At1g67190 Antibody

What is the At1g67190 antibody and what does it target?

At1g67190 antibody is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana At1g67190 protein. This antibody specifically targets proteins expressed by the At1g67190 gene in Arabidopsis thaliana (Mouse-ear cress). It's primarily designed for research applications and should not be used for diagnostic or therapeutic procedures . The antibody is typically supplied in liquid form with preservation buffer containing 0.03% Proclin 300 and storage constituents of 50% Glycerol and 0.01M PBS at pH 7.4 .

What are the primary applications for At1g67190 antibody?

The At1g67190 antibody has been validated for several standard laboratory techniques:

• Enzyme-linked immunosorbent assay (ELISA)

• Western blotting (WB)

These applications enable researchers to detect, quantify, and characterize the At1g67190 protein in plant tissue samples . Similar to approaches used with other plant antibodies, researchers could potentially explore additional applications such as immunofluorescence, though specific validation would be required .

What is the recommended storage and handling procedure?

Upon receipt, the At1g67190 antibody should be stored at -20°C or -80°C to maintain its activity and specificity. Researchers should avoid repeated freeze-thaw cycles as this can degrade antibody quality and performance . For working solutions, aliquoting the antibody before freezing is recommended to minimize freeze-thaw cycles. The antibody is typically shipped with cold packs, similar to other research antibodies , and proper storage upon arrival is crucial for maintaining its efficacy.

What controls should be included when using At1g67190 antibody?

When designing experiments with the At1g67190 antibody, researchers should incorporate appropriate controls to validate results:

• Positive control: Tissue or cell extracts known to express At1g67190 (preferably Arabidopsis thaliana samples)

• Negative control: Samples from organisms not expressing the target protein

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

• Blocking peptide competition assay: Using the immunizing peptide to confirm specificity

These controls help distinguish true positive signals from background or non-specific binding . Studies with other antibodies have demonstrated that lack of proper controls can lead to misinterpretation of results, particularly with polyclonal antibodies .

How can I validate the specificity of the At1g67190 antibody for my experimental system?

Antibody validation is critical, especially given known issues with commercial antibodies. A comprehensive validation approach should include:

• Western Blot Analysis: Verify the antibody detects bands of expected molecular weight in tissues known to express the target protein .

• Knockout/Knockdown Validation: If available, use genetic knockout or RNAi knockdown tissues where At1g67190 expression is absent or reduced. The antibody should show corresponding reduction or absence of signal .

• Overexpression Studies: Test tissues or cells overexpressing At1g67190 to confirm increased signal intensity .

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide before application to samples; specific signals should be blocked or reduced .

• Cross-reactivity Assessment: Test the antibody on closely related species to evaluate potential cross-reactivity .

This multi-faceted approach is particularly important given findings that some commercial antibodies may produce identical immunostaining patterns regardless of target protein presence, as demonstrated with AT1 receptor antibodies .

What are the potential cross-reactivity concerns with At1g67190 antibody?

While the At1g67190 antibody is designed to be specific for Arabidopsis thaliana, potential cross-reactivity with proteins from other plant species or with structurally similar proteins within Arabidopsis remains a concern. Studies on other antibodies have revealed that even well-characterized antibodies may detect proteins unrelated to their intended targets .

Researchers should conduct cross-reactivity tests when applying this antibody to:

• Non-Arabidopsis plant species

• Novel experimental conditions

• Different tissue types than previously validated

The lesson from studies with AT1 receptor antibodies, where six commercially available antibodies showed non-specific binding to proteins of similar molecular weight to their intended targets, underscores the importance of cross-reactivity assessment .

How does experimental design affect the reliability of At1g67190 antibody results?

Experimental design significantly impacts the reliability of antibody-based results. Researchers should consider implementing robust experimental designs such as:

How can I optimize Western blot protocols for At1g67190 antibody?

Optimizing Western blot protocols for the At1g67190 antibody requires systematic testing of several parameters:

• Sample Preparation:

  • Test different extraction buffers to optimize protein solubilization

  • Include protease inhibitors to prevent degradation

  • Determine optimal protein loading amounts (typically 20-50 μg total protein)

• Blocking Conditions:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Optimize blocking time and temperature

• Antibody Dilution:

  • Create a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

  • Determine optimal primary antibody incubation time and temperature

• Detection System:

  • Compare chemiluminescence vs. fluorescence detection

  • Optimize exposure times for imaging

This methodical approach helps identify conditions that maximize specific signal while minimizing background, similar to optimization procedures used for other plant antibodies .

What protein extraction methods are most effective for At1g67190 detection?

Effective protein extraction is crucial for successful At1g67190 detection. The following methods have proven effective for plant protein extraction:

• RIPA Buffer Extraction:

  • Composition: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS

  • Add protease inhibitor cocktail freshly before use

  • Most suitable for general protein extraction

• Native Protein Extraction:

  • Composition: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40

  • Preserves protein structure and interactions

  • Suitable for co-immunoprecipitation studies

• Subcellular Fractionation:

  • Sequential extraction of cytosolic, membrane, nuclear, and cytoskeletal fractions

  • Helps determine protein localization

  • Reduces sample complexity

The extraction method should be selected based on the downstream application and the cellular localization of the At1g67190 protein. Each method should be validated specifically for Arabidopsis thaliana tissue samples to ensure optimal results.

How can I resolve discrepancies between antibody-based detection methods and gene expression data?

Discrepancies between antibody-based protein detection and gene expression data are common in biological research. To resolve these discrepancies:

• Verify antibody specificity: As discussed in question 2.1, confirm the antibody is detecting the correct target .

• Consider post-transcriptional regulation: mRNA levels may not correlate with protein levels due to:

  • Differences in mRNA stability

  • Translational efficiency variations

  • Post-translational modifications

  • Protein degradation rates

• Employ multiple detection methods:

  • Compare results from different antibody-based techniques (Western blot, ELISA, immunofluorescence)

  • Use mass spectrometry for antibody-independent protein identification

  • Implement genetic approaches (reporter gene fusions)

• Temporal considerations: Examine whether time differences between mRNA expression and protein production could explain discrepancies.

Studies examining AT1 receptor antibodies have demonstrated that reliance on a single detection method can lead to misinterpretation, emphasizing the importance of using complementary approaches .

What statistical approaches are most appropriate for analyzing At1g67190 antibody data?

• For Western Blot Quantification:

  • Normalize target protein signal to loading controls

  • Use ANOVA for comparing multiple treatment groups

  • Apply non-parametric tests (Mann-Whitney U test) for non-normally distributed data

• For ELISA Data:

  • Generate standard curves using purified protein when possible

  • Apply four-parameter logistic regression for standard curve fitting

  • Use t-tests or ANOVA with appropriate post-hoc tests for group comparisons

• For Reproducibility Assessment:

  • Calculate coefficient of variation (CV) between technical and biological replicates

  • Report confidence intervals rather than just p-values

  • Perform power analysis to determine appropriate sample sizes

As noted in phase 1 clinical research, descriptive statistics including arithmetic mean, standard deviation, coefficient of variation, median, minimum, and maximum are typically calculated for all parameters when appropriate .

How can I troubleshoot common problems with At1g67190 antibody applications?

This troubleshooting guide addresses common issues encountered with antibody applications, drawing on principles that have been documented with other antibody systems .

What emerging technologies might complement or replace At1g67190 antibody-based detection?

Several emerging technologies offer complementary or alternative approaches to traditional antibody-based detection:

• CRISPR/Cas9 Epitope Tagging:

  • Endogenous tagging of At1g67190 with HA, FLAG, or GFP tags

  • Enables detection using highly validated tag-specific antibodies

  • Preserves native expression levels and regulation

• Proximity Labeling Technologies:

  • BioID or TurboID fusion proteins to identify interaction partners

  • Maps protein microenvironments in living cells

  • Complements traditional co-immunoprecipitation approaches

• Mass Spectrometry-Based Proteomics:

  • Label-free quantification of At1g67190

  • Parallel detection of post-translational modifications

  • Antibody-independent validation approach

• Nanobody and Aptamer Technologies:

  • Development of single-domain antibodies or DNA/RNA aptamers

  • Potentially higher specificity than conventional antibodies

  • Better penetration into complex tissues

These technologies address some limitations of traditional antibodies, such as the specificity concerns documented with AT1 receptor antibodies .

How might At1g67190 antibody research contribute to broader understanding of plant biology?

Research utilizing At1g67190 antibodies contributes to plant biology in several ways:

• Functional Characterization:

  • Determination of protein expression patterns across tissues and developmental stages

  • Correlation with phenotypic changes under various conditions

  • Assessment of protein-protein interactions and complex formation

• Stress Response Studies:

  • Examination of protein expression changes under abiotic stresses

  • Investigation of post-translational modifications during stress adaptation

  • Comparison with other plant species' stress responses

• Evolutionary Biology:

  • Comparative studies across related plant species

  • Examination of protein conservation and divergence

  • Understanding of functional adaptations in different ecological niches

The methodological approaches developed for At1g67190 antibody research may also inform broader antibody validation practices in plant biology, addressing the challenges of antibody specificity documented in other research areas .