At3g61590 Antibody

What is At3g61590 and why is it significant in plant research?

At3g61590 is a gene in Arabidopsis thaliana that encodes a protein involved in important protein-protein interactions, notably with ASK18 (AT5G49000). Research indicates that At3g61590 may play a role in arsenate/arsenite signaling pathways and could be part of the plant's response to arsenic exposure . Understanding this gene's function provides insights into plant stress responses and adaptation mechanisms to toxic elements.

How do At3g61590 antibodies facilitate protein interaction studies?

At3g61590 antibodies serve as essential tools for detecting and isolating the target protein in various experimental contexts. When studying protein interactions, such as the documented interaction between At3g61590 and ASK18, these antibodies can be used in co-immunoprecipitation assays to pull down protein complexes from plant extracts . The antibodies allow researchers to verify physical associations between proteins that may form functional complexes involved in signaling cascades or transcriptional regulation.

What are the key considerations when selecting an At3g61590 antibody for immunolocalization studies?

When selecting an At3g61590 antibody for immunolocalization:

• Specificity: Ensure the antibody recognizes only At3g61590 without cross-reactivity to related proteins

• Sensitivity: The antibody should detect physiological levels of the protein

• Compatibility: Verify compatibility with your fixation protocols (paraformaldehyde vs. glutaraldehyde)

• Type: Consider whether polyclonal or monoclonal antibodies better suit your research needs

• Validation: Look for antibodies validated specifically in Arabidopsis thaliana tissues

Similar to approaches used with other plant proteins like ATG6, optimal immunolocalization requires testing different antibody dilutions and fixation conditions to maximize signal-to-noise ratio .

How can protein-specific nanobodies be developed for At3g61590 research?

Developing nanobodies against At3g61590 would follow a similar approach to the one used for other challenging proteins like PRL-3:

• Immunization: Alpacas or llamas are immunized with purified At3g61590 recombinant protein

• B-cell isolation: B-cells are harvested from the immunized animals

• Library construction: VHH (variable domain of heavy chain antibodies) genes are amplified and cloned

• Selection: Phage display techniques identify nanobodies with high affinity and specificity

• Validation: Selected nanobodies are tested for their ability to specifically recognize At3g61590 in plant extracts

Nanobodies offer advantages over conventional antibodies, including smaller size (~15 kDa vs ~150 kDa), improved tissue penetration, and the ability to recognize unique epitopes . Their single-domain nature makes them particularly useful for studying proteins like At3g61590 in their native cellular environment.

What techniques can resolve contradictory results when using At3g61590 antibodies in co-localization studies?

When faced with contradictory co-localization results:

• Antibody validation: Perform Western blot analysis using wild-type and knockout/knockdown plants to confirm specificity

• Multiple antibody approach: Use antibodies targeting different epitopes of At3g61590

• Tagged protein verification: Compare results with fluorescently tagged At3g61590 (similar to ATG6-mCherry approaches)

• Super-resolution microscopy: Employ techniques like STED or PALM for higher resolution imaging

• Proximity ligation assays: Confirm protein interactions with higher sensitivity than traditional co-localization

• FRET analysis: Measure actual protein-protein proximity rather than optical co-localization

Researchers should also consider fixation artifacts and implement appropriate controls using known interaction partners like ASK18 .

How can researchers quantitatively analyze At3g61590 protein levels under arsenite stress conditions?

For quantitative analysis of At3g61590 protein levels under arsenite stress:

When exposing Arabidopsis to arsenite stress (60 μM As(III)), researchers should collect samples at multiple timepoints (0, 3, 6, and 24 hours) to capture dynamic changes in protein levels, similar to the approach used when studying arsenite's effects on PHT1;1 expression .

What immunoprecipitation protocols are most effective for isolating At3g61590 complexes?

For optimal immunoprecipitation of At3g61590 complexes:

• Sample preparation: Harvest plant tissue (seedlings exposed to relevant conditions) and grind in liquid nitrogen

• Buffer selection: Use a non-denaturing lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) supplemented with protease inhibitors

• Pre-clearing: Incubate lysate with protein A/G beads to reduce non-specific binding

• Antibody binding: Incubate cleared lysate with At3g61590 antibody (2-5 μg per mg of total protein)

• Complex capture: Add protein A/G beads and incubate with gentle rotation (4°C, 2-4 hours)

• Washing: Perform stringent washes (at least 5) with decreasing salt concentrations

• Elution: Use gentle elution with peptide competition or more harsh conditions with SDS

The success of this procedure can be verified by subsequent mass spectrometry analysis to identify interacting partners beyond the known ASK18 interaction .

How does phosphorylation status affect At3g61590 antibody recognition?

Post-translational modifications, particularly phosphorylation, can significantly impact antibody recognition of At3g61590:

• Epitope masking: Phosphorylation near antibody binding sites may alter accessibility

• Conformational changes: Phosphorylation can induce structural changes affecting distant epitopes

• Antibody selection: Phospho-specific antibodies recognize only the modified form, while other antibodies may show reduced binding to phosphorylated protein

When studying At3g61590 in arsenite response pathways, researchers should consider using both phospho-specific and total protein antibodies, as arsenite exposure may trigger signaling cascades involving protein phosphorylation . Validating antibody recognition under different conditions using phosphatase treatments can help resolve inconsistent detection issues.

What methods best characterize the interaction between At3g61590 and ASK18?

To characterize the At3g61590-ASK18 interaction:

• Yeast Two-Hybrid (Y2H): Confirms direct interaction as demonstrated in previous research

• Bimolecular Fluorescence Complementation (BiFC): Visualizes interaction in plant cells

• FRET-FLIM: Measures interaction distance with high precision

• Surface Plasmon Resonance (SPR): Determines binding kinetics and affinity constants

• Pull-down assays: Verifies interaction using recombinant proteins

• Co-immunoprecipitation: Confirms interaction in native plant extracts

When implementing these methods, researchers should include positive controls (known interacting proteins) and negative controls (non-interacting proteins) to validate their findings. The Y2H approach has already successfully demonstrated the At3g61590-ASK18 interaction , but complementary methods provide stronger evidence and additional structural/functional insights.

How can researchers investigate the role of At3g61590 in arsenite signaling using antibody-based approaches?

To investigate At3g61590's role in arsenite signaling:

• Chromatin Immunoprecipitation (ChIP): Identify DNA binding sites if At3g61590 has transcription factor activity

• Proximity-dependent Biotin Identification (BioID): Discover proximal proteins in arsenite response

• Immunofluorescence time-course: Track protein localization changes during arsenite exposure

• Phospho-specific western blotting: Monitor phosphorylation changes in response to arsenite

• Protein stability assays: Measure At3g61590 degradation rates with cycloheximide chase

Researchers should design experiments with appropriate arsenite concentrations (30-60 μM) and timepoints (0-24 hours) that align with previous studies investigating arsenite signaling components . Antibody-based detection can reveal how At3g61590 responds to arsenite similar to how WRKY6 expression changes have been documented.

What epitope mapping strategies help optimize At3g61590 antibody design?

Effective epitope mapping strategies include:

• Peptide array analysis: Synthesize overlapping peptides spanning At3g61590 sequence to identify reactive regions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Identify surface-exposed regions suitable for antibody recognition

• Computational prediction: Use algorithms to identify antigenic determinants based on hydrophilicity, flexibility, and accessibility

• Structural analysis: If crystal structure is available, target accessible surface loops

• Evolutionary conservation analysis: Target less conserved regions for specificity

When designing antibodies against At3g61590, researchers should avoid regions that might be involved in the interaction with ASK18 if they want antibodies that can detect the protein regardless of its interaction status.

How can researchers troubleshoot non-specific binding issues with At3g61590 antibodies?

To troubleshoot non-specific binding:

• Antibody validation: Test antibodies in tissues from knockout/knockdown plants

• Blocking optimization: Evaluate different blocking agents (BSA, non-fat milk, casein) at various concentrations

• Stringency adjustment: Modify salt concentration and detergent levels in wash buffers

• Pre-adsorption: Incubate antibody with recombinant protein or peptide from non-target but cross-reactive proteins

• Antibody dilution optimization: Test serial dilutions to find the optimal signal-to-noise ratio

• Secondary antibody selection: Choose secondary antibodies with minimal cross-reactivity to plant proteins

Non-specific binding can also be addressed through proper experimental design, including parallel experiments with isotype control antibodies and competitive blocking with the immunizing peptide.

What considerations are important when designing CRISPR/Cas9 epitope tagging strategies for At3g61590 antibody validation?

When designing CRISPR/Cas9 epitope tagging of At3g61590:

• Tag position: Consider N-terminal versus C-terminal tagging based on protein function

• Tag selection: Choose between common epitopes (FLAG, HA, Myc) or fluorescent proteins (GFP, mCherry)

• Linker design: Incorporate flexible linkers (GGGGS)n to minimize functional interference

• Guide RNA selection: Target sequences near termini with minimal off-target effects

• Validation strategy: Plan for both tag antibody detection and native At3g61590 antibody comparison

• Functional complementation: Verify tagged protein retains interaction with ASK18

Creating epitope-tagged endogenous At3g61590 through CRISPR/Cas9 helps validate antibodies against the native protein and provides a powerful tool for studying protein dynamics in planta, similar to approaches used with ATG6-mCherry fusion proteins .