At2g39530 Antibody

What is At2g39530 protein and what is its significance in plant biology?

At2g39530 is identified as a Casp-Like Protein in Arabidopsis thaliana (Mouse-ear cress), comprising 178 amino acids. The protein is encoded by the At2g39530 gene and has a UniProt accession number of Q8GWD5 . While the complete functional characterization of this protein continues to evolve, Casp-Like proteins generally play important roles in plant cellular processes. The study of At2g39530 contributes to our understanding of plant cellular organization, stress responses, and developmental pathways in model organisms. This protein's investigation offers insights into fundamental plant biology mechanisms that may have broader implications across plant species.

What are the key specifications of the At2g39530 Antibody?

The At2g39530 Antibody (product code CSB-PA811476XA01DOA) is a polyclonal antibody raised in rabbits against a recombinant Arabidopsis thaliana At2g39530 protein . It is purified using antigen affinity methods and supplied in liquid form. The antibody is stored in a preservative buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 . This non-conjugated IgG antibody is specifically reactive to Arabidopsis thaliana and has been validated for ELISA and Western Blot applications to ensure proper identification of the target antigen . The detailed specifications are summarized in the table below:

How should the At2g39530 Antibody be stored to maintain optimal activity?

The At2g39530 Antibody requires careful storage conditions to maintain its activity and specificity. Upon receipt, the antibody should be stored at either -20°C or -80°C . It is critical to avoid repeated freeze-thaw cycles as these can degrade the antibody and diminish its performance in experimental applications . For laboratories conducting ongoing research with this antibody, it is advisable to create working aliquots upon receipt to minimize freeze-thaw cycles. Each aliquot should contain sufficient volume for single experiments. When handling the antibody during experiments, it should be kept on ice and returned to frozen storage promptly after use. Proper storage ensures the antibody maintains its binding capacity and specificity, which are essential for generating reliable experimental results in plant molecular biology research.

What experimental applications has the At2g39530 Antibody been validated for?

The At2g39530 Antibody has been validated specifically for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications . For Western Blotting, the antibody can be used to detect the native At2g39530 protein in plant tissue extracts, providing information about protein expression levels and molecular weight confirmation. In ELISA applications, the antibody enables quantitative detection of the At2g39530 protein in various sample preparations. While these are the validated applications, researchers may potentially adapt this antibody for other immunological techniques such as immunohistochemistry or immunoprecipitation, though additional validation would be required. For any application, it is essential to include proper controls to ensure specific detection of the target protein and minimize background or non-specific binding.

How can researchers validate the specificity of the At2g39530 Antibody in their experimental systems?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For the At2g39530 Antibody, several complementary approaches should be employed. First, researchers should perform a Western blot with positive controls containing recombinant At2g39530 protein alongside wild-type Arabidopsis thaliana extracts . A specific band at the expected molecular weight (corresponding to the 178 amino acid protein) should be observed. Additionally, negative controls using At2g39530 knockout/knockdown plant lines should be included, where the specific band should be absent or significantly reduced .

Peptide competition assays provide another validation method, where pre-incubating the antibody with excess purified At2g39530 protein or peptide immunogen should block specific binding in subsequent assays . For definitive validation, researchers can employ mass spectrometry to confirm the identity of immunoprecipitated proteins. Parallel detection with a second antibody targeting a different epitope of At2g39530 can further confirm specificity. These validation procedures should be documented with appropriate controls and quantified to establish threshold criteria for specific versus non-specific binding.

What are the optimal conditions for using At2g39530 Antibody in Western Blot analyses?

Optimizing Western blot conditions for the At2g39530 Antibody requires careful consideration of multiple parameters. Sample preparation should begin with efficient protein extraction from Arabidopsis tissues using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitors . For gel electrophoresis, 10-12% SDS-PAGE gels are recommended to achieve optimal resolution of the At2g39530 protein.

After transfer to a PVDF or nitrocellulose membrane, blocking should be performed with 2.5-5% skimmed milk in TBST for two hours at room temperature . The primary antibody (At2g39530 Antibody) should be diluted in the range of 1:500 to 1:2000 in blocking solution, with incubation overnight at 4°C. Following thorough washing with TBST, an appropriate HRP-conjugated secondary anti-rabbit antibody (typically 1:5000-1:10000 dilution) should be applied for 1-2 hours at room temperature.

Signal detection sensitivity can be enhanced using ECL chemiluminescence reagents appropriate for the expected expression level of At2g39530. Exposure times should be optimized based on signal strength, typically starting with 30 seconds to 5 minutes. For quantitative analysis, multiple exposure times should be collected to ensure measurements are made within the linear range of detection.

How can researchers incorporate At2g39530 Antibody into studies investigating plant stress responses?

The At2g39530 Antibody can be a valuable tool for investigating plant stress responses by enabling researchers to monitor changes in At2g39530 protein levels under various stress conditions. To integrate this antibody effectively, researchers should first establish baseline expression levels in normal growth conditions using quantitative Western blot analysis. Then, systematic experiments can be designed to expose Arabidopsis plants to various stressors, including abiotic stresses (drought, salt, temperature extremes, light stress) and biotic stresses (pathogen infection).

Tissue-specific expression analysis can be particularly informative, as stress responses often vary between plant tissues. Researchers should collect samples from different plant tissues (roots, leaves, stems, flowers) at multiple time points post-stress induction to capture the dynamic nature of stress responses . Western blot analysis using the At2g39530 Antibody can then reveal tissue-specific and temporal changes in protein expression.

To gain deeper insights, At2g39530 protein levels should be correlated with physiological parameters (e.g., photosynthetic rate, reactive oxygen species accumulation) and molecular markers of stress response pathways. Complementary approaches such as RT-qPCR for At2g39530 transcript levels can help distinguish between transcriptional and post-translational regulation. This multi-parameter analysis provides a comprehensive understanding of At2g39530's role in plant stress response mechanisms.

What considerations are important when using At2g39530 Antibody in co-immunoprecipitation experiments?

When using the At2g39530 Antibody for co-immunoprecipitation (Co-IP) experiments to identify protein interaction partners, several critical factors must be considered. First, the antibody must be validated for immunoprecipitation efficiency using recombinant At2g39530 protein prior to experimental use. The selection of lysis buffer is crucial—a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, and protease inhibitors often preserves protein-protein interactions while effectively extracting membrane-associated proteins like At2g39530 .

The antibody-to-lysate ratio requires optimization, typically starting with 2-5 μg of antibody per 500 μg of total protein. Pre-clearing the lysate with Protein A/G beads can reduce non-specific binding. For the immunoprecipitation itself, incubating the antibody with the lysate overnight at 4°C provides sufficient time for antigen capture while minimizing degradation . Washing conditions must balance removing non-specific binders while preserving specific interactions—typically 3-5 washes with decreasing salt concentrations.

For elution, sample buffer with SDS and DTT (dithiothreitol) is effective for subsequent SDS-PAGE analysis. Mass spectrometry analysis of the co-immunoprecipitated proteins provides the most comprehensive identification of interaction partners. Throughout the experiment, appropriate controls must be included: a non-specific IgG control, an input sample, and ideally a negative control from At2g39530 knockout lines to distinguish specific from non-specific interactions.

What protocol is recommended for using At2g39530 Antibody in ELISA?

For ELISA applications using the At2g39530 Antibody, a detailed methodological approach is essential. Begin by coating high-binding 96-well plates with purified recombinant At2g39530 protein (for indirect ELISA) or capture antibody (for sandwich ELISA) at 1-10 μg/mL in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C. After washing with PBS-T (PBS with 0.05% Tween-20), block non-specific binding sites with 3% BSA in PBS-T for 2 hours at room temperature.

For indirect ELISA, add serially diluted test samples containing At2g39530 protein and incubate for 2 hours at room temperature. For sandwich ELISA, add samples after blocking, followed by detection using the At2g39530 Antibody diluted 1:500-1:2000 in blocking buffer . Incubate for 2 hours at room temperature or overnight at 4°C. After washing, add an appropriate HRP-conjugated secondary antibody (anti-rabbit IgG) at 1:5000-1:10000 dilution and incubate for 1 hour at room temperature.

Develop the assay using TMB substrate and stop the reaction with 2N H₂SO₄ after appropriate color development (typically 15-30 minutes). Read absorbance at 450 nm with 570 nm as reference wavelength. Always include a standard curve using purified recombinant At2g39530 protein (0.1-1000 ng/mL) and appropriate negative controls. This protocol enables sensitive and specific quantification of At2g39530 protein in experimental samples.

How can researchers optimize protein extraction for detecting At2g39530 in different plant tissues?

Optimizing protein extraction for detecting At2g39530 across different plant tissues requires consideration of tissue-specific characteristics and the protein's subcellular localization. For Arabidopsis leaves, a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail is effective . For root tissue, which contains more interfering compounds, consider adding 1% polyvinylpolypyrrolidone (PVPP) to absorb phenolic compounds and 5 mM DTT to prevent oxidation.

Extraction methods should be adapted based on At2g39530's putative membrane association as a Casp-like protein. For membrane-rich samples, inclusion of 0.5% SDS or stronger detergents may improve extraction efficiency, though care should be taken to ensure antibody compatibility with these detergents in downstream applications. The tissue-to-buffer ratio should be optimized (typically 1:3-1:5 w/v) to ensure efficient extraction without over-dilution.

Mechanical disruption methods also vary by tissue type: for leaves, mortar and pestle grinding in liquid nitrogen is effective; for seeds or siliques, bead-beating systems may be necessary. Following extraction, centrifugation at 20,000 × g for 20 minutes at 4°C helps remove debris. When comparing At2g39530 levels across different tissues, normalization to total protein concentration (determined by Bradford or BCA assay) is essential for accurate quantification. Additionally, western blot loading controls such as actin or GAPDH should be included, though the choice of control should consider tissue-specific expression variations.

What controls and standards are essential when working with At2g39530 Antibody?

When working with the At2g39530 Antibody, implementing rigorous controls and standards is essential for generating reliable and interpretable data. The primary positive control should be recombinant At2g39530 protein, which allows verification of antibody function and provides a reference for the expected molecular weight . Wild-type Arabidopsis thaliana tissue extracts serve as biological positive controls, while At2g39530 knockout/knockdown lines provide critical negative controls to confirm antibody specificity.

For immunoblotting applications, loading controls are essential - housekeeping proteins such as actin, tubulin, or GAPDH should be detected on the same membrane to normalize for protein loading variations. A technical negative control using non-specific rabbit IgG at the same concentration as the At2g39530 Antibody helps identify non-specific binding.

Quantitative standards should be included when measuring At2g39530 protein levels. For Western blots, a dilution series of recombinant At2g39530 protein (e.g., 1, 5, 10, 25, 50 ng) allows creation of a standard curve for protein quantification . For ELISA, a similar standard curve should be prepared using purified recombinant protein. When comparing samples from different experimental conditions, replicate analysis (minimum triplicate biological replicates) is necessary to account for biological variability and enable statistical analysis. These controls and standards ensure experimental rigor and facilitate meaningful interpretation of results across different experimental systems.

How can researchers integrate At2g39530 Antibody data with other molecular techniques for comprehensive protein characterization?

Integrating At2g39530 Antibody data with complementary molecular techniques provides a comprehensive understanding of this protein's function and regulation. Researchers should begin by correlating protein expression data from Western blots with transcript levels measured by RT-qPCR, which can reveal post-transcriptional regulation mechanisms. For spatial expression analysis, immunohistochemistry using the At2g39530 Antibody can be combined with in situ hybridization to compare protein localization with mRNA distribution patterns at the tissue and cellular levels .

For functional characterization, data from immunoprecipitation using the At2g39530 Antibody can be integrated with mass spectrometry to identify protein interaction partners . These protein-protein interactions can be further validated and characterized using techniques such as bimolecular fluorescence complementation (BiFC) or Förster resonance energy transfer (FRET). To understand the protein's role in specific pathways, researchers can correlate changes in At2g39530 protein levels with alterations in physiological parameters and other pathway components under various experimental conditions.

Integration with genetic approaches is also valuable - comparing antibody-detected protein levels in wild-type plants versus mutant lines can reveal regulatory mechanisms. This multi-disciplinary approach can be visualized and integrated using pathway mapping tools that incorporate protein expression data, interaction networks, and genetic dependencies, providing a systems-level understanding of At2g39530 function in plant biology.

What troubleshooting strategies should be employed when unexpected results occur with At2g39530 Antibody?

When encountering unexpected results with the At2g39530 Antibody, systematic troubleshooting approaches are necessary to identify and resolve issues. For weak or absent signals in Western blots, researchers should first verify antibody activity using a recombinant At2g39530 protein positive control . Optimization of primary antibody concentration (testing dilutions from 1:250 to 1:2000) and incubation conditions (4°C overnight versus room temperature for 2 hours) can significantly improve detection sensitivity. Ensure the extraction method effectively solubilizes At2g39530 protein by testing alternative extraction buffers with different detergents (RIPA versus NP-40 based buffers).

For high background or non-specific banding, increase blocking stringency (5% BSA instead of milk, or vice versa), optimize secondary antibody dilution (typically 1:5000 to 1:20000), and increase washing durations and frequencies (5-6 washes of 10 minutes each). Consider pre-adsorption of the antibody with Arabidopsis proteins from At2g39530 knockout lines to remove antibodies that bind non-specifically.

When results differ from published literature, verify experimental conditions match published protocols exactly, including plant growth conditions, developmental stage, and tissue selection, as At2g39530 expression may vary with these factors. Cross-validation with a second detection method (e.g., mass spectrometry) can confirm unexpected findings. Always document troubleshooting experiments comprehensively, including all parameters and controls, to identify patterns that may reveal the source of unexpected results.

How should researchers quantify and statistically analyze At2g39530 protein expression data?

Quantitative analysis of At2g39530 protein expression requires rigorous methodological approaches and appropriate statistical treatment. For Western blot quantification, researchers should use dedicated image analysis software (e.g., ImageJ, Image Lab) to measure band intensities, ensuring images are captured in the linear dynamic range of detection . Normalization to loading controls (actin, tubulin, or total protein via Ponceau S staining) is essential to account for loading variations. For ELISA data, standard curves should be fitted using appropriate regression models (typically four-parameter logistic for immunoassays) with R² values exceeding 0.98 for reliable quantification.

Statistical analysis should begin with testing for normality (Shapiro-Wilk test) and homogeneity of variance (Levene's test) to determine appropriate statistical tests. For comparing At2g39530 expression across multiple experimental conditions, ANOVA followed by appropriate post-hoc tests (Tukey's HSD for all pairwise comparisons or Dunnett's test when comparing to a control) should be applied with significance typically set at p<0.05. For time-course experiments, repeated measures ANOVA or mixed-effects models are more appropriate to account for temporal dependencies.

Power analysis should be conducted to determine adequate sample sizes, typically requiring a minimum of 3-5 biological replicates with 2-3 technical replicates each. Effect sizes should be reported (Cohen's d or similar metrics) alongside p-values to indicate biological significance. When analyzing correlations between At2g39530 levels and other parameters, Pearson's or Spearman's correlation coefficients (depending on data normality) should be calculated and tested for significance.

What approaches can be used to analyze At2g39530 protein modifications using the antibody?

Analyzing post-translational modifications (PTMs) of At2g39530 protein requires specialized approaches that extend beyond basic antibody applications. Researchers should first employ 2D gel electrophoresis followed by Western blotting with the At2g39530 Antibody to detect potential charge variants indicating phosphorylation, acetylation, or other modifications . Comparing migration patterns under different treatment conditions can reveal condition-specific modifications.

For phosphorylation analysis, samples can be treated with lambda phosphatase prior to Western blotting; a mobility shift after phosphatase treatment suggests phosphorylation of At2g39530. Alternatively, Phos-tag™ acrylamide gels specifically retard the migration of phosphorylated proteins, allowing separation of phosphorylated from non-phosphorylated forms when followed by Western blotting with the At2g39530 Antibody.

Immunoprecipitation with the At2g39530 Antibody followed by mass spectrometry provides the most comprehensive analysis of PTMs . This approach can identify specific modified residues and quantify modification stoichiometry. For targeted analysis of suspected modifications, immunoprecipitated At2g39530 can be probed with modification-specific antibodies (anti-phospho, anti-ubiquitin, etc.) on Western blots.

When analyzing glycosylation, treatment with PNGase F or other glycosidases before Western blotting can reveal glycosylated forms through mobility shifts . For all modification analyses, appropriate controls must be included: positive controls using proteins known to carry the modification of interest, and negative controls where the modification is enzymatically removed or prevented through mutation of target residues.

How might the At2g39530 Antibody contribute to understanding plant-environment interactions?

The At2g39530 Antibody offers significant potential for advancing our understanding of plant-environment interactions, particularly in the context of stress responses and adaptation mechanisms. As a Casp-like protein, At2g39530 may play important roles in cell wall organization or membrane function, which are critical components of plant responses to environmental challenges . Researchers can design comprehensive studies examining At2g39530 protein expression across diverse environmental conditions, including drought, temperature extremes, pathogen exposure, and nutrient limitations.

Particularly valuable would be time-course experiments that track At2g39530 protein levels at multiple intervals after stress onset, potentially revealing whether this protein is involved in early signaling events or in longer-term adaptive responses. The antibody could be used in comparative studies across Arabidopsis ecotypes with differing stress tolerances to determine if variation in At2g39530 regulation correlates with adaptive traits.

Integration with emerging plant phenotyping technologies would allow correlation of At2g39530 expression patterns with physiological parameters measured in real-time, such as photosynthetic efficiency, stomatal conductance, or growth rates under stress conditions. Additionally, using the antibody to examine At2g39530 in transgenic plants with modified expression of known stress-response regulators could position this protein within established signaling networks. Through these approaches, the At2g39530 Antibody could contribute to developing more stress-tolerant crops by identifying novel components of plant adaptation mechanisms.

What emerging technologies might enhance the utility of At2g39530 Antibody in plant research?

Emerging technologies are poised to dramatically expand the utility of the At2g39530 Antibody in plant research. Single-cell proteomics techniques, when coupled with the At2g39530 Antibody, could reveal cell-type specific expression patterns that are masked in whole-tissue analyses . This approach would be particularly valuable for understanding developmental regulation and tissue-specific functions of the At2g39530 protein.

Proximity labeling methods such as BioID or TurboID, combined with immunoprecipitation using the At2g39530 Antibody, would enable identification of the protein's proximal interactome, providing insights into its functional role within specific subcellular compartments. For spatial analysis, clearing techniques like CLARITY or iDISCO adapted for plant tissues, followed by whole-mount immunostaining with the At2g39530 Antibody, could generate comprehensive 3D maps of protein distribution across intact plant organs.

Advanced microscopy approaches, including super-resolution techniques like STORM or PALM, would allow visualization of At2g39530 at nanometer resolution when using fluorescently-labeled antibodies. Integration with emerging genetic tools is also promising—combining the antibody with CRISPR-edited plant lines containing tagged or modified versions of At2g39530 could elucidate structure-function relationships.

Finally, computational approaches including machine learning algorithms could integrate antibody-generated protein expression data with transcriptomics and metabolomics datasets to predict functional networks and identify new research directions. These technological advances will maximize the research impact of the At2g39530 Antibody beyond its current applications.