At1g59620 Antibody

What is At1g59620 and what role does it play in plant immunity?

At1g59620, also known as CW9, is a gene in Arabidopsis thaliana that encodes a disease resistance protein belonging to the CC-NBS-LRR (coiled-coil nucleotide-binding site leucine-rich repeat) class family . This protein is part of the plant's innate immune system that recognizes pathogen effectors and triggers defense responses. NBS-LRR proteins function as immune receptors that detect pathogen invasion and initiate signaling cascades leading to disease resistance. The CC domain is particularly important for signaling and protein-protein interactions during the immune response. Studies of CC-NBS-LRR proteins like At1g59620 are crucial for understanding the molecular basis of plant disease resistance and developing strategies to enhance crop protection against pathogens .

What types of At1g59620 antibodies are commercially available for research?

Several types of At1g59620 antibodies are available for research purposes, targeting different regions of the protein:

• Mouse monoclonal antibody combinations targeting:

  • N-terminus region (X-Q19HX1-N)

  • C-terminus region (X-Q19HX1-C)

  • Middle region (X-Q19HX1-M)

• Rabbit polyclonal antibody (CSB-PA864811XA01DOA) raised against recombinant Arabidopsis thaliana At1g59620 protein

Each antibody type has been validated for specific applications such as ELISA and Western blotting, with the monoclonal combinations showing ELISA titers of approximately 10,000, corresponding to detection sensitivity of about 1 ng of target protein on Western blots .

How do I properly store and handle At1g59620 antibodies to maintain their activity?

Proper storage and handling of At1g59620 antibodies are critical for maintaining their specificity and sensitivity over time. Store antibodies at -20°C or -80°C immediately upon receipt . Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of antibody activity . When working with the antibodies:

• Aliquot the stock solution into smaller volumes before freezing to minimize freeze-thaw cycles

• When thawing, allow the antibody to slowly warm to room temperature by placing it on ice

• Use sterile techniques when handling to prevent contamination

• If diluting for use, prepare working solutions in fresh buffer containing a carrier protein (such as BSA) and preservative

• Store working dilutions at 4°C for short-term use (1-2 weeks) or re-freeze in aliquots for long-term storage

• Follow the manufacturer's recommendations for storage buffer conditions (typically containing 50% glycerol and 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as a preservative)

How does the function of At1g59620 relate to other NLR immune receptors in the Arabidopsis genome?

At1g59620 functions within the complex network of NLR immune receptors that collectively provide resistance against diverse pathogens. Recent research indicates that NLR gene expression is carefully balanced within plant cells, with some NLRs being positively regulated while others are suppressed . The PHD-finger protein EDM2 has been shown to play a critical role in this balancing act, acting as both an activator and suppressor of different NLR genes .

In the context of this regulatory network, At1g59620 may be subject to similar fine-tuning mechanisms that control its expression levels. The balance between different NLR proteins appears to be critical for optimal immune function while minimizing fitness costs to the plant. The specific regulatory elements controlling At1g59620 expression likely include epigenetic marks such as H3K9me2, which has been shown to influence NLR gene expression through mechanisms involving alternative polyadenylation .

What epigenetic factors influence At1g59620 expression and how can antibodies help investigate these mechanisms?

Epigenetic regulation plays a significant role in controlling NLR gene expression, including At1g59620. Key epigenetic factors influencing this regulation include:

• Histone modifications, particularly H3K9me2 (dimethylation of lysine 9 on histone H3), which is associated with transcriptional repression

• DNA methylation patterns, especially in CHG contexts, which are linked to H3K9me2 via cytosine methyltransferases CMT2 and CMT3

• Alternative polyadenylation mechanisms that can produce either full-length functional transcripts or truncated non-coding variants

Antibodies against At1g59620 can be powerful tools for investigating these epigenetic regulatory mechanisms through techniques such as:

• Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) to identify regions where the protein interacts with specific chromatin marks

• RNA Immunoprecipitation (RIP) to detect associations between At1g59620 protein and specific RNA transcripts

• Immunoprecipitation followed by mass spectrometry (IP-MS) to identify protein interaction partners involved in epigenetic regulation

These approaches can reveal how At1g59620 expression and function are integrated into broader epigenetic regulatory networks that control plant immunity .

How do post-translational modifications affect At1g59620 function and antibody recognition?

Post-translational modifications (PTMs) of At1g59620 likely play crucial roles in regulating its function as an immune receptor. For CC-NBS-LRR proteins, known PTMs include:

• Phosphorylation: Often regulates protein activation state and signaling capacity

• Ubiquitination: Can influence protein stability, localization, and activity

• SUMOylation: May affect protein-protein interactions and subcellular localization

• Glycosylation: Can impact protein folding, stability, and recognition properties

These modifications can significantly influence antibody recognition of the target protein. Researchers should consider the following when using At1g59620 antibodies:

• Antibodies raised against recombinant proteins (like CSB-PA864811XA01DOA ) may not recognize certain PTMs present in the native protein

• Some antibodies may preferentially bind to modified or unmodified forms of the protein

• PTMs might mask epitopes, reducing antibody binding efficiency in certain cellular contexts

• Using multiple antibodies targeting different regions (N-terminus, C-terminus, and middle region ) can help detect the protein regardless of some PTM states

When interpreting experimental results, researchers should consider how cellular conditions that alter PTM status (such as pathogen infection or abiotic stress) might affect antibody detection efficiency and potentially lead to false negative results.

What are the optimal conditions for Western blot detection of At1g59620?

For optimal Western blot detection of At1g59620, follow these methodological guidelines:

Sample Preparation:

• Extract total protein from plant tissue using a buffer containing protease inhibitors to prevent degradation

• Include phosphatase inhibitors if investigating phosphorylation states

• Use fresh tissue whenever possible; if using frozen tissue, minimize freeze-thaw cycles

SDS-PAGE Conditions:

• Use 8-10% polyacrylamide gels to effectively resolve the 343 amino acid At1g59620 protein

• Load 20-50 μg of total protein per lane

• Include positive controls (recombinant At1g59620) and negative controls (extracts from knockout lines)

Transfer and Blocking:

• Transfer proteins to a PVDF membrane (recommended over nitrocellulose for plant proteins)

• Block with 5% non-fat dry milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature

Antibody Incubation:

• Primary antibody dilution: 1:1000 to 1:2000 for monoclonal antibodies , 1:500 to 1:1000 for polyclonal antibodies

• Incubate overnight at 4°C with gentle agitation

• Wash thoroughly (4 × 5 minutes) with TBST

• Secondary antibody (anti-mouse for monoclonal , anti-rabbit for polyclonal ): 1:5000 to 1:10000 dilution

• Incubate for 1 hour at room temperature

Detection:

• Use enhanced chemiluminescence (ECL) for sensitive detection

• Exposure time may vary depending on protein abundance (typically 30 seconds to 5 minutes)

• For quantitative analysis, use a digital imaging system rather than film

These conditions should be optimized based on specific experimental requirements and antibody characteristics.

How can I validate the specificity of At1g59620 antibodies in my experimental system?

Validating antibody specificity is crucial for ensuring reliable experimental results. For At1g59620 antibodies, consider implementing the following validation strategies:

Genetic Controls:

• Test the antibody on samples from At1g59620 knockout or knockdown lines (expected: no signal or reduced signal)

• Use overexpression lines as positive controls (expected: enhanced signal)

Molecular Weight Verification:

• Confirm that the detected band corresponds to the expected molecular weight of At1g59620 (approximately 38 kDa for the 343 amino acid protein )

• Be aware that post-translational modifications may alter the apparent molecular weight

Cross-Reactivity Assessment:

• Test on related Arabidopsis CC-NBS-LRR proteins to assess potential cross-reactivity

• If working with other plant species, evaluate homologs with high sequence similarity

Blocking Peptide Competition:

• Pre-incubate the antibody with the immunizing peptide or recombinant protein

• This should abolish or significantly reduce the specific signal

Multiple Antibody Comparison:

• Compare results using antibodies targeting different regions of At1g59620 (N-terminus, C-terminus, and middle region )

• Consistent detection with multiple antibodies increases confidence in specificity

Mass Spectrometry Validation:

• Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the pulled-down protein

• This approach provides the highest level of validation for antibody specificity

Document all validation steps thoroughly as they will strengthen the credibility of your research findings.

What immunofluorescence protocols are recommended for studying At1g59620 subcellular localization?

For immunofluorescence studies of At1g59620 subcellular localization in Arabidopsis, the following protocol is recommended:

Sample Preparation:

• Fix plant tissues (protoplasts or thin tissue sections) in 4% paraformaldehyde in PBS (pH 7.4) for 30 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 in PBS for 15 minutes

• Block with 3% BSA in PBS for 1 hour

Antibody Incubation:

• Primary antibody: Use At1g59620-specific antibodies at 1:100 to 1:200 dilution

• Incubate overnight at 4°C in a humid chamber

• Wash 3 × 10 minutes with PBS

• Secondary antibody: Use fluorophore-conjugated secondary antibodies (Alexa Fluor series recommended) at 1:500 dilution

• Incubate for 2 hours at room temperature

• Wash 3 × 10 minutes with PBS

Nuclear Counterstaining:

• Stain with DAPI (4′,6-diamidino-2-phenylindole) at 1 μg/mL for 10 minutes

• Wash briefly with PBS

Mounting and Imaging:

• Mount in an anti-fade medium

• Image using confocal microscopy with appropriate laser settings for the selected fluorophores

Controls to Include:

• Primary antibody omission control

• Secondary antibody only control

• Unrelated primary antibody control

• Tissues from At1g59620 knockout plants

This protocol can be adapted for co-localization studies by including additional primary antibodies against known subcellular markers (e.g., ER, Golgi, plasma membrane, or nuclear markers) and using secondary antibodies with non-overlapping emission spectra.

Why might I observe inconsistent At1g59620 detection in Western blots?

Inconsistent detection of At1g59620 in Western blots can stem from multiple factors. Here's a troubleshooting guide to address common issues:

It's worth noting that At1g59620 expression, like other CC-NBS-LRR proteins, may be influenced by pathogen exposure or abiotic stress conditions, which could explain some variability in detection between experiments .

How can I minimize cross-reactivity when studying At1g59620 in plant tissues?

Cross-reactivity can be a significant challenge when studying CC-NBS-LRR proteins like At1g59620 due to sequence similarities within this protein family. To minimize cross-reactivity:

• Antibody Selection:

  • Choose antibodies raised against unique regions of At1g59620 rather than conserved domains

  • Consider using the X-Q19HX1-N antibody targeting the N-terminus, which may have lower cross-reactivity than antibodies targeting the more conserved NBS domain

• Protocol Optimization:

  • Increase antibody dilution (1:2000 or higher) to reduce non-specific binding

  • Use more stringent washing conditions (higher salt concentration or addition of 0.1% SDS)

  • Extend blocking time to 2 hours or overnight with 5% BSA instead of milk

  • Pre-absorb antibodies with extracts from At1g59620 knockout plants

• Genetic Controls:

  • Always include samples from At1g59620 mutant or knockout lines as negative controls

  • Use transgenic lines expressing tagged versions of At1g59620 as positive controls

• Buffer Additives:

  • Add 0.1-0.5% non-ionic detergents (e.g., Tween-20) to reduce hydrophobic interactions

  • Include 150-300 mM NaCl to minimize electrostatic interactions

  • Consider adding 1-5% polyethylene glycol to reduce non-specific binding

• Alternative Approaches:

  • Use epitope-tagged At1g59620 expressed under its native promoter when possible

  • Consider immunoprecipitation followed by mass spectrometry to confirm antibody specificity

These approaches can significantly improve the specificity of At1g59620 detection in complex plant samples.

What factors might affect At1g59620 protein levels during pathogen infection studies?

Several factors can influence At1g59620 protein levels during pathogen infection studies, complicating the interpretation of immunoblot or immunolocalization results:

• Transcriptional Regulation:

  • NLR gene expression is dynamically regulated during immune responses

  • EDM2 and similar regulators may alter At1g59620 transcription in response to pathogen recognition

  • Alternative polyadenylation mechanisms can generate different transcript variants with varying stability and translation efficiency

• Post-transcriptional Control:

  • microRNA-mediated silencing may be activated during infection

  • RNA-binding proteins might affect transcript stability or translation efficiency

  • Alternative splicing patterns could change in response to pathogen signals

• Protein Stability Regulation:

  • Ubiquitin-proteasome pathway activation during infection may alter protein turnover rates

  • Pathogen effectors might directly target At1g59620 for degradation

  • Protein stabilization through interaction with defense-related chaperones

• Technical Considerations:

  • Timing of sample collection is critical (protein levels may peak and then decline)

  • Different pathogens may elicit different expression patterns

  • Plant age and developmental stage affect baseline expression

  • Environmental conditions (light, temperature, humidity) can influence both basal expression and pathogen-induced changes

• Experimental Design Recommendations:

  • Include a detailed time-course analysis (0, 3, 6, 12, 24, 48, and 72 hours post-infection)

  • Compare compatible and incompatible pathogen interactions

  • Use both mock-inoculated and untreated controls

  • Consider employing protein synthesis inhibitors to distinguish between new synthesis and protein stability effects

Understanding these factors is essential for correctly interpreting changes in At1g59620 protein levels observed during infection studies.

What are the future research directions for At1g59620 antibody applications?

The study of At1g59620 using antibody-based approaches has significant potential for advancing our understanding of plant immunity. Future research directions include:

• Systems Biology Integration:

  • Using At1g59620 antibodies in multiplexed protein detection systems to understand its dynamics within broader immune networks

  • Combining antibody-based detection with transcriptomics and metabolomics for a comprehensive view of defense responses

• Structural and Functional Studies:

  • Employing antibodies as tools for protein purification to enable structural studies (X-ray crystallography or cryo-EM)

  • Using antibody-mediated protein depletion to assess functional roles in vivo

  • Developing antibodies against specific post-translationally modified forms of At1g59620

• Crop Improvement Applications:

  • Translating knowledge from Arabidopsis to develop antibodies against orthologous proteins in crop species

  • Using antibodies to screen for desired protein expression levels in breeding programs

  • Monitoring NLR protein accumulation in response to various agricultural treatments

• Technical Advancements:

  • Development of single-domain antibodies (nanobodies) against At1g59620 for in vivo imaging

  • Creating biosensors using At1g59620 antibodies to monitor plant health in real-time

  • Establishing proximity-labeling techniques with At1g59620 antibodies to identify transient protein interactions during immune signaling

• Epigenetic Regulation:

  • Further exploring the connections between chromatin modifications and At1g59620 expression using ChIP-seq approaches with both histone mark antibodies and At1g59620 antibodies

These research directions will benefit from continued development and characterization of highly specific antibodies targeting different epitopes and modified forms of At1g59620.

How do findings from At1g59620 research contribute to broader understanding of plant immunity?

Research on At1g59620 and related CC-NBS-LRR proteins has made significant contributions to our understanding of plant immunity in several key areas:

• Immune Receptor Diversity and Evolution:

  • Studies of At1g59620 contribute to our understanding of how NLR gene families diversify and evolve to recognize new pathogen threats

  • The specific structural features of At1g59620 provide insights into the molecular basis of pathogen recognition

• Balanced Regulation of Defense Responses:

  • At1g59620 research supports the emerging concept that plants maintain careful balance in NLR expression to optimize defense while minimizing fitness costs

  • The involvement of epigenetic regulators like EDM2 in controlling NLR expression demonstrates sophisticated regulatory mechanisms for immune homeostasis

• Integration of Epigenetic and Transcriptional Control:

  • Studies linking H3K9me2 marks to alternative polyadenylation in NLR genes reveal novel mechanisms for gene expression control in plants

  • This research helps explain how plants can rapidly modulate defense gene expression during pathogen challenges

• Translational Implications:

  • Understanding At1g59620 regulation and function provides targets for genetic improvement of disease resistance in crops

  • Knowledge of NLR protein dynamics informs strategies for deploying durable resistance in agricultural systems

• Methodological Advances:

  • Development and characterization of At1g59620 antibodies establishes protocols that benefit research on other plant immune receptors

  • Validation strategies for these antibodies contribute to improved standards in plant molecular biology research