At1g61190 Antibody

What is the At1g61190 protein and why is it significant for plant immunity research?

At1g61190 (also known as F11P17.9; F11P17_9) is classified as a probable disease resistance protein in Arabidopsis thaliana containing LRR and NB-ARC domains, which are typical features of plant resistance (R) proteins . These domains are crucial for pathogen recognition and subsequent immune response activation in plants.

The significance of At1g61190 lies in its potential role in plant innate immunity, particularly in disease resistance signaling pathways. By studying this protein through antibody-based approaches, researchers can better understand plant-pathogen interactions and potentially develop strategies to enhance crop resistance to various pathogens.

How should I select the appropriate At1g61190 antibody for my research needs?

When selecting an At1g61190 antibody, consider these methodological criteria:

• Antibody type: Determine whether polyclonal (recognizing multiple epitopes) or monoclonal (specific to one epitope) antibodies better suit your experimental needs. Polyclonal antibodies often provide higher sensitivity but potentially lower specificity .

• Validation status: Always use antibodies validated for your specific application. An antibody validated for Western blotting may not perform adequately in immunohistochemistry or flow cytometry .

• Host species: Consider the host species (rabbit for commercial At1g61190 antibodies) in relation to your secondary detection system and to avoid cross-reactivity in your experimental setup .

• Application compatibility: Confirm the antibody has been validated for your intended application (ELISA, Western blot, etc.) .

• Epitope information: Request information about the epitope region to understand potential cross-reactivity with related proteins and to predict accessibility in different experimental conditions .

What controls should I include when using At1g61190 antibodies in experiments?

Proper experimental controls are critical when working with At1g61190 antibodies:

• Negative control tissue: Include samples from tissues where At1g61190 is not expressed or use At1g61190 knockout/knockdown plants if available .

• Isotype control: Use a matched isotype control (same immunoglobulin class as the primary antibody) with no known specificity to assess background staining .

• Secondary antibody control: Perform a control with only the labeled secondary antibody to detect non-specific binding .

• Blocking validation: Test different blocking agents (typically 5-10% normal serum from the same host species as the secondary antibody) to minimize background .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm specificity, as signal elimination indicates specific binding .

What are the recommended protocols for using At1g61190 antibodies in Western blotting?

For optimal Western blot results with At1g61190 antibodies:

• Sample preparation: Extract total protein from Arabidopsis tissues using a buffer containing 20 mM Tris pH 7.5, 5 mM MgCl₂, 2.5 mM DTT, 300 mM NaCl, and 0.1% NP-40 with protease inhibitors .

• Protein separation: Use 6-10% SDS-PAGE gels for optimal resolution of At1g61190 (expected MW approximately 100-130 kDa based on similar resistance proteins) .

• Transfer conditions: Transfer to nitrocellulose or PVDF membranes (0.45 μm pore size) using standard wet transfer protocols .

• Blocking: Block with 5% non-fat milk in TBS-T (0.1% Tween-20) for 1 hour at room temperature .

• Antibody dilution: Start with a 1:1000 to 1:5000 dilution of the primary antibody in blocking buffer. Incubate overnight at 4°C for optimal results .

• Washing: Wash 3-5 times with TBS-T before and after secondary antibody incubation .

• Detection: Use HRP-conjugated anti-rabbit secondary antibody (typically 1:10,000 dilution) and ECL detection reagents .

How can I optimize immunoprecipitation experiments using At1g61190 antibodies?

For successful immunoprecipitation with At1g61190 antibodies:

• Cell lysis optimization: Use a gentle lysis buffer (e.g., 20 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40 or Triton X-100, 1 mM EDTA) supplemented with protease inhibitors to preserve protein-protein interactions .

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding .

• Antibody binding: Incubate cleared lysates with 2-5 μg of At1g61190 antibody for 2-4 hours at 4°C .

• Bead incubation: Add protein A/G beads and incubate for another 1-2 hours at 4°C .

• Washing stringency: Perform at least 3-5 washes with decreasing salt concentrations to maintain specific interactions while removing background .

• Elution method: Consider native elution with competitive peptides if downstream functional assays are planned, or use standard SDS elution for mass spectrometry analysis .

• Validation: Confirm successful immunoprecipitation by Western blot using the same or different At1g61190 antibody that recognizes a separate epitope .

How should I validate the specificity of an At1g61190 antibody?

A comprehensive validation strategy for At1g61190 antibodies includes:

• Genetic controls: Test the antibody on tissues from At1g61190 knockout/knockdown plants, which should show significantly reduced or absent signal .

• Heterologous expression system: Express At1g61190 in a system that doesn't normally express this protein (e.g., bacterial or mammalian cells) and confirm antibody detection .

• Immunoblotting pattern: Verify that the antibody detects a band of the expected molecular weight (~100-130 kDa based on similar R proteins) .

• Cross-reactivity assessment: Test against related LRR-NB-ARC proteins to ensure specificity within the protein family .

• Multiple detection methods: Validate across different techniques (Western blot, immunofluorescence, ELISA) to confirm consistent detection .

• Mass spectrometry confirmation: Following immunoprecipitation, use mass spectrometry to confirm the identity of the precipitated protein .

What are the common pitfalls in At1g61190 antibody validation and how can I avoid them?

Common validation pitfalls and their solutions include:

• Overlooking tissue-specific expression: At1g61190 expression may vary across tissues and conditions. Validate using tissues with confirmed expression and include appropriate controls .

• Inadequate blocking: Insufficient blocking can lead to high background. Optimize blocking conditions using 5-10% normal serum from the secondary antibody host species .

• Cross-reactivity with related proteins: The LRR-NB-ARC protein family shares structural similarities. Validate specificity against closely related family members and consider epitope mapping .

• Application mismatch: An antibody validated for Western blot may fail in immunofluorescence. Validate specifically for each application rather than assuming cross-application performance .

• Batch-to-batch variation: Different lots of the same antibody may perform differently. Request validation data for the specific lot and maintain internal validation controls .

• Poor experimental design: Insufficient controls can lead to misinterpretation. Always include positive and negative controls, isotype controls, and secondary-only controls .

How can I use At1g61190 antibodies for studying protein-protein interactions in plant immunity?

For investigating At1g61190 protein interactions:

• Co-immunoprecipitation (Co-IP): Use At1g61190 antibodies to pull down the protein complex, followed by Western blotting with antibodies against suspected interaction partners or mass spectrometry for unbiased discovery .

• Proximity ligation assay (PLA): Combine At1g61190 antibodies with antibodies against potential interaction partners to visualize and quantify protein proximity (<40 nm) in situ .

• Chromatin immunoprecipitation (ChIP): If At1g61190 is suspected to interact with DNA or chromatin-associated proteins, ChIP can identify genomic binding regions .

• Immunofluorescence co-localization: Use dual-labeling immunofluorescence to examine co-localization of At1g61190 with other proteins during pathogen response .

• FRET-based approaches: Combine antibody detection with fluorescence resonance energy transfer to examine dynamic interactions in living cells .

A strategic approach would include:

• Initial Co-IP screening for potential interaction partners

• Validation using reciprocal Co-IP experiments

• Confirmation with orthogonal methods like PLA or FRET

• Functional validation through genetic manipulation of identified partners

How can I use At1g61190 antibodies to study protein modifications during plant immune responses?

To investigate At1g61190 modifications during immune responses:

• Phosphorylation analysis: Use phospho-specific antibodies alongside the general At1g61190 antibody to detect activation-dependent phosphorylation events .

• Ubiquitination detection: Perform immunoprecipitation with At1g61190 antibodies followed by Western blotting with anti-ubiquitin antibodies to detect regulation through the ubiquitin-proteasome system .

• SUMOylation assessment: Similar to ubiquitination analysis, but using anti-SUMO antibodies to detect this modification which can alter protein function or localization .

• Time-course experiments: Examine changes in At1g61190 modifications at different timepoints after pathogen exposure to map the temporal dynamics of activation .

• Subcellular fractionation: Combine with immunoblotting to detect location-specific modifications that may relate to protein translocation during immune signaling .

• Mass spectrometry analysis: Following immunoprecipitation, use mass spectrometry to identify and quantify specific modification sites and their changes during immune responses .

What approaches can I use to study At1g61190 localization dynamics during pathogen infection?

To track At1g61190 localization during infection:

• Immunofluorescence microscopy: Use At1g61190 antibodies with appropriate fixation protocols (typically 4% paraformaldehyde) to visualize protein distribution in tissue sections .

• Subcellular fractionation with immunoblotting: Separate cellular compartments (cytosol, membrane, nucleus, etc.) and detect At1g61190 distribution using Western blot .

• Time-resolved imaging: Perform immunofluorescence at different timepoints after pathogen challenge to track dynamic relocalization .

• Co-localization studies: Combine At1g61190 antibodies with markers for specific subcellular compartments to precisely determine localization .

• Super-resolution microscopy: Techniques like STED or STORM can provide nanoscale resolution of At1g61190 distribution when used with fluorescently labeled antibodies .

• Live-cell imaging: While not directly using antibodies, complementary approaches using fluorescently tagged At1g61190 can provide real-time localization data that can be validated with antibody-based fixed-cell approaches .

How can I troubleshoot weak or absent signal when using At1g61190 antibodies?

When facing detection issues with At1g61190 antibodies:

• Sample preparation optimization:

  • Ensure complete protein extraction with appropriate lysis buffers

  • Add protease inhibitors to prevent degradation

  • Avoid freeze-thaw cycles of samples

  • Consider native vs. denaturing conditions based on epitope accessibility

• Antibody optimization:

  • Perform titration experiments to determine optimal concentration

  • Try longer incubation times (overnight at 4°C rather than 1 hour at room temperature)

  • Test different blocking agents (BSA, milk, normal serum)

  • Consider alternative buffer formulations

• Detection enhancement:

  • Use signal amplification systems (biotinylated secondary antibodies with streptavidin-HRP)

  • Increase exposure time for Western blots

  • Try more sensitive substrates for HRP detection

  • Consider tyramide signal amplification for immunofluorescence

• Epitope retrieval methods:

  • For fixed tissues, test antigen retrieval methods (heat-induced, protease-based)

  • For Western blots, ensure complete denaturation of proteins

What strategies can address non-specific binding or high background with At1g61190 antibodies?

To reduce background and non-specific binding:

• Optimized blocking:

  • Increase blocking time and concentration (up to 5% BSA or 10% normal serum)

  • Use the blocking serum from the same species as the secondary antibody

  • Add 0.1-0.3% Triton X-100 or 0.05-0.1% Tween-20 to reduce hydrophobic interactions

• Antibody dilution and incubation:

  • Increase antibody dilution (perform titration experiments)

  • Perform all antibody incubations at 4°C to increase specificity

  • Pre-absorb the antibody with control tissue lysates to remove cross-reactive antibodies

• Washing optimization:

  • Increase washing steps (5-6 washes of 5-10 minutes each)

  • Use higher salt concentration in wash buffers (up to 500 mM NaCl)

  • Add 0.05-0.1% Tween-20 to wash buffers

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Reduce secondary antibody concentration

  • Include a secondary-only control to assess background

• Sample preparation:

  • For cell or tissue preparations, ensure >90% viability as dead cells can contribute to non-specific binding

  • Use appropriate fixation methods (too harsh fixation can increase background)

Can At1g61190 antibodies be used to develop disease-resistance markers in crop plants?

At1g61190 antibodies could potentially be used to develop disease-resistance markers:

• Cross-species reactivity assessment: First determine if the antibody recognizes homologous proteins in crop species of interest . Since At1g61190 has predicted functional homologs in other plant species, antibodies might show cross-reactivity with related R proteins.

• Validation in crop systems: Test antibody specificity in crop tissues using Western blot, comparing resistant and susceptible varieties .

• Marker development approach:

  • Analyze protein expression in different varieties following pathogen challenge

  • Correlate protein levels/modifications with disease resistance phenotypes

  • Develop quantitative assays (e.g., ELISA) that could be used in breeding programs

• Practical implementation considerations:

  • Standardize tissue sampling and processing protocols

  • Develop high-throughput assay formats suitable for screening large populations

  • Correlate antibody-based markers with genetic markers for integrated breeding approaches

• Limitations to consider:

  • Post-translational modifications might vary between species

  • Expression patterns may differ in crop plants compared to Arabidopsis

  • Multiple homologs might exist in polyploid crop species, complicating interpretation

What is the potential for using At1g61190 antibodies in studying systemic acquired resistance in plants?

At1g61190 antibodies could provide valuable insights into systemic acquired resistance (SAR) mechanisms:

• Monitoring protein dynamics during SAR:

  • Track At1g61190 protein levels in both local and systemic tissues after pathogen challenge

  • Examine changes in protein modification status during SAR establishment

  • Correlate protein behavior with expression of SAR marker genes

• Interaction studies during SAR signaling:

  • Use co-immunoprecipitation to identify differential protein interactions in SAR-induced versus naive plants

  • Examine interactions with known SAR components like NPR1, EDS1, or PAD4

  • Identify potential roles in long-distance signal perception or transduction

• Tissue-specific expression analysis:

  • Compare At1g61190 protein levels in vascular tissues versus mesophyll cells during SAR

  • Examine correlation with mobile SAR signals like pipecolic acid or azelaic acid

  • Study potential roles in signal generation versus signal perception

• Experimental design considerations:

  • Include time-course analyses spanning early (hours) to late (days) SAR responses

  • Compare responses in different plant organs (leaves, stems, roots)

  • Analyze protein behavior under different SAR-inducing conditions (biotic vs. chemical inducers)

By applying these methodological approaches, researchers can potentially uncover novel roles for At1g61190 in plant immunity and disease resistance, contributing to our fundamental understanding of plant-pathogen interactions and potentially informing crop improvement strategies.