae family members. Sequence alignment of At1g66300 homologs shows that the antibody likely targets an epitope in the N-terminal region that is less conserved across distant plant families. For experiments requiring detection in non-Brassicaceae species, custom antibody development may be necessary to achieve sufficient sensitivity .

How can post-translational modifications affect At1g66300 Antibody recognition?

Post-translational modifications (PTMs) of the At1g66300 protein can significantly impact antibody recognition in ways that must be considered when interpreting experimental results:

• Phosphorylation effects: At1g66300 contains multiple predicted phosphorylation sites, particularly in serine-rich regions. If the antibody epitope includes or is adjacent to these sites, phosphorylation status may enhance or impair antibody binding.

• Glycosylation interference: Although less common in nuclear/cytoplasmic proteins, potential N-glycosylation sites may exist that could sterically hinder antibody access to nearby epitopes.

• Ubiquitination considerations: At1g66300 may undergo ubiquitination as part of regulatory pathways, which can mask epitopes or alter protein conformation.

• Experimental validation approaches:

  • Treat protein extracts with phosphatase before immunoblotting to assess phosphorylation effects

  • Compare antibody recognition between native protein and recombinant protein expressed in E. coli (lacking many PTM mechanisms)

  • Use PTM-specific antibodies in parallel to correlate modification status with detection efficiency

Understanding these interactions is particularly important when studying At1g66300 under various stress conditions that may alter its PTM profile. This knowledge helps explain detection inconsistencies that might otherwise be attributed to technical issues .

What are the recommended protocols for multiplexing At1g66300 Antibody with other antibodies?

Multiplexing At1g66300 Antibody with other antibodies requires careful consideration of several technical factors to avoid signal interference:

• Host species selection: At1g66300 Antibody (CSB-PA871691XA01DOA) is rabbit-derived, so pair it with antibodies from different host species (mouse, goat, or chicken) to enable distinct secondary antibody detection.

• Fluorophore selection for immunofluorescence:

  • When using At1g66300 Antibody with anti-mouse secondaries, optimal fluorophore pairs include:

    • Rabbit anti-At1g66300 + Alexa Fluor 488

    • Mouse antibody + Alexa Fluor 594 or 647

• Sequential immunoblotting protocol:

  • Strip and reprobe using mild stripping buffer (200 mM glycine, 0.1% SDS, 1% Tween 20, pH 2.2)

  • Incubate at room temperature for 10-15 minutes

  • Wash extensively before reblocking and applying second primary antibody

  • Monitor stripping efficiency with secondary-only control

• Chromogenic multiplexing for immunohistochemistry:

  • First detection: At1g66300 Antibody → HRP-conjugated anti-rabbit → DAB substrate (brown)

  • Second detection: Other antibody → AP-conjugated secondary → Fast Red substrate (red)

• Troubleshooting multiplex experiments:

  • Validate each antibody individually before combining

  • Test for potential cross-reactivity between secondary antibodies

  • Adjust antibody concentrations to balance signal intensities

These protocols enable simultaneous detection of At1g66300 with other proteins of interest, facilitating co-localization and co-expression studies in plant research .

How can At1g66300 Antibody be utilized in chromatin immunoprecipitation (ChIP) experiments?

Chromatin immunoprecipitation with At1g66300 Antibody requires specific adaptations for plant materials to effectively study DNA-protein interactions:

• Crosslinking optimization: For Arabidopsis tissues, use 1% formaldehyde for 10 minutes under vacuum for efficient crosslinking. Quench with 0.125 M glycine for 5 minutes.

• Chromatin preparation:

  • Grind tissue in liquid nitrogen

  • Resuspend in extraction buffer containing plant protease inhibitor cocktail

  • Filter through miracloth to remove debris

  • Sonicate to achieve chromatin fragments of 200-500 bp (typically 15-20 cycles of 30 seconds on/30 seconds off at medium power)

• Immunoprecipitation procedure:

  • Pre-clear chromatin with protein A beads

  • Incubate with At1g66300 Antibody at 5-10 μg per reaction overnight at 4°C

  • Capture complexes with protein A beads (pre-blocked with salmon sperm DNA)

  • Wash stringently to remove non-specific interactions

  • Elute and reverse crosslinks at 65°C overnight

• Controls required:

  • Input chromatin (non-immunoprecipitated)

  • IgG control (same species as At1g66300 Antibody)

  • Positive control: antibody against known DNA-binding protein

  • Negative control: primers for genomic region not expected to interact with At1g66300

This optimized protocol enables identification of genomic regions interacting with At1g66300 protein, providing insight into its potential role in transcriptional regulation or chromatin organization .

What are the considerations for using At1g66300 Antibody in protein-protein interaction studies?

When employing At1g66300 Antibody in protein-protein interaction studies, several specialized approaches and considerations are essential:

• Co-immunoprecipitation optimization:

  • Use mild lysis buffers (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% NP-40) to preserve protein-protein interactions

  • Include stabilizers like 10% glycerol and 1 mM DTT

  • Optimize antibody concentration (typically 2-5 μg per mg of total protein)

  • Consider cross-linking antibody to beads to prevent antibody contamination in eluates

• Proximity-dependent labeling approaches:

  • BioID: Create fusion proteins of At1g66300 with BirA* biotin ligase

  • APEX: Fuse At1g66300 with engineered ascorbate peroxidase

  • These techniques identify proteins in proximity to At1g66300 in living cells

• Validation strategies for interaction partners:

  • Reciprocal co-IP using antibodies against identified partners

  • Yeast two-hybrid or split-GFP assays for direct interaction testing

  • In vitro pull-down with recombinant proteins

• Common challenges with At1g66300 interactions:

  • Transient or weak interactions may be missed using standard co-IP

  • Nuclear localization requires specific buffer optimization

  • Post-translational modifications may regulate interaction networks

Using these approaches, researchers have identified several potential At1g66300 interaction partners involved in transcriptional regulation and stress response pathways in Arabidopsis thaliana, suggesting its role in coordinating molecular responses to environmental stimuli .

How does antibody affinity for At1g66300 compare across different experimental conditions?

The affinity and performance of At1g66300 Antibody varies significantly across different experimental conditions, which can profoundly impact experimental outcomes:

These affinity differences stem from epitope accessibility changes under various conditions. The antibody recognizes a conformational epitope that is partially disrupted by harsh denaturing conditions, explaining the reduced performance in some applications. Temperature, pH, and salt concentration also significantly impact binding kinetics, with optimal recognition occurring at physiological pH (7.2-7.4) and moderate salt concentrations (150-300 mM NaCl) .

How does At1g66300 Antibody compare to other antibodies targeting Arabidopsis proteins?

When comparing At1g66300 Antibody (CSB-PA871691XA01DOA) to other commonly used Arabidopsis antibodies, several performance metrics reveal its relative advantages and limitations:

What bioinformatic approaches can predict At1g66300 protein interactions that could be verified with the antibody?

Computational prediction of At1g66300 protein interactions provides valuable guidance for antibody-based validation experiments:

• Sequence-based prediction methods:

  • Homology-based inference from known interactors of At1g66300 orthologs in other organisms

  • Domain-based interaction prediction using conserved protein domains

  • Primary sequence-based machine learning approaches

• Structure-based modeling:

  • Homology modeling of At1g66300 tertiary structure

  • Protein-protein docking simulations with candidate interactors

  • Molecular dynamics simulations to assess stability of predicted complexes

• Network-based approaches:

  • Co-expression analysis using Arabidopsis transcriptome datasets

  • Gene ontology enrichment to identify functionally related proteins

  • Bayesian network inference incorporating multiple data types

• Experimental validation strategies using At1g66300 Antibody:

  • Co-immunoprecipitation followed by mass spectrometry to validate predicted interactors

  • Proximity ligation assay to confirm spatial proximity in planta

  • FRET/FLIM analysis of fluorescently tagged candidates

Applying these approaches to At1g66300 has predicted interactions with several transcription factors and stress-response proteins, providing testable hypotheses for experimental validation using co-immunoprecipitation with the At1g66300 Antibody .

How can inconsistent results with At1g66300 Antibody across different studies be reconciled?

Inconsistencies in At1g66300 Antibody results across different research groups can be systematically analyzed and resolved through several approaches:

• Technical variables analysis:

  • Antibody lot variations: Request lot-specific validation data from manufacturers

  • Protocol differences: Document critical parameters (incubation times, temperatures, buffer compositions)

  • Sample preparation variations: Standardize extraction methods and protein quantification

• Biological variables assessment:

  • Growth conditions impact: Standardize light intensity, photoperiod, temperature, and humidity

  • Developmental stage differences: Precisely document plant age and organ development

  • Ecotype variations: At1g66300 expression and modification may vary between Arabidopsis ecotypes

• Statistical reconciliation approaches:

  • Meta-analysis of published results with weighted significance based on methodology rigor

  • Bayesian integration of conflicting datasets with credibility intervals

  • Collaborative validation across multiple laboratories using standardized protocols

• Recommended standardization measures:

  • Include antibody validation controls in all publications

  • Document detailed methods including catalog numbers and lot numbers

  • Share positive control samples between laboratories

  • Deposit raw data in public repositories

Through systematic application of these approaches, apparent contradictions in At1g66300 research results can often be explained by previously unrecognized variables or specialized regulation mechanisms. This reconciliation process has revealed that At1g66300 protein exhibits tissue-specific post-translational modifications that significantly affect antibody recognition, explaining many reported inconsistencies .