At2g04810 Antibody

What is the At2g04810 protein and what cellular processes is it involved in?

At2g04810 encodes an autophagy-related protein in Arabidopsis thaliana, similar to the ATG8 protein family that plays crucial roles in autophagy processes in plants. The protein is involved in autophagosome formation, which is essential for nutrient recycling and cellular homeostasis in plants. The protein has a predicted molecular weight of approximately 15 kDa, consistent with other members of the ATG8 family. Understanding this protein's function is important for studying stress responses, senescence, and developmental processes in plants. When conducting research with At2g04810 antibodies, it's important to note that they recognize epitopes specific to this autophagy-related protein .

What are the typical applications for At2g04810 antibody in plant research?

At2g04810 antibody is primarily used in Western blotting (WB) and immunolocalization (IL) applications. In Western blotting, the antibody can detect the protein at its expected molecular weight of approximately 15 kDa. For Western blot applications, a typical dilution range of 1:1000 to 1:2000 is recommended for optimal results. For immunolocalization studies, which allow researchers to visualize the subcellular localization of At2g04810 protein within plant tissues, a dilution of 1:1000 is typically used . These applications enable researchers to study protein expression levels under various conditions and to determine the spatial distribution of the protein within plant cells and tissues.

Does the At2g04810 antibody show cross-reactivity with homologous proteins in other plant species?

Yes, antibodies raised against plant autophagy-related proteins like At2g04810 often demonstrate cross-reactivity with homologous proteins across multiple plant species due to the high conservation of these proteins. Based on similar antibodies such as anti-ATG8, reactivity has been confirmed in various species including Nicotiana benthamiana, Solanum lycopersicum (tomato), Zea mays (corn), and Populus trichocarpa. Additionally, predicted reactivity extends to other plant species including Oryza sativa (rice), Hordeum vulgare (barley), and Triticum aestivum (wheat) . When planning experiments with non-model plant species, it is advisable to perform preliminary validation tests to confirm antibody reactivity with your specific plant species of interest.

What is the recommended protocol for using At2g04810 antibody in Western blot analysis?

For optimal Western blot results with At2g04810 antibody, follow this methodological approach: Extract total proteins from plant samples using an appropriate lysis buffer similar to that described in Perez-Perez et al. (2010). Separate approximately 30 μg of total protein extract on a 15% SDS-PAGE gel. Transfer proteins to a nitrocellulose membrane using either semi-dry or tank transfer methods for approximately 1 hour. Block the membrane with 5% non-fat dry milk in TBST. Incubate with primary At2g04810 antibody at a dilution of 1:1000 to 1:2000 overnight at 4°C. Wash the membrane 3-5 times with TBST, then incubate with an appropriate HRP-conjugated secondary antibody. Develop using an enhanced chemiluminescence detection system. This protocol has been shown to effectively detect autophagy-related proteins in plant samples .

How should At2g04810 antibody be stored and handled to maintain its efficacy?

For optimal preservation of antibody activity, store lyophilized At2g04810 antibody at -20°C. After reconstitution with the recommended volume of sterile water (typically 50 μl), make smaller aliquots to avoid repeated freeze-thaw cycles, which can significantly degrade antibody quality and reduce specificity. Each aliquot should be stored at -20°C. Before using the antibody for experiments, remember to briefly spin the tubes to collect any material that might adhere to the cap or sides of the tube. When working with the antibody, maintain cold chain practices by keeping it on ice during experimental procedures. Properly stored and handled antibodies can maintain their reactivity and specificity for extended periods, ensuring consistent experimental results .

What controls should be included when using At2g04810 antibody in experiments?

When designing experiments with At2g04810 antibody, include these essential controls: 1) Positive control: Use samples from wild-type Arabidopsis thaliana known to express the target protein, or recombinant At2g04810 protein if available. 2) Negative control: Include samples from knockout or knockdown lines of At2g04810, if available, or from species known not to react with the antibody (similar to how Cuscuta chinensis and Symbiodinium sp. have been identified as non-reactive with certain plant antibodies) . 3) Loading control: Use antibodies against constitutively expressed proteins (like actin or tubulin) to normalize protein loading. 4) Secondary antibody-only control: Incubate a membrane section with only secondary antibody to detect non-specific binding. 5) Peptide competition assay: Pre-incubate the antibody with excess target peptide to confirm specificity by signal abolishment.

How can At2g04810 antibody be used to study autophagy flux in plant stress responses?

To study autophagy flux using At2g04810 antibody, implement this methodological approach: First, establish experimental conditions with appropriate stress treatments (e.g., nutrient starvation, drought, pathogen infection) alongside control groups. Extract proteins from both control and stress-induced samples using a lysis buffer optimized for preserving autophagy-related proteins. Separate proteins via SDS-PAGE and perform Western blot analysis using At2g04810 antibody at 1:1000-1:2000 dilution. The formation of At2g04810-PE (phosphatidylethanolamine) conjugates, visible as lower mobility bands, indicates autophagy induction. To measure flux specifically, include samples treated with autophagy inhibitors like concanamycin A or E-64d. An accumulation of At2g04810-PE conjugates in inhibitor-treated samples compared to non-inhibitor samples indicates active autophagy flux. This approach enables quantitative assessment of autophagy activity under various stress conditions .

How can contradictory results with At2g04810 antibody be troubleshooted and resolved?

When encountering contradictory results using At2g04810 antibody, implement this systematic troubleshooting approach: First, verify antibody quality through dot blot analysis with recombinant At2g04810 protein to confirm binding activity. Next, optimize protein extraction methods, as autophagy-related proteins may require specific buffer conditions to maintain native conformation. For Western blot inconsistencies, systematically test different blocking reagents (BSA vs. milk), membrane types (PVDF vs. nitrocellulose), and detection systems. Validate specificity by including knockout/knockdown samples or performing peptide competition assays. For contradictory localization results, compare fixation methods (paraformaldehyde vs. glutaraldehyde) and test different antigen retrieval techniques. When comparing results between species, consider evolutionary divergence in protein sequence that may affect epitope recognition. Document all experimental conditions meticulously to identify variables contributing to discrepancies. This methodical approach helps resolve contradictions and establish reliable protocols .

What expression systems are suitable for producing recombinant At2g04810 protein for antibody validation?

For recombinant At2g04810 protein production, several expression systems offer distinct advantages. Bacterial systems (E. coli) provide high yield and cost-effectiveness, though they may lack post-translational modifications relevant to plant proteins. For this approach, use pET or pGEX vectors with BL21(DE3) or Rosetta strains, inducing expression at lower temperatures (16-20°C) to enhance protein solubility. Plant-based expression systems provide more authentic post-translational modifications. Transient expression in Nicotiana benthamiana using Agrobacterium-mediated transformation offers a rapid approach with proper plant-specific modifications. For stable expression, transgenic Arabidopsis thaliana can be developed as demonstrated with other recombinant antibodies . Alternatively, cell-free expression systems allow rapid production without cellular constraints. The choice depends on experimental requirements for protein authenticity, yield, and downstream applications. Purification typically employs affinity tags (His, GST) followed by size exclusion chromatography to ensure protein homogeneity for antibody validation.

How can transgenic plants be used to express At2g04810-targeting antibodies?

Transgenic plants can be effectively utilized to express At2g04810-targeting antibodies through a methodology similar to that used for expressing therapeutic antibodies in plant systems. The process begins with designing expression constructs containing both heavy chain (HC) and light chain (LC) genes of the antibody under control of strong plant promoters. These constructs may include the KDEL ER retention signal for improved antibody accumulation, similar to the approach used for expressing mAb CO in Arabidopsis . Agrobacterium-mediated transformation is then used to introduce these constructs into Arabidopsis thaliana. Following selection of transformants, PCR and Western blot analyses confirm the insertion and expression of both HC and LC components. The antibodies can then be purified from plant biomass using affinity chromatography methods. This plant-based expression system offers advantages including proper protein folding, post-translational modifications, and cost-effective production compared to mammalian cell systems .

What are the current approaches for generating high-specificity antibodies against plant proteins like At2g04810?

Current approaches for generating high-specificity antibodies against plant proteins like At2g04810 include both traditional and advanced methodologies. Traditional approaches involve identifying unique epitopes within the At2g04810 protein sequence that distinguish it from other related proteins, typically using recombinant protein fragments as immunogens. Polyclonal antibodies, similar to those developed for ATG8 , provide broad epitope recognition but variable specificity. For higher specificity, monoclonal antibodies can be developed through hybridoma technology. More advanced approaches include phage display technology to select high-affinity antibody fragments from diverse libraries. Most recently, AI-driven antibody design platforms like MAGE (Monoclonal Antibody GEnerator) represent cutting-edge approaches, using sequence-based protein Large Language Models to generate paired variable heavy and light chain antibody sequences against specific targets . These AI approaches can design antibodies using only the antigen sequence as input, without requiring preexisting antibody templates, potentially revolutionizing rapid antibody development against specific plant proteins .

How can artificial intelligence tools enhance analysis of At2g04810 antibody experimental data?

Artificial intelligence tools can significantly enhance At2g04810 antibody experimental data analysis through multiple approaches: Advanced image analysis algorithms can automatically quantify immunofluorescence signals in complex plant tissues, improving objectivity and throughput compared to manual scoring. AI-based pattern recognition tools can identify subtle differences in protein localization patterns across different experimental conditions, potentially revealing functional insights not immediately apparent to human observers. Machine learning models can integrate Western blot quantification data with other experimental parameters to identify complex relationships between At2g04810 protein levels and plant phenotypes. For high-throughput screening applications, AI tools similar to those used in clinical research output validation can automate the quality control process, significantly reducing analysis time while maintaining data integrity. Additionally, natural language processing can extract relevant information about At2g04810 from published literature to contextualize new experimental findings. These AI-enhanced analytical approaches transform raw experimental data into deeper biological insights while improving reproducibility and efficiency .

What are the most sensitive detection methods when working with low-abundance At2g04810 protein?

For detecting low-abundance At2g04810 protein, several high-sensitivity methods can be employed: Enhanced chemiluminescence (ECL) with signal amplification systems can increase Western blot sensitivity, allowing detection of protein amounts in the low picogram range. Tyramide signal amplification (TSA) can be applied to both Western blots and immunohistochemistry, amplifying signals through deposition of multiple tyramide molecules at antibody binding sites, increasing sensitivity 10-100 fold over standard methods. For mass spectrometry-based detection, selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) approaches enable targeted detection of specific At2g04810 peptides, even in complex protein mixtures. Proximity ligation assay (PLA) offers exceptional sensitivity for detecting protein-protein interactions involving At2g04810 by generating a fluorescent signal only when two antibodies bind in close proximity. Digital ELISA platforms, which can detect single protein molecules, represent the ultimate sensitivity for quantifying extremely low-abundance targets. These advanced methods enable reliable detection and quantification of At2g04810 under conditions where conventional approaches would fail.

How can computational approaches help predict epitopes for developing new At2g04810-specific antibodies?

Computational approaches significantly enhance epitope prediction for developing highly specific At2g04810 antibodies through several methodological strategies: Begin with sequence-based prediction algorithms that analyze protein primary structure to identify regions with high antigenicity, surface accessibility, and flexibility. Structural epitope prediction utilizes 3D protein models (either experimentally determined or computationally predicted) to identify surface-exposed regions likely to serve as antibody binding sites. For increased specificity, employ comparative genomics to identify regions in At2g04810 that differ from homologous proteins, reducing cross-reactivity potential. Machine learning algorithms can integrate multiple parameters (hydrophilicity, secondary structure, evolutionary conservation) to improve prediction accuracy. Most advanced approaches include AI-based systems like protein-specific Large Language Models that can design paired antibody sequences directly against the At2g04810 target, similar to the MAGE system developed for viral antigens . These computational methods significantly streamline antibody development by prioritizing optimal epitopes before experimental work begins, reducing development time and resources while enhancing specificity .

What are the essential validation tests to confirm At2g04810 antibody specificity?

To rigorously validate At2g04810 antibody specificity, implement the following comprehensive testing panel: First, conduct Western blot analysis using wild-type Arabidopsis extracts alongside genetic controls (knockout/knockdown lines) to confirm absence of signal in genetic null backgrounds. Perform a peptide competition assay by pre-incubating the antibody with excess immunogenic peptide, which should abolish specific signals if the antibody is properly target-specific. Test cross-reactivity against purified recombinant At2g04810 protein and closely related family members to assess discrimination capability. Evaluate reactivity across multiple plant species with known At2g04810 homologs to establish cross-species utility, similar to validation approaches used for ATG8 antibodies . For immunocytochemistry applications, compare antibody labeling patterns with fluorescent protein-tagged At2g04810 expression. Additionally, conduct immunoprecipitation followed by mass spectrometry to confirm that the antibody predominantly pulls down At2g04810 and associated proteins. This multi-method validation approach ensures high confidence in antibody specificity before application in critical research contexts .

How do storage conditions and freeze-thaw cycles affect At2g04810 antibody performance?

Storage conditions and freeze-thaw cycles significantly impact At2g04810 antibody performance through several mechanisms. Repeated freeze-thaw cycles cause protein denaturation, aggregation, and fragmentation, resulting in progressively diminished binding activity. Quantitative studies show that antibody activity can decrease by 5-20% with each freeze-thaw cycle, with potentially more severe effects for polyclonal antibodies against plant proteins. Temperature fluctuations during storage can accelerate degradation, even without complete thawing. Proper storage requires maintaining lyophilized antibody at -20°C until use, then reconstituting with sterile water and dividing into small single-use aliquots (typically 10-20 μl) . For working solutions, short-term storage (1-2 weeks) at 4°C with preservatives (0.02% sodium azide) minimizes degradation while avoiding freeze-thaw effects. For reconstituted antibodies, storage at -80°C rather than -20°C may further extend shelf-life. Implementing a quality control program with periodic testing of antibody performance using standardized samples helps monitor potential degradation over time .

What quantitative methods can verify batch-to-batch consistency of At2g04810 antibodies?

To ensure batch-to-batch consistency of At2g04810 antibodies, implement these quantitative validation methods: ELISA titration curves comparing new and reference batches provide sensitivity and EC50 values that should fall within established acceptance ranges (typically ±20% variation). Quantitative Western blotting using serial dilutions of standardized Arabidopsis extracts allows determination of detection limits and signal linearity across batches. Surface plasmon resonance (SPR) offers precise affinity measurements (Kd values) that should remain consistent between batches. Flow cytometry using fixed plant protoplasts can quantify binding characteristics through mean fluorescence intensity measurements. Imaging cytometry with automated analysis can assess immunolabeling patterns in standardized samples, with statistical comparison of signal distribution profiles. Establish a reference standard of purified At2g04810 protein for normalization across testing methods. Document all validation results in a comprehensive certificate of analysis for each batch, including acceptance criteria for each parameter. These rigorous quantitative approaches ensure experimental reproducibility when transitioning between antibody batches .