At1g46840 Antibody

What is At1g46840 and what protein does it encode?

At1g46840 is a gene in Arabidopsis thaliana (Mouse-ear cress) that encodes an F-box family protein according to the Araport11 database . The protein has the UniProt accession number Q9C629 . F-box proteins are components of SCF ubiquitin-ligase complexes that play critical roles in protein degradation via the ubiquitin-proteasome pathway, functioning in numerous cellular processes including cell cycle regulation, signal transduction, and developmental processes.

What are the key specifications of commercial At1g46840 Antibodies?

The At1g46840 Antibody is typically available as a polyclonal antibody raised in rabbits using recombinant Arabidopsis thaliana At1g46840 protein as the immunogen . Commercial preparations generally have the following specifications:

What validated applications exist for At1g46840 Antibody?

The At1g46840 Antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western blot applications based on manufacturer specifications . When using this antibody for Western blot applications, it is critical to ensure proper identification of the antigen by comparing with appropriate positive and negative controls. There is currently no published data in the search results indicating validation for immunohistochemistry, immunoprecipitation, or other applications.

What is the recommended storage protocol for At1g46840 Antibody?

For optimal antibody performance and longevity, store At1g46840 Antibody at -20°C or -80°C upon receipt . Avoid repeated freeze-thaw cycles as these can compromise antibody integrity and binding efficiency. For short-term use, aliquoting the antibody into smaller volumes before freezing can help minimize freeze-thaw cycles and preserve antibody functionality.

What is the recommended protocol for Western blot using At1g46840 Antibody?

While specific optimized protocols for At1g46840 Antibody are not provided in the search results, the following general protocol can be adapted:

• Sample Preparation:

  • Extract total protein from Arabidopsis tissues using appropriate buffer

  • Quantify protein concentration (Bradford or BCA assay)

  • Denature proteins in Laemmli buffer at 95°C for 5 minutes

• Gel Electrophoresis:

  • Load 10-30 μg protein per lane on SDS-PAGE

  • Include positive control (recombinant At1g46840) and negative control

• Transfer and Blocking:

  • Transfer proteins to PVDF or nitrocellulose membrane

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Antibody Incubation:

  • Dilute At1g46840 Antibody (optimization required, start with 1:1000)

  • Incubate overnight at 4°C

  • Wash 3× with TBST

• Detection:

  • Incubate with HRP-conjugated secondary antibody

  • Visualize using chemiluminescence

Since the expected molecular weight is not specified in the search results, researchers should be prepared to validate the specific band corresponding to At1g46840 protein in their experimental system.

How can I validate the specificity of At1g46840 Antibody?

Antibody specificity is crucial for accurate results, especially given known issues with commercial antibodies . To validate At1g46840 Antibody specificity:

• Genetic Controls: Compare signal between wild-type Arabidopsis and mutant lines lacking At1g46840 (similar to the AT1R knockout approach in search result )

• Blocking Peptide: Pre-incubate antibody with excess recombinant At1g46840 protein before application to samples; specific signals should be eliminated

• Protein Overexpression: Test antibody against tissues/cells with different levels of At1g46840 expression, expecting proportional signal intensity

• Multiple Antibodies: Compare results using different antibodies targeting different epitopes of At1g46840

• Mass Spectrometry: Confirm the identity of the immunoprecipitated protein band by mass spectrometry

Lack of specificity in commercial antibodies is a significant concern, as highlighted in search result , where antibodies for AT1R produced identical staining patterns in both wild-type and knockout tissues, indicating non-specific binding.

What are common issues when using At1g46840 Antibody and how can they be resolved?

Based on general antibody principles and the specificity issues noted in search result , researchers may encounter several challenges:

The experience with AT1R antibodies described in search result demonstrates that even when an antibody produces clear bands or staining patterns, these may not represent the target protein. Therefore, stringent validation using genetic controls is essential.

How can cross-reactivity be assessed for At1g46840 Antibody?

Cross-reactivity assessment is critical given the issues highlighted in search result :

• In silico analysis: Compare the immunogen sequence of At1g46840 with other F-box proteins in Arabidopsis to identify potential cross-reactive proteins

• Gene knockout approach: Test the antibody in Arabidopsis lines with At1g46840 knocked out; any remaining signal indicates cross-reactivity

• Heterologous expression systems: Express At1g46840 and related F-box proteins individually in a non-plant system (e.g., E. coli or yeast) and test antibody reactivity

• Peptide competition: Test whether peptides from related F-box proteins can compete with At1g46840 for antibody binding

The study in search result demonstrated that commercial antibodies for AT1R showed identical staining patterns in wild-type and AT1R knockout mice, revealing complete lack of specificity. This underscores the importance of rigorous validation for At1g46840 Antibody.

How do post-translational modifications of At1g46840 affect antibody recognition?

While specific information about post-translational modifications (PTMs) of At1g46840 is not provided in the search results, F-box proteins commonly undergo PTMs that could impact antibody recognition:

• Phosphorylation: May alter protein conformation and epitope accessibility

• Ubiquitination: Given the role of F-box proteins in ubiquitin-mediated processes, At1g46840 itself might be ubiquitinated, potentially masking epitopes

• Glycosylation: Could create steric hindrance for antibody binding

• Proteolytic processing: May generate fragments with different molecular weights, resulting in multiple bands

Researchers should consider these possibilities when interpreting Western blot results showing unexpected band patterns. To detect specific PTM forms, specialized antibodies targeting the modified epitopes would be required.

What are the comparative advantages of immunological versus genetic approaches for At1g46840 detection?

Given the documented issues with antibody specificity , a multi-method approach combining immunological detection with genetic approaches is recommended for comprehensive study of At1g46840.

How can flow cytometry be optimized for At1g46840 Antibody applications?

While specific flow cytometry protocols for At1g46840 Antibody are not described in the search results, general principles from result can be adapted:

• Cell preparation: Prepare single-cell suspensions from Arabidopsis protoplasts by enzymatic digestion of plant tissues

• Fixation and permeabilization: Required for intracellular proteins; use 4% paraformaldehyde followed by detergent (e.g., 0.1% Triton X-100)

• Antibody optimization:

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

  • Include appropriate isotype control (rabbit IgG)

  • Use fluorophore-conjugated secondary antibody with brightness appropriate for expected expression level

• Controls:

  • Negative control: Arabidopsis mutants lacking At1g46840

  • Positive control: Arabidopsis lines overexpressing At1g46840

  • Secondary-only control: To assess non-specific binding

• Analysis considerations:

  • Gate on intact single cells

  • Compensate for autofluorescence common in plant cells

  • Compare mean fluorescence intensity across samples

Flow cytometry allows quantitative assessment of At1g46840 expression at the single-cell level, enabling detection of cell-to-cell variation not apparent in bulk assays like Western blotting .

How might cryoEM approaches improve At1g46840 antibody characterization?

Based on search result , emerging cryoEM techniques offer promising approaches for antibody characterization:

• Structural characterization: CryoEM can determine the structure of antibody-antigen complexes, revealing precise epitope binding sites on At1g46840

• Polyclonal antibody analysis: CryoEM combined with next-generation sequencing can identify individual antibody sequences within polyclonal mixtures, potentially allowing selection of the most specific antibodies

• Epitope mapping: CryoEM structural data can identify specific binding regions, informing the design of more specific antibodies or enabling selection of non-competing antibody pairs for sandwich assays

• Heterogeneity analysis: CryoEM can assess the structural diversity within polyclonal antibody preparations, helping researchers understand variability in experimental results

This approach represents a significant advancement over traditional antibody characterization methods, potentially addressing the specificity issues highlighted in search result .