At1g06640 Antibody

What is the At1g06640 Antibody and what is its target protein in Arabidopsis thaliana?

The At1g06640 Antibody is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana At1g06640 protein. It specifically targets the At1g06640 protein (UniProt: Q9C5K7) in Arabidopsis thaliana (mouse-ear cress) . This antibody has been affinity-purified using the target antigen, which enhances its specificity for research applications. Polyclonal antibodies like this one contain a mixture of immunoglobulins that recognize different epitopes on the target protein, often providing robust detection even if some epitopes are altered or masked in experimental conditions.

What experimental applications has the At1g06640 Antibody been validated for?

Based on available data, the At1g06640 Antibody has been validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications . These validated applications allow researchers to detect and quantify the target protein in different experimental contexts. For Western blotting, this antibody can be used to identify the presence and relative abundance of the At1g06640 protein in complex protein mixtures separated by gel electrophoresis. In ELISA applications, it can be employed for quantitative detection of the target protein in solution-based samples.

What are the recommended storage and handling conditions for maintaining antibody activity?

The At1g06640 Antibody should be stored at -20°C or -80°C upon receipt to maintain its activity and specificity. Repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and reduce binding efficiency . The antibody is supplied in liquid form in a storage buffer containing 50% glycerol, 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative . When working with the antibody, it's advisable to aliquot it into smaller volumes before freezing to minimize freeze-thaw cycles and maintain optimal performance across multiple experiments.

What positive and negative controls should be incorporated when using the At1g06640 Antibody?

For rigorous experimental design with the At1g06640 Antibody, researchers should include:

Positive Controls:

• Recombinant At1g06640 protein (the immunogen)

• Wild-type Arabidopsis thaliana tissue known to express the target protein

• Overexpression systems with verified At1g06640 expression

Negative Controls:

• At1g06640 knockout/knockdown plant lines

• Non-plant tissues or unrelated protein samples

• Primary antibody omission controls

• Pre-immune serum controls (if available)

Inclusion of these controls helps validate signal specificity and distinguish true protein detection from background or non-specific binding. This becomes particularly important when working with polyclonal antibodies that may exhibit some level of cross-reactivity.

How should dilution optimization be performed for Western blot applications?

Determining the optimal antibody dilution is crucial for achieving the best signal-to-noise ratio. A systematic approach includes:

• Prepare a dilution series (typically 1:500, 1:1000, 1:2000, 1:5000) of the At1g06640 Antibody

• Run identical protein samples on multiple gels

• Process each membrane with a different antibody dilution

• Compare signal intensity and background levels

• Select the dilution that provides clear target detection with minimal background

For the At1g06640 Antibody, start with the manufacturer's recommended dilution ranges for Western blot applications and adjust based on your specific protein abundance and detection system. Remember that optimal dilutions may vary between different detection methods (chemiluminescence, fluorescence, or chromogenic).

What blocking strategies are most effective when using the At1g06640 Antibody?

Effective blocking is essential for reducing non-specific binding. For plant-derived antibodies like the At1g06640 Antibody, consider these blocking approaches:

Test different blocking agents to determine which provides the cleanest results with your specific samples. The optimal blocking strategy may vary depending on the plant tissue type and protein extraction method used.

How can researchers validate the specificity of the At1g06640 Antibody beyond manufacturer testing?

While the At1g06640 Antibody has been affinity-purified , additional validation is recommended for critical research applications:

• Peptide competition assay: Pre-incubate the antibody with excess purified At1g06640 protein or peptide before application to samples. Signal disappearance confirms specificity.

• Genetic validation: Compare signal between wild-type and At1g06640 knockout/knockdown lines. Signal reduction or elimination in knockout lines confirms specificity.

• Mass spectrometry validation: Immunoprecipitate proteins using the At1g06640 Antibody and analyze by mass spectrometry to confirm target enrichment.

• Orthogonal method comparison: Compare protein expression patterns detected by the antibody with mRNA expression data from RNA-seq or qRT-PCR.

These validation approaches provide multiple lines of evidence for antibody specificity and target recognition in experimental systems.

What are potential cross-reactivity concerns with polyclonal antibodies in plant research?

The At1g06640 Antibody is a polyclonal antibody , which means it contains a mixture of antibodies that recognize different epitopes on the target protein. While this provides robust detection, it can also introduce cross-reactivity concerns:

• Conserved protein domains: The At1g06640 protein may share conserved domains with other Arabidopsis proteins, potentially leading to off-target binding.

• Post-translational modifications: Different modifications on the target protein might affect epitope recognition.

• Splice variants: If the At1g06640 gene produces multiple splice variants, the antibody may detect some variants better than others depending on the immunogen design.

To address these concerns, researchers should:

• Examine protein sequence homology to identify potential cross-reactive proteins

• Validate results using complementary approaches

• Consider using more specific detection methods for critical experiments

How does the polyclonal nature of the At1g06640 Antibody affect experimental reproducibility?

As a polyclonal antibody generated against recombinant At1g06640 protein , lot-to-lot variation may occur. This variability can impact experimental reproducibility in several ways:

• Epitope coverage: Different lots may contain antibodies recognizing different epitope distributions.

• Relative affinities: The proportion of high-affinity vs. low-affinity antibodies may vary between lots.

• Background binding profiles: Non-specific binding characteristics might differ between production batches.

Recommendations to enhance reproducibility:

• Document lot numbers used in experiments

• Purchase sufficient quantities of a single lot for complete studies

• Revalidate new lots against previous lots before use in critical experiments

• Maintain consistent experimental conditions (protein extraction methods, blocking reagents, etc.)

What are common causes of weak or absent signals when using the At1g06640 Antibody, and how can they be addressed?

When experiencing detection issues with the At1g06640 Antibody, consider these potential causes and solutions:

For plant tissue specifically, consider that the extraction method significantly impacts protein integrity and antibody accessibility. Different extraction buffers may be required for different plant tissues to optimize detection of the At1g06640 protein.

How can sample preparation be optimized for detecting the At1g06640 protein in different Arabidopsis tissues?

Different plant tissues present unique challenges for protein extraction and antibody detection:

Leaf tissue preparation:

• Rapidly freeze in liquid nitrogen to prevent protein degradation

• Grind thoroughly to fine powder while maintaining frozen state

• Extract with buffer containing appropriate detergents (0.5-1% Triton X-100 or NP-40)

• Include protease inhibitors to prevent degradation

• Clear cellular debris by centrifugation at 12,000-15,000g

Root tissue preparation:

• Wash thoroughly to remove soil contaminants

• Consider stronger lysis buffers containing 6-8M urea for more complete protein extraction

• Extend extraction time to ensure complete tissue disruption

Seed tissue preparation:

• Use mechanical disruption (bead beating) for efficient breakdown of tough seed coats

• Consider specialized extraction buffers with higher detergent concentrations

• Perform additional centrifugation steps to remove lipid contaminants

Optimizing extraction conditions for each tissue type can significantly improve detection sensitivity and specificity when using the At1g06640 Antibody.

How should researchers quantify and normalize Western blot data when using the At1g06640 Antibody?

• Image acquisition: Capture images within the linear range of detection to avoid signal saturation

• Background subtraction: Apply consistent background subtraction methods across all samples

• Normalization approaches:

  • Normalize to total protein (using stain-free gels or Ponceau S staining)

  • Use established housekeeping proteins appropriate for plant tissues (e.g., actin, tubulin, GAPDH)

  • Verify that normalization controls remain stable under your experimental conditions

• Statistical analysis: Apply appropriate statistical tests to determine significance of observed differences

• Biological replicates: Include multiple biological replicates (minimum three) from independent experiments

Presenting both raw and normalized data enhances transparency and allows readers to evaluate the quality of normalization.

How can contradictory results between antibody-based detection and transcript analysis be reconciled?

When At1g06640 protein levels detected by antibody do not correlate with mRNA expression, consider these potential explanations:

• Post-transcriptional regulation: mRNA levels do not always correlate with protein abundance due to:

  • Variations in translation efficiency

  • Differences in protein stability and turnover rates

  • miRNA-mediated regulation

• Technical considerations:

  • Antibody may recognize specific protein conformations or modifications

  • Protein extraction efficiency may vary between samples

  • Detection sensitivity differences between protein and RNA methods

• Biological explanations:

  • Temporal delays between transcription and translation

  • Tissue-specific post-translational modifications affecting antibody recognition

  • Alternative splicing producing protein variants with different antibody epitopes

To reconcile contradictory results:

• Validate findings using multiple antibodies targeting different epitopes if available

• Employ complementary protein detection methods (mass spectrometry)

• Consider the biological context and potential regulatory mechanisms