At2g05970 Antibody

What is the At2g05970 antibody and what protein does it target?

The At2g05970 antibody is a polyclonal antibody developed against the Arabidopsis thaliana At2g05970 protein. It is generated in rabbits using a recombinant Arabidopsis thaliana At2g05970 protein as the immunogen. The antibody targets the protein encoded by the At2g05970 gene in Arabidopsis thaliana (Mouse-ear cress), with the UniProt accession number Q9ZUF1 . As with all research antibodies, it is intended for research use only and not for diagnostic or therapeutic applications. For optimal experimental design, researchers should consider that this antibody has been tested specifically for ELISA and Western blot applications to ensure proper identification of the target antigen .

How should the At2g05970 antibody be stored to maintain its functionality?

The At2g05970 antibody should be stored at -20°C or -80°C upon receipt to maintain its functionality and prevent degradation. Researchers should avoid repeated freeze-thaw cycles, as this can significantly impact antibody performance and lead to false negative results in experiments . The antibody is typically supplied in a liquid form containing preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) . When handling the antibody, it's recommended to aliquot it into smaller volumes before freezing to minimize freeze-thaw cycles. For short-term use (within one week), the antibody can be stored at 2-8°C. This storage protocol aligns with standard practices for preserving antibody reactivity, a critical consideration when designing long-term research projects requiring consistent antibody performance.

What are the recommended applications for the At2g05970 antibody?

The At2g05970 antibody has been tested and validated for ELISA and Western blot (WB) applications specifically for the identification of the target antigen . When designing experiments using this antibody, researchers should consider:

• For Western blot: The antibody can be used to detect the target protein from Arabidopsis thaliana samples, though specific working dilutions need to be optimized based on sample type and protein concentration.

• For ELISA: The antibody can serve as either a primary detection antibody or a capture antibody in sandwich ELISA formats, depending on the experimental design requirements .

To establish optimal working concentrations, researchers should perform titration experiments starting with the manufacturer's recommended dilution range. For novel applications beyond the tested methods, validation steps should include appropriate positive and negative controls to confirm antibody specificity before proceeding with full experimental series.

How can I validate the specificity of the At2g05970 antibody in my experiments?

Validating antibody specificity is crucial for ensuring reliable research results. For the At2g05970 antibody, researchers should implement the following comprehensive validation approach:

• Genetic controls: If available, use samples from At2g05970 knockout/knockdown plants alongside wild-type controls. A specific antibody should show signal in wild-type samples but not in knockout samples, similar to validation protocols used for other plant antibodies .

• Western blot analysis: Perform western blot on plant tissue samples to confirm the expected molecular weight of the target protein. The antibody should detect bands at sizes consistent with the predicted molecular weight of the At2g05970 protein .

• Blocking peptide competition: Conduct a competition assay where the antibody is pre-incubated with the immunizing peptide before application to samples. Specific binding should be reduced or eliminated in these conditions .

• Cross-reactivity assessment: Test the antibody against samples from related plant species to assess potential cross-reactivity, especially important for evolutionary studies .

• Multiple antibody comparison: If available, compare results using independent antibodies targeting different epitopes of the same protein, as consistent patterns across antibodies increase confidence in specificity .

Remember that commercial antibodies may sometimes show lack of specificity as documented in multiple studies with other antibodies , making rigorous validation essential for each experimental system.

What factors might contribute to non-specific binding when using the At2g05970 antibody?

Several factors can contribute to non-specific binding when using the At2g05970 antibody, potentially confounding experimental results:

• Inadequate blocking: Insufficient blocking of membranes or plates can lead to high background signal. Optimize blocking conditions (concentration, buffer composition, duration) for your specific sample type .

• Cross-reactivity with similar proteins: The polyclonal nature of the At2g05970 antibody means it contains a mixture of antibodies that may recognize similar epitopes present on other proteins. Studies of other antibodies have shown that commercial antibodies can sometimes cross-react with proteins unrelated to their intended targets .

• Sample preparation issues: Incomplete denaturation, excessive protein loading, or improper tissue fixation can all contribute to non-specific binding. Ensure sample preparation protocols are optimized for plant tissues .

• Secondary antibody cross-reactivity: When using indirect detection methods, the secondary antibody may contribute to non-specific binding. Include appropriate controls (primary antibody omitted) to identify secondary antibody-specific background .

• Buffer compatibility issues: Components in sample or wash buffers may affect antibody binding characteristics. Consider testing different buffer compositions if non-specific binding persists .

To address these challenges, researchers should systematically optimize experimental conditions and include appropriate controls, including knockout/knockdown samples when available, as has been recommended for other plant antibodies where specificity concerns exist .

What is the optimal experimental design for using At2g05970 antibody in Western blot applications?

For optimal Western blot results with the At2g05970 antibody, researchers should follow this methodological approach:

• Sample preparation:

  • Extract proteins using a buffer containing protease inhibitors to prevent degradation

  • Include positive controls (tissues known to express At2g05970) and negative controls

  • Consider using At2g05970 knockout tissues as definitive negative controls if available

• Electrophoresis conditions:

  • Load appropriate protein amounts (typically 20-50 μg per lane)

  • Use fresh SDS-PAGE gels (10-12% typically appropriate for most plant proteins)

  • Include molecular weight markers to verify target band size

• Transfer and blocking:

  • Optimize transfer conditions based on protein size

  • Block membranes with 5% non-fat dry milk or BSA in TBST (may need optimization)

  • Consider testing different blocking reagents if background is problematic

• Antibody incubation:

  • Start with 1:1000 dilution of the At2g05970 antibody and optimize if needed

  • Incubate overnight at 4°C for optimal sensitivity

  • Use appropriate HRP-conjugated secondary antibody (anti-rabbit IgG)

• Detection and analysis:

  • Use enhanced chemiluminescence (ECL) detection system

  • Perform quantitative analysis by densitometry if needed

  • Always include loading controls (e.g., housekeeping proteins)

This methodology incorporates precautions against potential antibody specificity issues that have been documented with other antibodies , ensuring more reliable experimental outcomes.

How can I incorporate the At2g05970 antibody into immunoprecipitation experiments?

While immunoprecipitation (IP) is not specifically listed among the tested applications for the At2g05970 antibody , researchers interested in adapting it for IP should consider this methodological framework:

• Preliminary assessment:

  • First validate the antibody in Western blot to confirm it recognizes the native protein

  • Consider performing a small-scale pilot IP to evaluate antibody performance

• Lysate preparation:

  • Use mild lysis buffers to preserve protein-protein interactions

  • Include protease and phosphatase inhibitors

  • Clear lysates by centrifugation to remove debris that might cause non-specific binding

• Antibody coupling:

  • Pre-couple the At2g05970 antibody to protein A/G beads or magnetic beads

  • Use approximately 2-5 μg of antibody per sample

  • Consider using crosslinkers to prevent antibody co-elution if needed

• IP procedure:

  • Incubate cleared lysate with antibody-bound beads (4-16 hours at 4°C)

  • Perform extensive washing (at least 3-5 washes) with decreasing salt concentrations

  • Elute using mild conditions to preserve protein integrity

• Validation controls:

  • Include IgG control to identify non-specific binding

  • Use At2g05970 knockout/knockdown tissue as negative control if available

  • Confirm results by Western blot of IP eluates

Be aware that IP experiments may require troubleshooting as antibodies successful in Western blot don't always perform well in IP. Studies have shown that immunoprecipitation can sometimes fail to identify expected proteins even with antibodies that show putative signals in other applications , necessitating thorough validation.

How can I optimize the At2g05970 antibody for immunohistochemistry experiments in plant tissues?

Although immunohistochemistry (IHC) is not listed among the validated applications for the At2g05970 antibody , researchers interested in adapting it for this purpose should consider the following optimization strategy:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde or another appropriate fixative

  • Consider testing multiple fixation protocols as overfixation can mask epitopes

  • Optimize embedding and sectioning methods (paraffin, cryo, or vibratome sectioning)

• Antigen retrieval:

  • Test multiple antigen retrieval methods (heat-induced in citrate buffer, enzymatic, etc.)

  • Monitor tissue integrity during retrieval process

  • Include positive control tissues with known expression patterns

• Antibody titration:

  • Perform a dilution series (typically starting at 1:100 - 1:500) to determine optimal concentration

  • Include appropriate negative controls (pre-immune serum, antibody omission)

  • If available, use tissues from At2g05970 knockout plants as definitive negative controls

• Detection system optimization:

  • Test different secondary antibody systems (fluorescent vs. enzymatic)

  • For fluorescent detection, select fluorophores compatible with plant tissue autofluorescence

  • Consider signal amplification methods (tyramide signal amplification) for low abundance proteins

• Validation criteria:

  • Compare localization patterns with published in situ hybridization or GFP-fusion data

  • Perform peptide competition assays to confirm specificity

  • Document all optimization steps meticulously

This approach acknowledges the challenges researchers have encountered with antibody specificity in similar contexts and emphasizes the importance of proper validation when adapting antibodies for new applications.

What strategies can be employed to troubleshoot unexpected results when using the At2g05970 antibody?

When encountering unexpected results with the At2g05970 antibody, implement this systematic troubleshooting approach:

• Unexpected band patterns in Western blot:

  • Verify protein extraction and loading consistency

  • Test multiple extraction methods to ensure complete protein solubilization

  • Consider post-translational modifications or alternative splicing of At2g05970

  • Perform phosphatase treatment if phosphorylation is suspected

  • Compare results with At2g05970 knockout tissues if available

• Signal inconsistency across experiments:

  • Evaluate antibody storage conditions and freeze-thaw history

  • Prepare fresh working dilutions from concentrated stock

  • Test antibody from different lots if available

  • Standardize lysate preparation and protein quantification methods

• High background or non-specific binding:

  • Optimize blocking conditions (agent, concentration, time)

  • Increase washing stringency with higher salt or detergent concentrations

  • Test alternative incubation temperatures and durations

  • Perform pre-absorption with non-specific proteins

• No signal despite expected expression:

  • Confirm protein expression using alternative methods (RT-PCR, RNA-seq data)

  • Verify that epitope is accessible and not masked by protein interactions

  • Test alternative extraction methods to ensure protein is solubilized

  • Consider epitope retrieval methods if working with fixed tissues

• Contradictory results across different applications:

  • Recognize that antibodies may perform differently across applications

  • Verify protein identity using mass spectrometry if possible

  • Consider that the antibody may recognize proteins other than At2g05970

This troubleshooting framework addresses the complexities of antibody-based experiments and incorporates lessons learned from studies demonstrating antibody cross-reactivity issues in the literature .

How should I interpret Western blot results when multiple bands appear using the At2g05970 antibody?

When multiple bands appear in Western blots using the At2g05970 antibody, careful interpretation is required:

• Expected band patterns:

  • The predicted molecular weight of the At2g05970 protein should be your primary reference point

  • Consider post-translational modifications that may alter migration (phosphorylation, glycosylation)

  • Alternative splicing may produce multiple legitimate isoforms

• Distinguishing specific vs. non-specific binding:

  • Compare observed bands with theoretical protein size

  • Evaluate consistency of band patterns across different samples

  • Check if bands disappear in knockout/knockdown samples, the gold standard for specificity

  • Consider peptide competition assays to identify specific bands

• Analysis of potential artifacts:

  • Protein degradation often produces lower molecular weight fragments

  • Incomplete denaturation can result in oligomeric forms at higher weights

  • Cross-reactivity with similar proteins may produce consistent but non-target bands

• Quantification considerations:

  • Only quantify bands validated as specific to the target protein

  • Use appropriate normalization controls

  • Report all visible bands in publications with transparent discussion of specificity

Studies have demonstrated that commercially available antibodies may detect multiple proteins not related to their intended targets . For example, research on AT1R antibodies showed that each antibody tested recognized distinct unknown proteins of diverse molecular sizes , highlighting the importance of rigorous validation of band specificity.

What comparative analysis approaches can help validate At2g05970 antibody results?

To strengthen confidence in results obtained with the At2g05970 antibody, researchers should implement these comparative analysis approaches:

• Orthogonal technique validation:

  • Complement antibody-based detection with transcript analysis (RT-PCR, RNA-seq)

  • Compare protein levels with transcript levels to identify discrepancies

  • Consider mass spectrometry-based protein identification for critical findings

  • Use GFP-tagged At2g05970 constructs to compare localization patterns

• Multiple antibody comparison:

  • If available, use independent antibodies targeting different epitopes of At2g05970

  • Consistent results across different antibodies significantly increase confidence

  • Discrepancies between antibodies warrant further investigation

• Genetic manipulation approaches:

  • Compare antibody signals between wild-type, knockdown, and knockout samples

  • Utilize overexpression systems to confirm signal proportionality

  • Consider CRISPR-edited plants with epitope tags on the endogenous At2g05970

• Cross-species comparison:

  • Test the antibody on closely related plant species with homologous proteins

  • Evaluate evolutionary conservation of the epitope recognized by the antibody

  • Consider species-specific post-translational modifications

• Data integration framework:

  Validation Method	Advantages	Limitations	Implementation Notes
  Knockout controls	Gold standard for specificity	Requires genetic resources	Essential for definitive validation
  Orthogonal techniques	Independent confirmation	May measure different parameters	Use to correlate protein/mRNA levels
  Multiple antibodies	Confirms epitope accessibility	Requires additional resources	Increases confidence in results
  Peptide competition	Simple to implement	Limited specificity information	Good initial validation step
  Mass spectrometry	Direct protein identification	Technical complexity	Critical for novel findings

This comprehensive validation framework addresses the known challenges with antibody specificity documented in the scientific literature and provides a systematic approach to ensure reliable research findings.

What are the best practices for reporting results using the At2g05970 antibody in scientific publications?

When reporting results obtained using the At2g05970 antibody in scientific publications, researchers should adhere to these best practices:

• Detailed antibody information:

  • Provide complete antibody identification (manufacturer, catalog number, lot number)

  • Specify the host species, clonality, and immunogen used for antibody production

  • Clearly state that the antibody is directed against the Arabidopsis thaliana At2g05970 protein

• Methodology transparency:

  • Detail all experimental conditions (dilutions, incubation times/temperatures)

  • Describe blocking agents, buffers, and detection systems

  • Specify image acquisition parameters and any post-acquisition processing

  • Include all antibody validation steps performed

• Control documentation:

  • Report all controls used to validate specificity

  • Include images of negative controls (knockout tissues, primary antibody omission)

  • Describe any discrepancies or unexpected results honestly

• Result interpretation:

  • Show representative full blots/images, not just cropped regions of interest

  • Indicate molecular weight markers on all blots

  • Present quantification methods and statistical analyses transparently

  • Acknowledge limitations of antibody-based techniques

• Reproducibility considerations:

  • State the number of independent experiments performed

  • Address potential batch effects or variability between experiments

  • Consider providing raw data in supplementary materials or repositories

These reporting standards align with growing concerns in the scientific community about antibody specificity and aim to improve reproducibility in plant molecular biology research. By transparently documenting all aspects of antibody use and validation, researchers contribute to the collective reliability of findings in the field.