At5g44550 Antibody

What is the At5g44550 antibody and what gene product does it target?

At5g44550 antibody (product code CSB-PA875455XA01DOA) is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana At5g44550 protein . The antibody specifically recognizes the protein encoded by the At5g44550 gene in Arabidopsis thaliana. This gene corresponds to the UniProt accession number Q9FI10, which is essential for establishing the protein's identity and functional characteristics in scientific research . Unlike related proteins such as the Late embryogenesis abundant protein family (encoded by At5g44310), the At5g44550 antibody offers specificity for targeted experimental analysis of its corresponding protein's expression and localization patterns.

What are the optimal storage conditions for maintaining At5g44550 antibody activity?

For optimal preservation of antibody activity, the At5g44550 antibody should be stored at -20°C or -80°C immediately upon receipt . Repeated freeze-thaw cycles should be strictly avoided as they can significantly compromise antibody functionality and binding capacity. The antibody is supplied in liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . This formulation helps maintain stability during storage periods. For routine laboratory use, researchers should consider preparing small working aliquots to minimize freeze-thaw cycles. Documentation of storage conditions, including temperature logs and freeze-thaw events, is essential for troubleshooting unexpected experimental results and ensuring research reproducibility.

What applications has the At5g44550 antibody been validated for?

The At5g44550 antibody has been specifically validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications . These techniques allow researchers to detect and quantify the At5g44550 protein in various experimental contexts. When designing experiments, it's important to note that the antibody's application validation ensures reliable antigen identification in these specific methodologies. For Western blotting, researchers should optimize protein extraction protocols specifically for plant tissues, as Arabidopsis samples often contain compounds that can interfere with protein separation and detection. Similarly, ELISA protocols may require optimization of blocking agents and antibody dilutions to minimize background and maximize specific signal detection when working with plant samples.

How should researchers design proper controls when using At5g44550 antibody in Western blot experiments?

A robust experimental design for Western blot applications with At5g44550 antibody requires multiple controls. Researchers should include:

• Positive control: Recombinant At5g44550 protein or known At5g44550-expressing tissues

• Negative control: Samples from At5g44550 knockout mutant plants if available

• Loading control: Detection of a housekeeping protein (e.g., actin or tubulin)

• Secondary antibody-only control: To assess non-specific binding

• Pre-absorption control: Antibody pre-incubated with the immunizing peptide

Drawing from methodologies used with similar plant antibodies, researchers should consider membrane blocking with 5% non-fat milk or BSA in TBST buffer . Signal development should be carefully optimized, as plant samples often require longer exposure times than animal samples. When analyzing results, the specificity of the antibody should be confirmed by the presence of a single band at the expected molecular weight, with minimal background staining. Quantification should be normalized to loading controls and statistically validated across biological replicates.

What protein extraction protocols are optimal for detecting At5g44550 in Arabidopsis tissues?

For optimal detection of At5g44550 protein in Arabidopsis tissues, researchers should employ extraction protocols that preserve protein integrity while minimizing interference from plant-specific compounds. The following protocol is recommended based on methodologies used for similar plant antibody applications:

It's crucial to consider that Arabidopsis tissues contain phenolic compounds and secondary metabolites that can interfere with protein extraction and subsequent immunodetection. Adding polyvinylpolypyrrolidone (PVPP) at 2% (w/v) to the extraction buffer can help adsorb these interfering compounds. For difficult tissues, a TCA/acetone precipitation step may improve protein purity. Sample preparation should be tailored to the specific tissue being analyzed, as protein expression and extraction efficiency can vary significantly between roots, leaves, flowers, and seeds.

How can researchers optimize ELISA protocols for At5g44550 antibody to improve sensitivity and specificity?

To optimize ELISA protocols using At5g44550 antibody, researchers should systematically adjust several parameters to enhance both sensitivity and specificity:

• Antibody titration: Perform a checkerboard titration of primary antibody (At5g44550) concentrations ranging from 1:500 to 1:10,000 to determine optimal dilution that maximizes specific signal while minimizing background .

• Blocking optimization: Test multiple blocking agents including 3-5% BSA, 5% non-fat dry milk, or commercial blocking buffers designed for plant samples. Blocking time should be optimized between 1-3 hours at room temperature.

• Sample preparation: For plant tissues, consider using specialized extraction buffers containing detergents like 0.1% Tween-20 or 0.5% Triton X-100 to improve protein solubilization while maintaining antibody-epitope recognition.

• Incubation conditions: Compare antibody binding efficiency at different temperatures (4°C overnight vs. room temperature for 1-2 hours) and determine if gentle agitation improves results.

• Detection system: For enhanced sensitivity, consider using streptavidin-biotin amplification systems or high-sensitivity substrates such as chemiluminescent reagents rather than colorimetric detection.

A systematic validation approach should include analysis of linearity, detection limits, and reproducibility across multiple biological replicates. Researchers should document all optimization steps in their methods section when publishing results, as these details are crucial for reproducibility in the field.

How can At5g44550 antibody be used in immunoprecipitation experiments to study protein-protein interactions?

Immunoprecipitation (IP) using At5g44550 antibody can reveal novel protein-protein interactions and protein complexes. Drawing from methodologies used with similar plant proteins like AtSerpin1, researchers should:

• Cross-link proteins in vivo: Use formaldehyde (1% for 10 minutes) or DSP (dithiobis-succinimidyl propionate) to stabilize transient protein interactions before cell lysis.

• Optimize lysis conditions: Use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM EDTA with protease inhibitors, adjusting detergent concentration to balance solubilization with preservation of protein interactions.

• Pre-clear lysates: Incubate with protein A/G beads to reduce non-specific binding.

• Antibody immobilization: Covalently couple At5g44550 antibody to beads using crosslinking reagents to prevent antibody co-elution, similar to approaches used with AtSerpin1 antibodies .

• Sequential elution strategy: Use a pH gradient or increasing salt concentration to differentially elute proteins based on binding strength.

For identifying interaction partners, samples should be analyzed by mass spectrometry following tryptic digestion, comparable to the liquid chromatography-nanospray tandem mass spectrometry approach used in AtSerpin1 studies . Validation of potential interactors should include reciprocal IP experiments and functional assays. The significance of these interactions can be assessed through physiological assays in Arabidopsis plants with altered expression of At5g44550 or its interacting partners.

What approaches can researchers use to study At5g44550 localization in plant cells using this antibody?

For subcellular localization studies of At5g44550, researchers can employ multiple complementary approaches:

• Immunofluorescence microscopy:

  • Fix Arabidopsis tissues with 4% paraformaldehyde

  • Permeabilize cell walls with enzymes (cellulase/macerozyme) or detergents

  • Block with 3% BSA in PBS containing 0.1% Triton X-100

  • Incubate with At5g44550 antibody (1:100 to 1:500 dilution)

  • Detect with fluorophore-conjugated secondary antibodies

  • Co-stain with organelle markers for colocalization analysis

• Subcellular fractionation combined with Western blotting:

  • Isolate different cellular compartments (nucleus, chloroplast, mitochondria, etc.)

  • Verify fraction purity using compartment-specific marker proteins

  • Perform Western blot analysis with At5g44550 antibody on each fraction

  • Quantify relative distribution across compartments

• Electron microscopy immunogold labeling:

  • Embed tissues in appropriate resins

  • Prepare ultrathin sections

  • Incubate with At5g44550 antibody

  • Detect with gold-conjugated secondary antibodies

  • Quantify gold particle distribution across cellular compartments

To validate localization findings, researchers should complement antibody-based approaches with fluorescent protein fusion studies. Constructing transgenic Arabidopsis lines expressing At5g44550-GFP/RFP fusions under native promoters provides an independent method to confirm subcellular localization. Comparison between fixed and live imaging approaches can reveal potential fixation artifacts or dynamic localization patterns in response to environmental stimuli or developmental stages.

How can researchers use At5g44550 antibody to study protein expression changes during plant development or stress responses?

To investigate dynamic changes in At5g44550 protein expression during development or stress responses, researchers should implement a systematic approach combining multiple techniques:

• Developmental time course analysis:

  • Collect tissues at defined developmental stages (embryo, seedling, vegetative, reproductive)

  • Perform Western blot analysis with standardized loading (based on fresh weight, total protein, or reference protein)

  • Quantify relative expression levels normalized to stable reference proteins

  • Correlate protein expression with developmental markers or morphological changes

• Stress response profiling:

  • Expose plants to controlled stress conditions (drought, salt, heat, cold, pathogens)

  • Sample tissues at multiple time points (0, 1, 3, 6, 12, 24, 48 hours)

  • Perform Western blots with At5g44550 antibody

  • Compare protein levels to transcriptome changes (RT-qPCR or RNA-seq)

• Tissue-specific expression mapping:

  • Dissect different plant organs and tissues

  • Perform immunohistochemistry with At5g44550 antibody

  • Compare with in situ hybridization for mRNA localization

  • Document cell type-specific expression patterns

For quantitative analysis, researchers should employ digital image analysis of Western blots using appropriate software (ImageJ, etc.) with statistical validation across biological replicates (minimum n=3). The correlation between protein abundance and transcript levels should be assessed to identify potential post-transcriptional regulation. Additionally, researchers should consider post-translational modifications by analyzing band pattern changes or using modification-specific detection methods such as Phos-tag gels for phosphorylation analysis.

What are common issues when using At5g44550 antibody in Western blots and how can they be resolved?

Researchers may encounter several challenges when using At5g44550 antibody in Western blots. This table outlines common problems, potential causes, and recommended solutions:

For persistent issues, researchers should consider comparing different blocking agents (milk vs. BSA), adjusting membrane pore size (PVDF vs. nitrocellulose), and optimizing incubation temperatures. Documentation of all optimization steps is essential for method reproducibility and troubleshooting future experiments.

How can researchers validate the specificity of At5g44550 antibody in their experimental system?

Validating antibody specificity is crucial for ensuring reliable experimental results. For At5g44550 antibody, researchers should implement multiple complementary validation approaches:

• Genetic validation:

  • Test antibody reactivity in At5g44550 knockout or knockdown lines

  • Analyze overexpression lines for increased signal intensity

  • Use CRISPR/Cas9-edited plants with epitope alterations

• Biochemical validation:

  • Perform peptide competition assays by pre-incubating antibody with immunizing antigen

  • Conduct immunodepletion experiments to verify signal reduction

  • Confirm molecular weight of detected proteins matches theoretical predictions

• Cross-reactivity assessment:

  • Test antibody against recombinant closely-related proteins

  • Analyze tissues from different plant species with varying sequence homology

  • Examine multiple tissues with different protein expression profiles

• Orthogonal techniques:

  • Compare antibody detection with mass spectrometry protein identification

  • Correlate protein detection with mRNA expression (RT-qPCR)

  • Validate localization using fluorescent protein fusions

Researchers should document all validation results in publications, including images of full Western blots with molecular weight markers, positive and negative controls, and quantitative analyses where appropriate. This comprehensive validation approach ensures that experimental findings accurately reflect the biological behavior of At5g44550 rather than technical artifacts.

How should researchers interpret conflicting results between transcript levels and protein expression detected with At5g44550 antibody?

Discrepancies between mRNA and protein levels of At5g44550 are common biological phenomena rather than technical artifacts. When investigating such inconsistencies, researchers should consider multiple biological mechanisms:

• Post-transcriptional regulation:

  • Analyze mRNA stability using actinomycin D treatment to block transcription

  • Examine alternative splicing patterns through RT-PCR with isoform-specific primers

  • Investigate miRNA-mediated regulation by identifying potential miRNA binding sites

• Translational control:

  • Perform polysome profiling to assess translation efficiency

  • Analyze 5' and 3' UTR sequences for regulatory elements affecting translation

  • Consider codon optimization and its impact on translation rates

• Protein stability regulation:

  • Conduct cycloheximide chase experiments to measure protein half-life

  • Investigate ubiquitination status using immunoprecipitation followed by ubiquitin blotting

  • Examine potential proteolytic processing using inhibitors against different protease classes

• Technical considerations:

  • Verify antibody specificity for all potential protein isoforms

  • Consider developmental timing and tissue-specific regulation

  • Assess subcellular localization and potential compartment-specific degradation

When interpreting these complex relationships, researchers should avoid simplistic correlations between transcript and protein levels. Instead, consider the integration of multiple regulatory layers that affect the final protein abundance. Comprehensive analysis may reveal important insights into the regulatory mechanisms governing At5g44550 expression, potentially identifying novel research directions in plant molecular biology.

How does the application of At5g44550 antibody compare with other approaches for studying this protein in Arabidopsis?

Researchers studying At5g44550 protein have multiple methodological options, each with distinct advantages and limitations:

A comprehensive research strategy would integrate multiple approaches. For example, initial characterization with At5g44550 antibody can be followed by fluorescent protein fusion studies for dynamic analyses, and functional assessments using CRISPR-edited variants. This multi-faceted approach provides robust validation and deeper mechanistic insights than any single method alone.

What can researchers learn from comparing detection of At5g44550 protein with antibodies versus transcript analysis methods?

Comparative analysis between protein detection using At5g44550 antibody and transcript analysis provides insights into multiple layers of gene regulation:

• Temporal dynamics:

  • Protein accumulation often lags behind transcript induction

  • Transcripts may show rapid, transient responses while protein levels change more gradually

  • Antibody detection can reveal protein persistence after transcript degradation

• Spatial patterns:

  • Transcripts may localize to specific cellular domains before translation

  • Proteins can be trafficked to different compartments post-translation

  • Combining RNA in situ hybridization with immunolocalization reveals trafficking mechanisms

• Regulatory insights:

  • Discordant transcript-protein ratios suggest post-transcriptional regulation

  • Similar patterns indicate transcriptional control dominance

  • Stress-dependent changes in correlation may reveal condition-specific regulatory mechanisms

• Functional implications:

  • Active protein forms may require post-translational modifications detectable only by antibodies

  • Protein-protein interactions may stabilize or destabilize At5g44550 independently of transcript levels

  • Subcellular compartmentalization may affect protein function regardless of expression level

Researchers should design experiments that capture both transcript and protein data from the same samples when possible. Time-course experiments with parallel RT-qPCR and Western blot analysis can reveal the relationship between transcription, translation, and protein turnover rates. Statistical models correlating transcript and protein data across multiple conditions can identify factors affecting this relationship, potentially revealing novel regulatory mechanisms specific to At5g44550 or broadly applicable to plant biology.

How can researchers integrate At5g44550 antibody studies with functional genomics approaches to understand protein function?

Integrating At5g44550 antibody-based studies with functional genomics creates a comprehensive research framework:

• Genetic perturbation analysis:

  • Use At5g44550 antibody to confirm protein depletion in T-DNA insertion or CRISPR knockout lines

  • Assess protein level changes in RNAi or artificial microRNA lines

  • Quantify expression in overexpression or complementation lines

  • Correlate protein levels with phenotypic severity across allelic series

• Interactome mapping:

  • Perform immunoprecipitation with At5g44550 antibody followed by mass spectrometry

  • Validate interactions using reciprocal co-immunoprecipitation experiments

  • Map interaction domains through truncation constructs and domain-specific antibodies

  • Assess interaction dynamics under different environmental conditions

• Physiological and developmental phenotyping:

  • Correlate At5g44550 protein levels with specific phenotypes across tissues and developmental stages

  • Compare wild-type and mutant responses to biotic/abiotic stresses at both protein and phenotypic levels

  • Develop quantitative assays linking protein abundance to phenotypic outcomes

• Multi-omics integration:

  • Combine protein expression data with transcriptomics, metabolomics, and phenomics

  • Conduct network analysis to position At5g44550 within regulatory and metabolic pathways

  • Identify potential feedback mechanisms regulating At5g44550 expression

  • Develop predictive models of At5g44550 function in plant development or stress response

This integrated approach enables researchers to move beyond correlative observations toward mechanistic understanding of At5g44550 function. By systematically combining antibody-based detection with diverse functional genomics tools, researchers can establish causal relationships between protein levels, interactions, and biological outcomes, potentially revealing novel roles of At5g44550 in plant biology that could inform both basic science and agricultural applications.