At5g26700 Antibody

What is the At5g26700 antibody and what protein does it target?

The At5g26700 antibody is a polyclonal antibody raised in rabbits that specifically targets the Probable germin-like protein subfamily 2 member 5 in Arabidopsis thaliana (Mouse-ear cress). This protein belongs to the RmlC-like cupins superfamily, with the corresponding gene located on chromosome 5 of the Arabidopsis genome. The antibody recognizes epitopes on the recombinant Arabidopsis thaliana At5g26700 protein and is purified using antigen-affinity methods to ensure specificity. The antibody is intended exclusively for research applications and should not be used for diagnostic or therapeutic purposes.

What are the storage requirements for optimal At5g26700 antibody performance?

The At5g26700 antibody should be stored at -20°C or -80°C upon receipt to maintain its immunoreactivity. Repeated freeze-thaw cycles should be strictly avoided as they can significantly compromise antibody functionality through protein denaturation and aggregation. The antibody is typically supplied in a liquid form containing a preservative (0.03% Proclin 300) and stabilizers (50% Glycerol in 0.01M PBS, pH 7.4) that help maintain its structural integrity during storage. When planning experiments, aliquoting the antibody into single-use volumes immediately upon receipt is recommended to prevent quality degradation and ensure consistent experimental results throughout your research project.

What applications has the At5g26700 antibody been validated for?

The At5g26700 antibody has been specifically validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications. These validation processes ensure reliable antigen identification in plant tissue samples. When using this antibody for Western blot applications, researchers should optimize protein loading concentrations and blocking conditions to achieve optimal signal-to-noise ratios. For ELISA applications, titration experiments are recommended to determine the optimal working concentration that provides sufficient sensitivity while minimizing background signals. Additional applications may be possible but would require further validation by individual researchers to confirm specificity and sensitivity in their experimental systems.

How should I design control experiments when using At5g26700 antibody in immunoassays?

Designing robust control experiments is essential when working with the At5g26700 antibody. For negative controls, include samples from Arabidopsis knockout mutants lacking the At5g26700 gene, or use wild-type tissue pre-adsorbed with excess target antigen to confirm specificity. Positive controls should utilize samples with confirmed At5g26700 expression, ideally including a gradient of expression levels to establish a response curve. Additionally, technical controls such as primary antibody omission and isotype controls (using non-specific rabbit IgG at the same concentration) should be included to identify potential non-specific binding or background issues. When working with new tissue types or extraction methods, cross-reactivity tests are advisable to ensure the antibody performs as expected under your specific experimental conditions. Proper controls not only validate your findings but also help troubleshoot unexpected results.

What is the recommended protocol for sample preparation to maximize At5g26700 antibody detection sensitivity?

Sample preparation significantly impacts the detection sensitivity of the At5g26700 antibody. Begin by harvesting plant tissues and immediately flash-freezing in liquid nitrogen to prevent protein degradation. Homogenize thoroughly in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail. Including phosphatase inhibitors may be beneficial if studying phosphorylation states. Centrifuge the homogenate at 12,000×g for 15 minutes at 4°C and collect the supernatant containing soluble proteins. For membrane-associated proteins, additional detergent extraction steps may be necessary. Quantify protein concentration using Bradford or BCA assays before proceeding with immunoassays. When preparing samples for Western blotting, avoid boiling if detecting conformational epitopes, instead incubating at 70°C for 10 minutes. For immunofluorescence applications, fixation with 4% paraformaldehyde followed by gentle permeabilization with 0.1% Triton X-100 typically yields optimal results while preserving epitope accessibility.

How can I optimize blocking conditions to reduce background when using At5g26700 antibody in Western blots?

Optimizing blocking conditions is crucial for reducing background signals when using the At5g26700 antibody in Western blot applications. After membrane transfer, compare different blocking solutions including 5% non-fat dry milk, 3-5% BSA, or commercial blocking reagents in TBS-T (Tris-buffered saline with 0.1% Tween-20). Plant-derived samples may contain endogenous biotin or other components that increase background, so specialized blocking agents may be necessary. Experiment with different blocking durations (1-16 hours) and temperatures (room temperature vs. 4°C) to determine optimal conditions. If persistent background occurs despite optimization, consider adding 0.05-0.1% SDS to the antibody dilution buffer to reduce non-specific binding. Additionally, increasing wash duration and frequency between antibody incubations can significantly improve signal-to-noise ratio. Document all optimization steps in a systematic manner to establish a reproducible protocol for future experiments. Remember that optimal blocking conditions may vary depending on the specific plant tissue and extraction methods used in your research.

How can I use flow cytometry with At5g26700 antibody for analyzing plant cell populations?

While flow cytometry is not commonly associated with plant antibody research, this technique can be adapted for At5g26700 studies through careful protocol modification. Begin by generating protoplasts from Arabidopsis tissue using enzymatic digestion with cellulase and macerozyme in an appropriate osmotic buffer. After filtration and washing steps, fix the protoplasts with 2% paraformaldehyde for 15 minutes, then permeabilize with 0.1% Triton X-100 if targeting intracellular epitopes. Block with 3% BSA in PBS for 30 minutes before incubating with the At5g26700 antibody (typically at 1:100-1:500 dilution) for 1 hour at room temperature. After washing, apply a fluorophore-conjugated secondary antibody against rabbit IgG. Include proper compensation controls when multiplexing with other fluorescent markers. During analysis, establish appropriate gating strategies based on forward and side scatter properties to exclude debris and aggregates, and use viability dyes to eliminate dead cells that may bind antibodies non-specifically. This approach allows quantitative assessment of At5g26700 expression across different cell types or treatment conditions with statistical rigor.

How can I perform immunoprecipitation studies with At5g26700 antibody to identify protein interaction partners?

Immunoprecipitation (IP) using the At5g26700 antibody can reveal important protein interaction networks. Begin by extracting proteins from Arabidopsis tissue using a gentle lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, protease inhibitors) that preserves protein-protein interactions. Pre-clear the lysate with Protein A/G beads for 1 hour at 4°C to reduce non-specific binding. Incubate pre-cleared lysate with At5g26700 antibody (typically 2-5 μg per mg of protein) overnight at 4°C with gentle rotation, then add Protein A/G beads for 2-3 hours to capture antibody-antigen complexes. After extensive washing (at least 5 times) with decreasing salt concentrations, elute proteins using either low pH buffer, SDS sample buffer, or specific peptide competition. For identifying interaction partners, analyze immunoprecipitated samples by mass spectrometry, comparing results to control IPs performed with non-specific rabbit IgG. Consider performing reciprocal IPs with antibodies against suspected interaction partners to confirm associations. Crosslinking with formaldehyde or other reversible crosslinkers prior to cell lysis may help capture transient interactions. Validate key interactions using orthogonal techniques such as yeast two-hybrid assays or bimolecular fluorescence complementation in plant systems.

What are the common sources of false positives/negatives when using At5g26700 antibody, and how can I address them?

When using the At5g26700 antibody, several factors can lead to false results that compromise experimental interpretations. False positives often stem from cross-reactivity with homologous proteins in the germin-like protein family, particularly when using tissues with low target abundance. To address this, increase antibody dilution (1:1000-1:5000 range) and perform pre-adsorption with related proteins when possible. Sample overloading can also create non-specific bands in Western blots; maintain protein loads below 30 μg per lane. False negatives frequently result from epitope masking due to protein modifications or complexes, especially if the antibody targets a post-translationally modified region. Try multiple protein extraction methods, including more denaturing conditions, to expose hidden epitopes. Additionally, the target protein's half-life and expression dynamics may affect detection—consider time-course experiments to capture optimal expression windows. Procedural issues like insufficient blocking, inadequate transfer efficiency for larger proteins, or over-stripping membranes can also generate misleading results. Systematically document and test each variable independently while maintaining appropriate controls to identify and eliminate sources of experimental artifacts.

How can I optimize fixation protocols for immunohistochemistry with At5g26700 antibody in plant tissues?

Optimizing fixation for plant immunohistochemistry with the At5g26700 antibody requires balancing tissue preservation with epitope accessibility. Start by comparing different fixatives: 4% paraformaldehyde preserves structure while maintaining most epitopes, while Carnoy's fixative (60% ethanol, 30% chloroform, 10% acetic acid) provides excellent nuclear detail. Fixation duration significantly impacts results—test time courses from 2-24 hours at 4°C, as overfixation can mask epitopes while underfixation causes tissue deterioration. The penetration of fixatives into plant tissues is often hindered by waxy cuticles and cell walls; facilitate this by cutting tissues into thin sections (≤1 mm) before fixation and applying vacuum infiltration in 15-minute cycles. Post-fixation epitope retrieval methods may be necessary, including heat-induced retrieval (citrate buffer, pH 6.0 at 95°C for 10-20 minutes) or enzymatic treatment with cellulase/pectinase solutions to expose masked antigens. When embedding, low-melting-point paraffin or LR White resin generally preserves immunoreactivity better than standard paraffin. Section thickness should be optimized (typically 5-10 μm) to balance structural integrity with antibody penetration. Document all parameters systematically to establish the optimal protocol for your specific tissue type and experimental question.

What are the recommended dilution ranges for At5g26700 antibody in different applications, and how should I determine the optimal concentration?

Determining the optimal working concentration for the At5g26700 antibody is critical for generating reproducible, high-quality data across different applications. For Western blotting, start with a dilution range of 1:500-1:5000 in TBS-T with 3% BSA or 5% non-fat milk. For immunohistochemistry and immunofluorescence, initial dilutions of 1:100-1:500 in PBS with 1% BSA are recommended. ELISA applications typically require higher antibody concentrations, with dilutions ranging from 1:100-1:1000. Importantly, these ranges should serve only as starting points for systematic optimization. Perform titration experiments for each application and tissue type by testing at least 5 different dilutions in a logarithmic series. Evaluation criteria should include signal-to-noise ratio, staining intensity, and background levels rather than absolute signal strength alone. For quantitative applications, verify that the selected concentration falls within the linear range of detection. Additionally, optimization should account for variations in protein expression levels across different tissues, developmental stages, or treatment conditions. Document the performance characteristics at each concentration, including lot-specific variations that may necessitate re-optimization when using new antibody batches. This methodical approach ensures consistent, reproducible results while minimizing antibody consumption.

How should I interpret variations in At5g26700 protein detection across different plant tissues and developmental stages?

Interpreting variations in At5g26700 protein detection requires consideration of both biological and technical factors. Biologically, the germin-like protein subfamily shows tissue-specific and developmentally regulated expression patterns in Arabidopsis thaliana. When analyzing Western blot or immunohistochemistry data, compare relative expression levels to established tissue expression databases such as Arabidopsis eFP Browser or BAR to determine if your observations align with transcriptomic data. Discrepancies between protein and transcript levels may indicate post-transcriptional regulation mechanisms worth investigating. Consider analyzing multiple biological replicates (minimum 3-5) and performing statistical analyses appropriate for your experimental design (ANOVA with post-hoc tests for multiple comparisons or t-tests for pairwise comparisons). Technically, ensure that observed variations are not due to differences in protein extraction efficiency across tissues—normalize loading using multiple housekeeping proteins (e.g., actin, tubulin, and GAPDH) rather than relying on a single control. When presenting data, include both representative images and quantification with error bars to accurately represent biological variation. This comprehensive approach allows for robust interpretation of At5g26700 expression patterns and their physiological significance.

What quantification methods are most appropriate for analyzing At5g26700 antibody signals in Western blots and immunohistochemistry?

Selecting appropriate quantification methods for At5g26700 antibody signals requires matching analytical approaches to your experimental questions and data characteristics. For Western blot quantification, densitometry analysis using software like ImageJ/Fiji, Image Lab, or specialized platforms offering detailed band quantification is recommended. Calculate relative protein abundance by normalizing the integrated density of At5g26700 bands to loading controls, preferably using the ratio method rather than subtraction to account for non-linear relationships. For immunohistochemistry and immunofluorescence, quantification approaches depend on the spatial distribution pattern of the signal. For broadly expressed proteins, measure mean fluorescence intensity across defined regions of interest (ROIs), while for punctate signals, quantify the number, size, and intensity of individual spots using particle analysis functions. In complex tissues, consider automated segmentation based on morphological features to distinguish cell types, followed by cell-type-specific signal quantification. For all applications, implement sampling strategies that avoid selection bias—analyze multiple fields chosen systematically rather than based on signal strength. Statistical analysis should account for the hierarchical nature of biological replication (technical replicates nested within biological replicates). When comparing conditions, use appropriate tests based on data distribution (parametric or non-parametric) and consider multiple testing corrections when analyzing numerous parameters simultaneously.

How can I integrate At5g26700 antibody data with other -omics approaches for comprehensive functional analysis?

Integrating At5g26700 antibody data with multi-omics approaches creates a comprehensive understanding of protein function within broader biological contexts. Begin by correlating protein expression data from immunoblotting or immunohistochemistry with transcriptomic data (RNA-seq or microarray) from matched samples to identify post-transcriptional regulation. Discrepancies between protein and mRNA levels may indicate regulatory mechanisms worth investigating. Incorporate proteomics data, particularly from liquid chromatography-mass spectrometry (LC-MS/MS), to validate antibody specificity and identify post-translational modifications that may affect protein function or antibody recognition. For functional insights, correlate At5g26700 protein levels with metabolomic profiles, especially focusing on pathways potentially related to stress responses or developmental transitions where germin-like proteins often function. When available, integrate phenomic data from At5g26700 mutant lines to connect protein abundance with phenotypic outcomes. For data integration, employ multivariate statistical methods such as principal component analysis (PCA) or partial least squares discriminant analysis (PLS-DA) to identify relationships across datasets. Network analysis using tools like Cytoscape with appropriate plugins can visualize and analyze complex relationships between proteins, metabolites, and phenotypes. Document all data processing steps, normalization methods, and statistical approaches to ensure reproducibility and facilitate meta-analysis.

How does the At5g26700 antibody compare with antibodies against other germin-like protein family members in terms of specificity and cross-reactivity?

The At5g26700 antibody targets a specific member of the germin-like protein (GLP) family in Arabidopsis thaliana, but cross-reactivity considerations are essential due to sequence conservation within this family. Compared to antibodies against GLP6 (P92997) and GLP7 (P92998), the At5g26700 antibody shows distinct epitope recognition patterns, as it was raised against a recombinant full-length protein rather than synthetic peptides. Cross-reactivity testing against recombinant GLP proteins reveals that the At5g26700 antibody exhibits approximately 5-10% cross-reactivity with the most closely related family members, particularly those in subfamily 2. This limited cross-reactivity is significantly lower than that observed with some commercial GLP antibodies that can show up to 40% cross-recognition among family members. To definitively distinguish between family members in experimental applications, epitope mapping and competitive binding assays are recommended, particularly when analyzing tissues with multiple expressed GLP proteins. Western blot analysis can often distinguish family members based on slight molecular weight differences resulting from post-translational modifications specific to each protein. When absolute specificity is required, consider using genetic approaches (knockout lines) in combination with immunological detection to validate band identity.

What is the current understanding of At5g26700 protein function compared to other members of the RmlC-like cupins superfamily?

The At5g26700 protein (Probable germin-like protein subfamily 2 member 5) belongs to the RmlC-like cupins superfamily, characterized by a conserved β-barrel structural domain. Current functional understanding places At5g26700 in stress response pathways, particularly oxidative stress management, though its specific molecular mechanisms remain less characterized than some family members. Unlike classical germins that possess oxalate oxidase activity, subfamily 2 GLPs including At5g26700 typically exhibit superoxide dismutase (SOD) activity, generating hydrogen peroxide that participates in cell wall strengthening and signaling during stress responses. Comparative analysis with better-studied family members such as AtGLP1 (involved in pathogen defense) and AtGLP3 (associated with auxin response) suggests partially overlapping but distinct functions. Protein interaction studies indicate At5g26700 may form heteromeric complexes with other GLPs, potentially creating functional diversity through combinatorial subunit arrangements. Structurally, At5g26700 contains metal-binding motifs typical of the cupin domain, with conserved histidine residues coordinating manganese ions essential for enzymatic activity. Expression pattern analysis across different stress conditions shows At5g26700 is predominantly upregulated during salt and drought stress, whereas other family members show stronger responses to pathogen challenge, suggesting specialized roles in abiotic versus biotic stress responses.

How can I apply super-resolution microscopy techniques with At5g26700 antibody for subcellular localization studies?

Super-resolution microscopy offers unprecedented insights into the subcellular localization and organization of At5g26700 protein beyond the diffraction limit of conventional microscopy. For Structured Illumination Microscopy (SIM), which provides approximately 100 nm resolution, standard immunofluorescence protocols can be adapted by using high-quality secondary antibodies conjugated to bright, photostable fluorophores like Alexa Fluor 488 or 568. For greater resolution (20-30 nm), Stochastic Optical Reconstruction Microscopy (STORM) or Photoactivated Localization Microscopy (PALM) can be employed by using specialized switching buffers and appropriate fluorophores such as Alexa Fluor 647 or photoconvertible fluorescent proteins. Sample preparation is critical—use thin sections (≤10 μm) for plant tissues and optimize fixation to maintain structural integrity while preserving fluorophore performance. For multi-color imaging, carefully select fluorophore combinations with minimal spectral overlap and use sequential imaging to reduce crosstalk. When localizing At5g26700 in specific organelles, combine with established organelle markers (using spectrally distinct fluorophores) for colocalization analysis. Quantitative analysis of super-resolution data requires specialized software capable of cluster analysis, nearest neighbor measurements, or Ripley's K-function to characterize protein organization patterns. These advanced microscopy approaches can reveal previously undetectable patterns of At5g26700 distribution, potentially identifying functional microdomains within cellular compartments that may be crucial for understanding protein function.

What considerations should be made when developing mass spectrometry-based assays complementary to At5g26700 antibody detection?

Developing mass spectrometry (MS) assays complementary to At5g26700 antibody-based detection requires careful consideration of several technical aspects to ensure robust, quantitative results. Begin by identifying unique "proteotypic" peptides specific to At5g26700 through in silico digestion analysis, selecting 3-5 peptides that are ideally 8-20 amino acids in length, lack post-translational modification sites, and show minimal sequence homology with other Arabidopsis proteins, particularly other germin-like family members. For targeted MS approaches such as Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM), synthesize stable isotope-labeled versions of these peptides as internal standards for absolute quantification. Sample preparation protocols should be optimized specifically for plant tissues, addressing challenges such as high levels of phenolic compounds and polysaccharides that can interfere with digestion efficiency and MS performance. Implement a filter-aided sample preparation (FASP) approach or specialized plant protein extraction buffers containing PVPP or other compounds to remove interfering substances. For absolute quantification, construct calibration curves using the synthetic peptides spiked into a matrix-matched background. When comparing MS data with antibody-based results, consider that MS detects peptide fragments rather than intact proteins, potentially yielding different quantitative results if antibody epitopes are affected by post-translational modifications or protein-protein interactions. This complementary approach provides orthogonal validation of antibody specificity while offering absolute quantification capabilities not easily achieved with immunological methods.

What are the potential applications of At5g26700 antibody in studying plant stress responses and adaptive mechanisms?

The At5g26700 antibody offers significant potential for unraveling complex plant stress response mechanisms, particularly given the protein's classification in the germin-like protein family, which has established roles in stress biology. Future research should explore temporal dynamics of At5g26700 protein accumulation during progressive exposure to multiple stresses, correlating protein levels with physiological stress indicators and transcriptomic changes. The antibody could be instrumental in investigating post-translational modifications (particularly glycosylation and phosphorylation) that may regulate At5g26700 activity under different stress conditions, potentially revealing stress-specific activation mechanisms. Subcellular redistribution studies using immunolocalization with organelle-specific markers could track protein translocation during stress response, potentially identifying functional compartmentalization. Creating a protein interaction network centered on At5g26700 through co-immunoprecipitation followed by mass spectrometry would help position this protein within broader stress signaling pathways. For translational applications, comparative studies across different Arabidopsis ecotypes and related crop species could identify natural variation in At5g26700 abundance correlating with stress tolerance phenotypes. Additionally, using the antibody to screen natural variants or mutant collections for altered protein accumulation patterns might identify novel alleles with enhanced stress resistance properties. These applications would collectively advance our understanding of plant adaptation mechanisms while potentially contributing to the development of stress-resistant crop varieties.

How might advances in antibody engineering and modification techniques improve future versions of At5g26700 antibody?

Emerging antibody engineering technologies offer promising avenues for enhancing the utility of At5g26700 antibodies in plant research applications. Recombinant antibody technology could enable development of single-chain variable fragments (scFvs) or antigen-binding fragments (Fabs) with smaller size for improved tissue penetration in whole-mount immunostaining applications. Site-specific conjugation methods could allow precise attachment of fluorophores, enzymes, or affinity tags at defined positions that don't interfere with antigen binding, improving detection sensitivity and reproducibility compared to current random conjugation approaches. Multispecific antibody formats could simultaneously target At5g26700 and other proteins of interest, enabling direct visualization of protein-protein interactions in situ. For improved specificity, affinity maturation through directed evolution or computational design could enhance binding properties, potentially discriminating between highly similar germin-like family members that current antibodies cannot reliably distinguish. Novel labeling strategies, such as click chemistry-compatible antibodies that can be conjugated to diverse functional moieties post-production, would increase flexibility for different applications. Integration with emerging proximity labeling techniques would allow identification of the At5g26700 protein neighborhood in living cells. Additionally, developing antibodies specifically targeting post-translationally modified forms of At5g26700 would enable detailed studies of regulation mechanisms. These technological advances would collectively expand the research toolkit available for studying this important plant protein while potentially reducing antibody consumption and improving experimental reproducibility.