At4g19050 Antibody

What is the At4g19050 protein and why is it significant for plant research?

At4g19050 is a locus in the Arabidopsis thaliana genome that encodes a putative disease resistance protein. According to NCBI annotation, it is classified as an NB-ARC domain-containing disease resistance protein with a molecular weight of approximately 136,827 Da . Initially, the protein was wrongly predicted to encode a protein of 1416 amino acids, but this prediction has been revised . At4g19050 belongs to the family of plant disease resistance (R) proteins that play crucial roles in plant immunity against pathogens. These proteins typically contain specific domains that recognize pathogen effectors and trigger defense responses, making them significant targets for understanding plant immunity mechanisms and developing disease-resistant crops.

What are the key characteristics of commercially available At4g19050 antibodies?

The primary type of At4g19050 antibody available for research is a rabbit polyclonal antibody generated against recombinant Arabidopsis thaliana At4g19050 protein . These antibodies are typically supplied in liquid form, preserved with 0.03% Proclin 300 and formulated in 50% Glycerol, 0.01M PBS at pH 7.4 . The polyclonal nature means these antibodies recognize multiple epitopes on the At4g19050 protein. The antibody is designed for research use only and is not intended for diagnostic procedures . When properly stored and handled, these antibodies maintain their reactivity for detecting At4g19050 in various experimental applications.

What applications have been validated for At4g19050 antibodies?

At4g19050 antibodies have been validated for several key research applications:

The antibody has been specifically confirmed to recognize Arabidopsis thaliana (Mouse-ear cress) proteins, with its predominant application being in Western blot and ELISA experimental designs .

What is the optimal protein extraction protocol for detecting At4g19050 in plant tissues?

For effective detection of At4g19050, researchers should employ extraction protocols that account for the protein's characteristics as a disease resistance protein:

• Tissue selection and preparation:

  • Choose appropriate tissues where disease resistance proteins are typically expressed (leaves, roots)

  • Flash-freeze harvested tissue in liquid nitrogen

  • Grind tissue to a fine powder while maintaining frozen state

• Extraction buffer composition:

  • Base buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA

  • Detergents: Add 1% Triton X-100 or 0.5% NP-40 to solubilize membrane-associated proteins

  • Protease inhibitors: Complete protease inhibitor cocktail to prevent degradation

  • Phosphatase inhibitors: Add if studying phosphorylation status (10 mM NaF, 1 mM Na3VO4)

  • Reducing agents: 1 mM DTT or 5 mM β-mercaptoethanol to maintain protein structure

• Extraction procedure:

  • Add cold extraction buffer to ground tissue (3-5 ml per gram)

  • Vortex vigorously and incubate with gentle rotation at 4°C for 30 minutes

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration using Bradford or BCA assay

This method promotes efficient extraction while preserving protein integrity, critical for subsequent immunodetection applications.

What are the recommended protocols for immunolocalization of At4g19050 in plant tissues?

For immunolocalization of At4g19050 in Arabidopsis tissues, researchers can adapt protocols similar to those used for PIN proteins, with modifications appropriate for disease resistance proteins:

• Sample fixation:

  • Fix seedlings or tissue sections with 3% (w/v) paraformaldehyde and 0.02% Triton X-100 in MTSB buffer (7.5 g/L PIPES, 0.95 g/L EGTA, 0.66 g/L MgSO4, 2.5 g/L KOH, pH 7.0) for 45 minutes

  • Wash three times with distilled water

• Tissue permeabilization:

  • Incubate samples in 0.15% (w/v) driselase and 0.15% (w/v) macerozyme in 10 mM MES (pH 5.3) for 30 minutes at 37°C

  • Wash 4 times with MTSB

  • Treat twice with 10% (v/v) DMSO, 3% (v/v) Nonidet-P40 in MTSB for 20 minutes each

  • Wash 5 times with MTSB

• Blocking and antibody incubation:

  • Block with 3% BSA in MTSB for 1 hour

  • Incubate with At4g19050 primary antibody (1:400) in 3% BSA for 4 hours at room temperature

  • Wash 7 times with MTSB

  • Apply fluorophore-conjugated secondary antibody (e.g., goat anti-rabbit A555-conjugated, 1:600) for 3 hours

  • Wash 10 times with MTSB

• Mounting and imaging:

  • Mount samples in Prolong Gold antifade reagent containing DAPI for nuclear visualization

  • Image using confocal microscopy with appropriate excitation wavelengths (543 nm for A555 and 730 nm two-photon for DAPI)

  • Detect emission at >575 nm for A555 and 435-485 nm for DAPI

This protocol enables precise localization of At4g19050 protein within plant tissues while maintaining cellular architecture.

How should Western blot protocols be optimized for detecting At4g19050?

The high molecular weight of At4g19050 (approximately 136 kDa) requires specific optimization of Western blot protocols:

• Gel preparation and electrophoresis:

  • Use lower percentage gels (7-8% acrylamide) to facilitate separation of high molecular weight proteins

  • Load adequate protein (30-50 μg per lane) to ensure detection

  • Run at lower voltage (80-100V) for better resolution of large proteins

• Transfer optimization:

  • Select PVDF membrane (0.45 μm pore size) rather than nitrocellulose

  • Perform wet transfer at 30V overnight at 4°C to ensure complete transfer of large proteins

  • Add SDS (0.1%) to transfer buffer to improve large protein transfer

  • Verify transfer efficiency with reversible staining before blocking

• Antibody incubation:

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

  • Incubate with At4g19050 primary antibody at 1:1000 dilution overnight at 4°C

  • Wash thoroughly (4-5 times, 5 minutes each) with TBST

  • Apply HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour

• Detection considerations:

  • Use enhanced chemiluminescence substrates for sensitive detection

  • Consider longer exposure times (1-5 minutes) for optimal visualization

  • Include molecular weight markers spanning 100-250 kDa range

These optimizations address the challenges associated with detecting large proteins like At4g19050 while maintaining specificity and sensitivity.

How can At4g19050 antibody be utilized to study protein-protein interactions in plant immunity?

The At4g19050 antibody can be leveraged to investigate protein-protein interactions critical for plant immunity through several methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Prepare protein extracts using mild lysis buffers (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA)

  • Pre-clear lysates with Protein A/G beads

  • Incubate cleared lysates with At4g19050 antibody (2-5 μg per mg protein) overnight at 4°C

  • Add Protein A beads and incubate for 2-3 hours

  • Wash beads with increasing stringency buffers

  • Elute bound proteins and analyze by Western blot or mass spectrometry

• Proximity-dependent labeling:

  • Combine At4g19050 antibody with biotin-phenol and hydrogen peroxide for proximity labeling

  • Identify nearby proteins through streptavidin pulldown and mass spectrometry

  • Verify specific interactions with targeted Western blot analysis

• Immunofluorescence co-localization:

  • Perform dual immunolabeling with At4g19050 antibody and antibodies against candidate interacting proteins

  • Analyze co-localization using confocal microscopy and quantitative image analysis

  • Apply stimuli (pathogen elicitors) to observe dynamic changes in interaction patterns

These methods enable researchers to identify components of At4g19050-containing protein complexes and understand how these interactions contribute to disease resistance mechanisms.

What approaches can resolve potential cross-reactivity issues with At4g19050 antibody?

Cross-reactivity can be a significant concern with antibodies against plant R proteins due to sequence similarities within protein families. Researchers can address this issue through these methodological approaches:

• Specificity validation:

  • Perform Western blot analysis comparing wild-type plants with At4g19050 knockout or knockdown lines

  • Conduct peptide competition assays by pre-incubating the antibody with excess immunizing peptide

  • Compare reactivity patterns across closely related Arabidopsis accessions with sequence variations

• Experimental controls:

  • Include primary antibody omission controls in all experiments

  • Use pre-immune serum at equivalent concentration to primary antibody

  • Incorporate tissues known to lack At4g19050 expression as negative controls

• Cross-reactivity minimization:

  • Perform antibody pre-adsorption against plant extracts lacking At4g19050

  • Use affinity purification against the specific immunogen

  • Apply more stringent washing conditions in immunodetection protocols

• Bioinformatic prediction and validation:

  • Identify potentially cross-reactive proteins through sequence alignment

  • Verify antibody specificity against recombinant proteins of closely related family members

Implementing these approaches ensures greater confidence in experimental results by distinguishing specific At4g19050 signals from potential cross-reactivity artifacts.

How can researchers use At4g19050 antibody to investigate post-translational modifications?

Post-translational modifications (PTMs) of disease resistance proteins like At4g19050 often regulate their activity and interactions. The following methodological approaches can be used to investigate PTMs:

• Immunoprecipitation-based approaches:

  • Immunoprecipitate At4g19050 using the specific antibody

  • Analyze by Western blot using antibodies against common PTMs (phospho-serine/threonine/tyrosine, ubiquitin, SUMO)

  • For detailed analysis, submit immunoprecipitated protein for mass spectrometry

• 2D gel electrophoresis:

  • Separate proteins first by isoelectric point, then by molecular weight

  • Detect At4g19050 by Western blot to identify multiple isoforms representing PTMs

  • Compare patterns before and after treatments (pathogen challenge, stress)

• PTM-specific detection methods:

  • Phos-tag SDS-PAGE for enhanced separation of phosphorylated forms

  • Use of phosphatase treatments to confirm phosphorylation status

  • Deubiquitinase treatments to verify ubiquitination

• Site-specific analysis:

  • Generate phospho-specific antibodies for key regulatory sites (if identified)

  • Perform site-directed mutagenesis in expression constructs to validate function

  • Correlate PTM status with protein activation, localization, or degradation

These approaches provide insights into the regulatory mechanisms controlling At4g19050 function during plant immune responses and developmental processes.

What methodological considerations are important when studying At4g19050 expression during pathogen challenge?

When investigating At4g19050 protein dynamics during pathogen infection, researchers should consider these methodological aspects:

• Experimental design:

  • Include appropriate time points (0, 3, 6, 12, 24, 48, 72 hours post-infection)

  • Compare compatible vs. incompatible interactions

  • Use multiple pathogen strains (virulent, avirulent)

  • Include mock-infected controls at each time point

• Sample collection considerations:

  • Separately analyze infected tissue and surrounding regions

  • Consider local vs. systemic responses

  • Maintain consistent harvesting protocols to minimize variability

  • Process samples immediately to preserve protein integrity

• Analytical approaches:

  • Quantitative Western blot with normalization to loading controls

  • Immunofluorescence to track protein relocalization during infection

  • Subcellular fractionation to monitor compartment-specific accumulation

  • Co-IP at different infection stages to identify dynamic interaction partners

• Data integration:

  • Correlate protein data with transcript levels (qRT-PCR)

  • Monitor parallel defense markers (PR proteins, ROS production)

  • Link protein dynamics to physiological defense responses

This systematic approach enables researchers to understand how At4g19050 protein levels, localization, and interactions change during pathogen challenge, providing insights into its role in disease resistance.

How can researchers address weak or inconsistent signals when using At4g19050 antibody?

When working with At4g19050 antibody, weak or inconsistent signals may occur due to various factors. These methodological approaches can help resolve such issues:

• Protein extraction optimization:

  • Test different extraction buffers (varying detergents, salt concentrations)

  • Ensure complete tissue disruption in liquid nitrogen

  • Add protease inhibitor cocktail to prevent degradation

  • Concentrate proteins if expression levels are low (TCA precipitation)

• Western blot protocol adjustments:

  • Increase protein loading (30-50 μg per lane)

  • Extend primary antibody incubation (overnight at 4°C)

  • Reduce antibody dilution (1:500 instead of 1:1000)

  • Use high-sensitivity detection reagents

  • Optimize transfer conditions for high molecular weight proteins

• Sample-specific considerations:

  • Verify protein expression in selected tissues (At4g19050 may have tissue-specific expression)

  • Consider developmental timing (protein levels may vary during development)

  • Test expression under stress conditions (many R proteins are stress-induced)

• Antibody handling:

  • Avoid repeated freeze-thaw cycles of antibody

  • Centrifuge antibody vial before use to collect liquid that may be trapped in the cap

  • Add 0.1% sodium azide for long-term storage

Implementing these optimizations systematically can significantly improve signal detection while maintaining specificity.

What controls are essential for validating At4g19050 antibody specificity?

To ensure confidence in results obtained with At4g19050 antibody, researchers should implement these essential controls:

• Genetic controls:

  • Compare protein detection in wild-type vs. At4g19050 knockout/knockdown lines

  • Include overexpression lines as positive controls

  • Use related mutants to assess specificity within the R protein family

• Antibody controls:

  • Perform peptide competition assays (pre-incubate antibody with immunizing peptide)

  • Include primary antibody omission control

  • Use pre-immune serum at equivalent concentration

  • Test multiple antibody lots if available

• Technical validation:

  • Verify single band of expected molecular weight (~136 kDa) in Western blot

  • Confirm that signal intensity correlates with protein loading amount

  • Demonstrate reproducibility across independent biological replicates

• Cross-validation with orthogonal methods:

  • Correlate protein detection with mRNA expression data

  • Compare with epitope-tagged At4g19050 detection (if available)

  • Confirm subcellular localization using fractionation and immunoblotting

This comprehensive validation strategy ensures that signals detected with the At4g19050 antibody truly represent the target protein, reducing the risk of data misinterpretation.

How should researchers interpret variations in At4g19050 protein levels across different experimental conditions?

Interpreting variations in At4g19050 protein levels requires careful consideration of multiple factors that influence R protein expression and stability:

By applying these analytical frameworks, researchers can distinguish meaningful biological variations from technical artifacts and extract valuable insights about At4g19050 regulation.

How can researchers integrate At4g19050 protein data with other types of experimental evidence?

A comprehensive understanding of At4g19050 function requires integration of protein-level data with other experimental approaches:

• Multi-omics integration:

  • Compare protein levels (Western blot) with transcript abundance (RNA-seq, qRT-PCR)

  • Analyze correlation or divergence between protein and mRNA levels

  • Incorporate proteomics data to identify post-translational modifications

  • Connect with metabolomic profiles during defense responses

• Phenotypic correlation:

  • Link protein expression/localization with disease resistance phenotypes

  • Analyze genetic interactions through protein expression in various mutant backgrounds

  • Correlate protein dynamics with cellular defense responses (callose deposition, ROS burst)

• Structural and functional relationships:

  • Map protein domains to specific functions through deletion/mutation analysis

  • Correlate protein-protein interaction data with functional outputs

  • Link subcellular localization to sites of action during defense

• Integration methods:

  • Use correlation analysis to identify relationships between datasets

  • Apply network analysis to position At4g19050 within broader defense pathways

  • Develop predictive models incorporating multiple data types

  • Present integrated data in unified visualizations

This integrative approach provides a systems-level understanding of At4g19050 function within plant immunity networks.

How can At4g19050 antibody contribute to studying systemic acquired resistance in plants?

At4g19050 antibody can be instrumental in investigating systemic acquired resistance (SAR), a form of broad-spectrum immunity triggered throughout a plant following localized pathogen exposure:

• Spatial-temporal dynamics:

  • Track At4g19050 protein accumulation in local vs. distal tissues

  • Monitor protein levels during SAR establishment and maintenance

  • Compare with known SAR marker proteins

  • Analyze correlation with mobile SAR signals (pipecolic acid, G3P, etc.)

• Signal transduction analysis:

  • Immunoprecipitate At4g19050 during SAR to identify interacting partners

  • Compare protein modification status in SAR-induced vs. non-induced tissues

  • Analyze At4g19050 protein complexes in SAR mutant backgrounds

  • Investigate subcellular relocalization during systemic immunity

• Genetic interaction studies:

  • Examine At4g19050 protein in SAR-deficient mutants

  • Analyze protein levels following application of SAR-inducing chemicals

  • Investigate impact of At4g19050 mutation on other SAR components

• Translational applications:

  • Use antibody-based assays to screen for SAR-inducing compounds

  • Develop protein markers for SAR establishment in crop plants

  • Monitor protein dynamics in field vs. controlled conditions

This approach can reveal whether At4g19050 functions within the SAR pathway and how its regulation contributes to whole-plant immunity.

What methodological approaches facilitate studying At4g19050 involvement in abiotic stress responses?

While At4g19050 is classified as a disease resistance protein, many R proteins have dual roles in biotic and abiotic stress responses. Researchers can investigate these connections using these approaches:

• Stress-specific expression analysis:

  • Analyze protein levels following various abiotic stresses (drought, salt, cold, heat)

  • Perform time-course analysis to determine immediate vs. adaptive responses

  • Compare protein accumulation across different stress intensities

  • Examine tissue-specific responses to stresses

• Subcellular dynamics:

  • Track protein relocalization during stress responses using immunofluorescence

  • Perform biochemical fractionation to quantify compartment-specific accumulation

  • Investigate stress-induced changes in membrane association

• Protein modification analysis:

  • Compare post-translational modification patterns between biotic and abiotic stresses

  • Analyze correlation between modifications and stress severity

  • Identify stress-specific interaction partners through IP-MS

• Functional integration:

  • Examine At4g19050 protein in known abiotic stress signaling mutants

  • Investigate cross-tolerance between biotic and abiotic stresses at the protein level

  • Analyze convergence points between stress response pathways

These approaches can reveal potential roles for At4g19050 beyond pathogen resistance and contribute to understanding how plants integrate responses to multiple stress types.

How can advanced imaging techniques enhance At4g19050 protein studies with the specific antibody?

Cutting-edge imaging techniques can significantly expand our understanding of At4g19050 localization, dynamics, and interactions:

• Super-resolution microscopy:

  • Apply techniques like STORM, PALM, or SIM to resolve At4g19050 localization below diffraction limit

  • Determine precise subcellular localization at 10-20 nm resolution

  • Analyze nanoscale organization and clustering during defense responses

  • Combine with other protein markers for detailed co-localization analysis

• Live-cell imaging optimization:

  • Develop protocols for antibody fragment labeling in live tissues

  • Use microinjection of fluorescently-labeled antibodies for dynamic studies

  • Combine with genetically encoded fluorescent markers for dual visualization

• Multi-dimensional imaging:

  • Perform 3D reconstruction of At4g19050 distribution throughout cell volumes

  • Implement time-lapse imaging to track relocalization during responses

  • Use spectral imaging to distinguish specific signal from autofluorescence

  • Apply correlative light and electron microscopy for ultrastructural context

• Quantitative image analysis:

  • Develop automated segmentation algorithms for protein distribution

  • Apply fluorescence correlation spectroscopy to analyze protein dynamics

  • Use FRET approaches to measure protein-protein interactions in situ

  • Implement machine learning for pattern recognition in complex datasets

These advanced imaging approaches provide unprecedented insights into the spatial organization and dynamics of At4g19050 during plant immunity processes.