ATL73 Antibody

What is ATL73 Antibody and what organism does it target?

ATL73 Antibody (product code CSB-PA872275XA01DOA) is a polyclonal antibody that specifically targets the ATL73 protein in Arabidopsis thaliana, commonly known as mouse-ear cress . This antibody is produced by immunizing rabbits with recombinant Arabidopsis thaliana ATL73 protein as the immunogen . ATL73 is a specific target protein in Arabidopsis, a model organism widely used in plant molecular biology research.

The antibody is purified using antigen affinity methods, which helps enhance its specificity toward the target protein . Because it is a polyclonal antibody, it contains a heterogeneous mixture of antibodies that recognize different epitopes on the ATL73 protein. This characteristic can provide robust detection capabilities across various experimental conditions, although it may also introduce variability between different batches or lots of the antibody.

As with all research antibodies, it is designated for research use only and not for diagnostic or therapeutic applications, in accordance with standard laboratory reagent classifications .

What are the recommended storage and handling protocols for ATL73 Antibody?

Upon receipt, ATL73 Antibody should be immediately stored at either -20°C or -80°C for optimal preservation of antibody activity . Researchers should avoid repeated freeze-thaw cycles as this can significantly diminish antibody functionality and lead to experimental inconsistencies. The antibody is supplied in liquid form in a storage buffer consisting of 50% glycerol, 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative .

For laboratory handling during experiments, it is advisable to aliquot the antibody into smaller volumes before freezing to minimize freeze-thaw cycles. When removing from storage, thaw the antibody gradually on ice rather than at room temperature to preserve epitope recognition capabilities. Once thawed, the antibody should be kept on ice during experimental procedures.

Long-term storage stability data suggests that properly stored antibodies can maintain their activity for extended periods, but researchers should document the date of receipt and track the number of freeze-thaw cycles to monitor potential degradation. Following these protocols is particularly important for polyclonal antibodies like ATL73, as their heterogeneous nature means activity loss may not be uniform across all antibody populations in the solution.

What validated applications exist for ATL73 Antibody?

For ELISA applications, the antibody can be used to detect and quantify the presence of ATL73 in various sample preparations from Arabidopsis thaliana. In Western Blotting, the antibody recognizes the denatured form of the protein, allowing for size-based confirmation of target specificity.

It is worth noting that contemporary antibody validation standards, as outlined in the "five pillars" framework, recommend multiple approaches to confirm antibody specificity and functionality . These include genetic strategies (using knockout or knockdown controls), orthogonal strategies (comparing antibody-dependent and independent techniques), multiple antibody strategies (using different antibodies against the same target), recombinant expression strategies, and immunocapture mass spectrometry . While the manufacturer provides basic validation for ELISA and WB, researchers are encouraged to perform additional characterization for their specific experimental conditions.

How should ATL73 Antibody be validated for specific experimental applications?

Comprehensive validation of ATL73 Antibody should ideally incorporate multiple complementary strategies as outlined in the "five pillars" framework for antibody validation . For plant research with ATL73 Antibody, these strategies can be adapted as follows:

First, genetic validation using Arabidopsis thaliana ATL73 knockout or knockdown mutants provides the most stringent control for antibody specificity. Researchers should observe absence or significant reduction of signal in these genetic controls compared to wild-type samples . This approach directly addresses whether the antibody is detecting the intended target.

Second, orthogonal validation comparing antibody-based detection with antibody-independent methods (such as mass spectrometry or RNA expression analysis) helps confirm whether the protein detected by the antibody correlates with other measures of ATL73 presence . Correlation between ATL73 protein levels detected by the antibody and transcript levels can provide additional confidence in antibody specificity.

Third, expression validation using recombinant expression systems to overexpress ATL73 in plant protoplasts or heterologous systems can demonstrate increased signal corresponding to increased target concentration . This approach is particularly valuable for confirming antibody sensitivity.

Fourth, performing careful titration experiments to determine optimal antibody concentrations for each application is essential, as is establishing appropriate positive and negative controls specific to the experimental system. Researchers should document all validation steps thoroughly to enable reproducibility and proper interpretation of results.

What experimental controls are essential when using ATL73 Antibody?

When designing experiments using ATL73 Antibody, multiple controls are necessary to ensure reliable and interpretable results. In Western blotting applications, a primary negative control should include Arabidopsis thaliana ATL73 knockout or knockdown plant material processed identically to experimental samples . This control addresses whether bands detected in experimental samples are specific to ATL73 or represent cross-reactivity with other proteins.

Additional essential controls include:

• Technical negative controls: Omitting primary antibody while maintaining all other aspects of the protocol helps identify non-specific binding from secondary antibodies.

• Loading controls: Using antibodies against constitutively expressed Arabidopsis proteins ensures equal sample loading and transfer efficiency.

• Peptide competition assays: Pre-incubating the antibody with excess recombinant ATL73 protein before sample application should diminish or eliminate specific signals.

• Recombinant protein standards: Including purified recombinant ATL73 protein at known concentrations provides a reference for signal quantification and confirmation of expected molecular weight.

The alarming estimate that approximately 50% of commercial antibodies fail to meet basic characterization standards underscores the importance of implementing robust controls . Furthermore, since antibody specificity is context-dependent, validation should be performed under the specific experimental conditions used in the research study .

How can researchers troubleshoot non-specific binding with ATL73 Antibody?

Non-specific binding is a common challenge with polyclonal antibodies like ATL73 Antibody. Systematic troubleshooting approaches can help address this issue and improve experimental specificity. When experiencing high background or unexpected bands in Western blots, researchers should first optimize blocking conditions by testing different blocking agents (such as BSA, non-fat dry milk, or commercial blocking solutions) at various concentrations and incubation times.

Adjusting antibody concentration is equally important; titrating from higher to lower dilutions (e.g., 1:500 to 1:5000) can help identify the optimal concentration that maximizes specific signal while minimizing background. Increasing washing stringency by extending wash times, adding detergents like Tween-20 at appropriate concentrations, or using higher salt concentrations in wash buffers can also reduce non-specific interactions.

For particularly challenging samples, pre-adsorption techniques can be employed where the ATL73 Antibody is pre-incubated with protein extracts from ATL73 knockout plants to deplete antibodies that bind to non-specific targets . This technique effectively removes cross-reactive antibodies from the polyclonal mixture.

Additionally, it is essential to remember that the heterogeneous nature of plant tissues may require tissue-specific optimization. The protein composition varies significantly between different plant tissues and developmental stages, potentially affecting background levels and cross-reactivity patterns of polyclonal antibodies.

What considerations should be made when interpreting ATL73 Antibody data across different ecotypes and experimental conditions?

Interpreting data generated using ATL73 Antibody across different Arabidopsis thaliana ecotypes requires careful consideration of several factors that could influence results and their interpretation. Protein sequence variations among ecotypes may affect epitope recognition, potentially resulting in differential binding affinity of the antibody. Researchers should compare ATL73 protein sequences across studied ecotypes to identify potential variations that might affect antibody binding.

Post-translational modifications represent another critical consideration. If ATL73 undergoes different modifications (such as phosphorylation, glycosylation, or ubiquitination) in different ecotypes or under different experimental conditions, antibody recognition may be affected. This is particularly relevant for polyclonal antibodies that recognize multiple epitopes, as some may be sensitive to modifications while others remain unaffected.

Environmental conditions and stress responses can significantly alter protein expression patterns, potentially introducing confounding variables when comparing antibody-based detection across different growth conditions. Standardizing growth conditions or including appropriate controls for each condition is essential for meaningful comparisons.

Additionally, researchers should consider developing a standardized positive control that can be included across experiments to normalize signals and enable more reliable cross-experimental comparisons. This might involve using a recombinant ATL73 protein standard curve in each experiment to calibrate detection sensitivity.

How does the polyclonal nature of ATL73 Antibody affect experimental reproducibility?

The polyclonal nature of ATL73 Antibody introduces specific reproducibility challenges that researchers must address through methodological approaches. Polyclonal antibodies like ATL73 contain mixtures of antibodies recognizing different epitopes on the target protein, resulting in potential batch-to-batch variation . Each production lot may contain different proportions of antibodies with varying affinities and epitope specificities, which can affect experimental outcomes.

To mitigate these challenges, researchers should implement several strategies:

• Lot-to-lot validation: When obtaining a new lot of ATL73 Antibody, perform side-by-side comparisons with previous lots using identical samples and protocols to assess consistency.

• Internal standards: Include consistent positive controls in each experiment to track potential variations in antibody performance over time.

• Detailed method documentation: Maintain comprehensive records of antibody lot numbers, dilutions, incubation conditions, and all experimental parameters to enable accurate replication.

• Multiple detection methods: When possible, verify key findings using orthogonal techniques that don't rely solely on antibody-based detection.

The reproducibility crisis in antibody-based research has resulted in estimated financial losses of $0.4–1.8 billion per year in the United States alone, highlighting the significance of these methodological considerations . By implementing these strategies, researchers can enhance the reliability and reproducibility of experiments using ATL73 Antibody despite its polyclonal nature.

What optimized protocols exist for using ATL73 Antibody in plant tissue immunolocalization?

While the manufacturer's data indicates ATL73 Antibody is primarily validated for ELISA and Western Blot applications , researchers interested in immunolocalization studies should develop and validate protocols specifically for this purpose. Based on best practices for antibody-based localization in plant tissues, the following optimized protocol framework is recommended:

Tissue Fixation and Embedding:

• Fix Arabidopsis tissue samples in 4% paraformaldehyde in PBS (pH 7.4) for 12-16 hours at 4°C

• Dehydrate through an ethanol series (30%, 50%, 70%, 85%, 95%, 100%)

• Clear with a xylene:ethanol series and embed in paraffin or consider cryosectioning for better epitope preservation

Antigen Retrieval and Immunostaining:

• Deparaffinize sections and rehydrate through an ethanol series

• Perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 10-15 minutes

• Block with 3-5% BSA in PBS with 0.1% Triton X-100 for 1-2 hours

• Incubate with ATL73 Antibody at 1:100 to 1:500 dilution in blocking buffer overnight at 4°C

• Wash extensively with PBS containing 0.1% Tween-20

• Apply fluorophore-conjugated secondary antibody

• Counterstain with DAPI for nuclear visualization

Critical Controls:

• Omit primary antibody (negative control)

• Use tissue from ATL73 knockout plants (specificity control)

• Pre-absorb antibody with recombinant ATL73 protein (competition control)

This protocol should be optimized for each specific tissue type and experimental question, with particular attention to fixation conditions that preserve both tissue morphology and antibody epitopes. Documentation of all optimization steps is essential for reproducibility.

What quantitative approaches should be used when analyzing ATL73 Antibody data?

Quantitative analysis of data generated using ATL73 Antibody requires careful consideration of several methodological factors to ensure accuracy and reproducibility. For Western blot quantification, researchers should establish a linear dynamic range for detection by creating a standard curve using known concentrations of recombinant ATL73 protein. This allows confirmation that experimental samples fall within the quantifiable range of the assay.

Statistical analysis must account for the inherent variability of antibody-based detection methods. Performing a minimum of three biological replicates (from independent plant samples) and technical replicates (repeated measurements of the same sample) allows calculation of variation and appropriate statistical testing. Reporting both the mean and measures of dispersion (standard deviation or standard error) is essential.

Digital image analysis should follow standardized protocols to avoid bias. Using analysis software that can identify band intensity while controlling for background levels provides more objective quantification than visual assessment. All image adjustments should be applied uniformly across the entire image and across all comparable images in a dataset.

How should researchers report experimental details when publishing research using ATL73 Antibody?

The reproducibility crisis in antibody-based research necessitates comprehensive reporting of experimental details when publishing work using ATL73 Antibody. Based on best practices in the field , researchers should include the following details in their publications:

Antibody Identification Information:

• Complete manufacturer information (company name, location)

• Catalog number (CSB-PA872275XA01DOA for ATL73 Antibody)

• Lot number (specific to the antibody used in the study)

• Clonality (polyclonal)

• Host species (rabbit)

• Immunogen details (recombinant Arabidopsis thaliana ATL73 protein)

Experimental Methods:

• Detailed protocols including antibody dilutions, incubation times and temperatures

• Buffer compositions and pH values

• Blocking reagents and concentrations

• Washing procedures (number of washes, duration, buffer composition)

• Detection methods and equipment settings

Validation Procedures:

• Description of controls used to verify specificity

• Results from validation experiments

• Any optimization procedures performed

• Reference to relevant validation publications if available

Proper reporting enables other researchers to evaluate the reliability of the results and to reproduce the experiments if needed. This level of detail is increasingly required by high-quality journals and funding agencies in response to the antibody reproducibility crisis that has been estimated to cost between $0.4–1.8 billion per year in the United States alone due to unreliable antibody usage .

How does using ATL73 Antibody compare with alternative detection methods?

When considering experimental design, researchers should evaluate how ATL73 Antibody-based detection compares with alternative approaches for studying ATL73 protein in Arabidopsis thaliana. Each method offers distinct advantages and limitations that should inform methodological choices.

Antibody Detection vs. Transcript Analysis: RT-qPCR or RNA-seq for ATL73 transcript quantification provides information about gene expression but does not capture post-transcriptional regulation or protein stability factors. The correlation between transcript and protein levels can be poor for many genes. ATL73 Antibody detection directly measures protein levels, capturing the complete biological outcome of gene expression, translation, and protein turnover.