At5g25050 Antibody

What is the At5g25050 antibody and what protein does it target?

The At5g25050 antibody is a polyclonal antibody raised in rabbits that specifically targets the At5g25050 protein from Arabidopsis thaliana. The antibody recognizes recombinant Arabidopsis thaliana At5g25050 protein as its immunogen. The target protein is associated with UniProt accession number Q5FV41. This antibody is designed specifically for research applications in understanding protein expression and function in Arabidopsis thaliana and is not intended for diagnostic or therapeutic procedures .

What are the validated applications for At5g25050 antibody?

The At5g25050 antibody has been validated for specific research applications including Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB). These applications allow researchers to detect and quantify the presence of At5g25050 protein in various experimental samples. The antibody undergoes rigorous validation to ensure identification of the target antigen in these applications . When designing experiments, researchers should consider the specific buffer conditions and dilution ratios optimized for each application type to maximize detection sensitivity and specificity.

How should the At5g25050 antibody be stored and handled for optimal performance?

For optimal performance and stability, the At5g25050 antibody should be stored at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be avoided as they can compromise antibody activity and specificity. The antibody is supplied in liquid form with a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . When handling the antibody, researchers should use sterile technique, aliquot the antibody to minimize freeze-thaw cycles, and maintain cold chain protocols during experimental procedures to preserve antibody function and reactivity.

What controls should be included when using At5g25050 antibody in experimental procedures?

When designing experiments with the At5g25050 antibody, several controls should be included to ensure valid and interpretable results:

• Positive control: Sample known to express At5g25050 protein (Arabidopsis thaliana wild-type tissue)

• Negative control: Sample known not to express At5g25050 (non-plant tissue or At5g25050 knockout line)

• Secondary antibody-only control: To assess non-specific binding of the secondary detection antibody

• Blocking peptide control: Pre-incubation of antibody with immunizing peptide to confirm specificity

• Loading control: Detection of a housekeeping protein to normalize expression levels

These controls help distinguish true signal from background noise and validate the specificity of antibody binding, particularly important when interpreting results from complex plant tissue samples with potential cross-reactivity.

How does the polyclonal nature of At5g25050 antibody affect epitope recognition and experimental design?

The At5g25050 antibody is a polyclonal antibody purified through antigen affinity methods . Unlike monoclonal antibodies that recognize a single epitope, polyclonal antibodies contain a heterogeneous mixture of immunoglobulins that recognize multiple epitopes on the target antigen. This characteristic has several important implications for research:

• Enhanced sensitivity: Recognition of multiple epitopes often increases detection sensitivity

• Robust to epitope denaturation: Some antibodies in the polyclonal mixture may retain binding ability even if certain epitopes are modified

• Batch variability: Different production lots may have slightly different epitope preferences

• Cross-reactivity considerations: Higher potential for cross-reactivity with structurally similar proteins

When designing critical experiments, researchers should validate new lots of the antibody against previous standards and consider epitope availability in different experimental conditions (native vs. denatured proteins). For quantitative comparisons across multiple experiments, using antibody from the same lot is recommended to minimize variability in epitope recognition patterns.

What approaches can resolve potential cross-reactivity issues with At5g25050 antibody in complex plant samples?

When working with complex plant samples, potential cross-reactivity of the At5g25050 antibody with other proteins is an important consideration. Several methodological approaches can help address this challenge:

• Pre-absorption: Incubate antibody with tissues lacking the target protein to remove antibodies binding to non-specific epitopes

• Knockout validation: Compare antibody reactivity between wild-type and At5g25050 knockout plants

• Peptide competition assays: Compete antibody binding with excess immunizing peptide

• Orthogonal detection methods: Validate findings using alternative detection methods (e.g., mass spectrometry)

• Sequential immunoprecipitation: Perform multiple rounds of immunoprecipitation to increase specificity

The purification method used for the At5g25050 antibody (antigen affinity purification) helps minimize cross-reactivity, but careful experimental design remains essential. When analyzing tissues with low target expression, additional validation steps may be necessary to distinguish specific signal from background.

How can the At5g25050 antibody be adapted for specialized research applications beyond standard ELISA and Western blotting?

While the At5g25050 antibody is validated for ELISA and Western blotting , researchers may adapt it for specialized applications with appropriate optimization and validation:

• Immunohistochemistry (IHC): Optimize fixation protocols to preserve epitope accessibility in plant tissues

• Immunoprecipitation (IP): Adjust buffer conditions to maintain antibody-antigen interaction in solution

• Chromatin immunoprecipitation (ChIP): If At5g25050 has DNA-binding properties, validate antibody for chromatin-bound protein detection

• Flow cytometry: For protoplast analysis, validate antibody performance with appropriate permeabilization

• Super-resolution microscopy: Test antibody performance with various fluorophore conjugations

Each adaptation requires systematic optimization of parameters including antibody concentration, incubation conditions, blocking agents, and detection systems. Preliminary experiments should establish specificity in the modified application before proceeding to full-scale studies. Researchers should document and report all optimization steps to facilitate reproducibility.

What are the critical experimental variables affecting At5g25050 antibody performance in immunoassays?

Several experimental variables significantly impact the performance of At5g25050 antibody in immunoassays:

For optimal results, these variables should be systematically tested and optimized for each specific experimental setup. The antibody's storage buffer (containing 50% glycerol, 0.01M PBS, pH 7.4) may need to be considered when calculating final buffer compositions for immunoassays.

How should epitope masking and retrieval be addressed when working with At5g25050 antibody in fixed plant tissues?

Epitope masking can significantly impact At5g25050 antibody binding in fixed plant tissues. Plant tissues present unique challenges due to cell wall components and abundant secondary metabolites. Consider these methodological approaches:

• Fixation optimization: Test different fixatives (paraformaldehyde, glutaraldehyde, ethanol) and durations

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER): Test different buffer systems (citrate, Tris-EDTA) and pH values

  • Enzymatic digestion: Use proteases (proteinase K) or cell wall-degrading enzymes (cellulase, pectinase)

• Permeabilization: Optimize detergent concentration (Triton X-100, Tween-20) for membrane permeabilization

• Clearing techniques: Consider tissue clearing methods for deep tissue imaging

• Section thickness: Adjust microtome settings for optimal antibody penetration

Since the At5g25050 antibody was generated against a recombinant protein , it may recognize both native and denatured epitopes, but this requires validation. Document successful protocols thoroughly to ensure reproducibility across experiments and research groups.

What strategies can enhance detection sensitivity when the At5g25050 protein is expressed at low levels?

Detecting low-abundance At5g25050 protein presents methodological challenges. Several approaches can enhance detection sensitivity:

• Signal amplification systems:

  • Tyramide signal amplification (TSA)

  • Polymer-based detection systems

  • Avidin-biotin amplification

• Sample preparation techniques:

  • Subcellular fractionation to concentrate target protein

  • Immunoprecipitation before Western blotting

  • Protein concentration methods (TCA precipitation, ultrafiltration)

• Detection optimization:

  • Extended substrate exposure times for chemiluminescence

  • High-sensitivity digital imaging systems

  • Fluorescent secondary antibodies with low detection thresholds

• Protocol refinements:

  • Reduced washing stringency (while maintaining specificity)

  • Extended primary antibody incubation (overnight at 4°C)

  • Optimized blocking to reduce background while preserving signal

The antibody's antigen affinity purification provides a good foundation for specific detection, but these additional methodological approaches may be necessary when working with samples containing low levels of At5g25050 protein.

How can the At5g25050 antibody be integrated into multiparameter analysis of Arabidopsis protein networks?

The At5g25050 antibody can be effectively integrated into multiparameter analyses to study protein interaction networks in Arabidopsis through several methodological approaches:

• Co-immunoprecipitation studies:

  • Use At5g25050 antibody to pull down protein complexes

  • Identify interaction partners through mass spectrometry

  • Validate interactions with reciprocal co-IP experiments

• Multiplexed immunodetection:

  • Combine with antibodies against other proteins of interest

  • Use spectrally distinct fluorophores for simultaneous detection

  • Establish staining protocols that preserve epitopes for all targets

• Sequential probing strategies:

  • Strip and reprobe membranes with multiple antibodies

  • Document complete stripping through negative controls

  • Account for potential signal carryover in analysis

• Correlation analyses:

  • Compare At5g25050 localization with other proteins across conditions

  • Quantify co-localization using appropriate statistical methods

  • Control for random co-localization through randomization controls

When designing multiplexed experiments, consider potential interference between detection systems and validate the specificity of each antibody individually before combining them in complex experimental setups.

What are the considerations for using At5g25050 antibody in comparative studies across different Arabidopsis ecotypes or mutant lines?

When using the At5g25050 antibody for comparative studies across different Arabidopsis backgrounds, several methodological considerations are critical:

• Epitope conservation analysis:

  • Verify sequence conservation of the immunogen region across ecotypes

  • Consider potential post-translational modifications that might differ

  • Validate antibody reactivity in each genetic background

• Expression normalization strategies:

  • Use multiple housekeeping proteins as loading controls

  • Consider that common housekeeping genes may vary between ecotypes

  • Implement absolute quantification methods when appropriate

• Experimental design considerations:

  • Process all samples in parallel to minimize technical variability

  • Include biological replicates from independent plant populations

  • Randomize sample processing order to avoid systematic bias

• Validation approaches:

  • Confirm findings with orthogonal methods (qPCR, mass spectrometry)

  • Use genetic complementation to verify phenotype-protein correlations

  • Consider creating epitope-tagged versions in different backgrounds

The species reactivity of the At5g25050 antibody is specifically for Arabidopsis thaliana , but variation within this species requires careful validation when extending to diverse ecotypes or heavily mutagenized lines.

How should researchers design time-course experiments to study dynamic changes in At5g25050 protein levels during plant development or stress responses?

Designing robust time-course experiments to study At5g25050 protein dynamics requires careful methodological planning:

• Sampling strategy optimization:

  • Determine appropriate time intervals based on expected dynamics

  • Consider circadian fluctuations in protein expression

  • Plan for both early (minutes/hours) and late (days/weeks) timepoints

• Sample preservation methods:

  • Flash-freeze tissues immediately to capture exact timepoints

  • Use consistent harvesting protocols to minimize variability

  • Consider preserving samples with protease and phosphatase inhibitors

• Quantification approaches:

  • Implement replicate sampling at each timepoint

  • Use quantitative Western blotting with standard curves

  • Consider multiplex detection to normalize against loading controls

• Data analysis considerations:

  • Apply appropriate statistical tests for time-series data

  • Consider time-dependent normalization if housekeeping proteins change

  • Use visualization methods that clearly communicate temporal dynamics

• Validation experiments:

  • Correlate protein levels with transcriptional changes

  • Consider pulse-chase experiments to determine protein turnover rates

  • Validate findings under various environmental conditions

Given the 14-16 week lead time for At5g25050 antibody production , researchers should plan experiments well in advance and consider ordering sufficient antibody to complete entire time-course studies with the same lot.

What quality control metrics should researchers apply when validating a new lot of At5g25050 antibody?

When receiving a new lot of At5g25050 antibody, implementing thorough quality control is essential:

• Sensitivity assessment:

  • Perform dilution series with known positive samples

  • Determine detection limit compared to previous lots

  • Document minimal detectable protein concentration

• Specificity validation:

  • Test against positive and negative control samples

  • Perform peptide competition assays

  • Consider testing against knockout/knockdown lines

• Signal-to-noise evaluation:

  • Quantify background under standardized conditions

  • Compare specific signal intensity to background

  • Document optimal dilution for maximal signal-to-noise ratio

• Cross-reactivity testing:

  • Test against related plant species if relevant

  • Evaluate potential cross-reactivity with similar proteins

  • Document any non-specific bands or signals

• Application-specific performance:

  • Validate in all intended applications (ELISA, WB)

  • Document optimal conditions for each application

  • Compare results to historical data with previous lots

The antigen affinity purification method used for At5g25050 antibody helps maintain consistent quality, but lot-to-lot variation remains an important consideration for critical experiments.

How can researchers effectively troubleshoot weak or absent signal when using At5g25050 antibody?

When encountering weak or absent signal with At5g25050 antibody, a systematic troubleshooting approach should be implemented:

For each troubleshooting step, change only one variable at a time and include appropriate controls to interpret results accurately. Document successful protocol modifications for future reference.

What emerging technologies might enhance the utility of At5g25050 antibody in future plant biology research?

Several emerging technologies show promise for expanding the research applications of At5g25050 antibody:

• Proximity labeling techniques:

  • Combine antibody with biotin ligase for identifying proximal proteins

  • Apply APEX2 or BioID systems for in vivo interaction studies

  • Develop protocols for plant-specific proximity labeling

• Single-cell proteomics integration:

  • Adapt antibody for use in single-cell Western blotting

  • Optimize for mass cytometry (CyTOF) applications in plant cells

  • Develop protocols for spatial proteomics in plant tissues

• Multiplexed detection systems:

  • Implement cyclic immunofluorescence with antibody elution/reapplication

  • Adapt for hyperplexed imaging technologies

  • Develop barcoded antibody systems for simultaneous detection

• Structural studies applications:

  • Optimize for proximity ligation assays to study protein conformation

  • Adapt for super-resolution microscopy techniques

  • Develop protocols for in situ protein structure analysis

The polyclonal nature of the At5g25050 antibody provides flexibility for adaptation to these emerging technologies, though each application will require careful validation and optimization.

The ongoing development of antibody engineering techniques, as seen in therapeutic bispecific antibodies and other advanced antibody formats , suggests future opportunities for enhancing the specificity and utility of research antibodies like At5g25050 antibody.