At1g57690 Antibody

What is AT1g57690 and what biological function does it serve?

AT1g57690 (UniProt: Q9FVT4) is a protein expressed in Arabidopsis thaliana that belongs to a family of plant-specific proteins. Based on current research, this protein is believed to be involved in plant stress responses and developmental processes. The protein has been identified through genomic and proteomic studies of Arabidopsis thaliana, which serves as a model organism for understanding fundamental plant biology processes.

The specific antibody targeting this protein (CSB-PA887851XA01DOA) allows researchers to investigate its expression patterns, subcellular localization, and potential interactions with other proteins in various physiological and experimental conditions . Understanding this protein's function contributes to our knowledge of plant cellular processes and potential applications in agricultural research and biotechnology.

What validation methods confirm AT1g57690 antibody specificity?

The specificity of AT1g57690 antibody should be validated through multiple complementary approaches to ensure reliable experimental results. The recommended validation workflow includes:

• Western blot analysis: Demonstrating a single band at the expected molecular weight in wild-type samples and absence of this band in knockout/knockdown lines

• Immunoprecipitation followed by mass spectrometry: Confirming the identity of the precipitated protein

• Immunofluorescence with appropriate controls: Showing expected localization patterns that disappear in knockout lines

• Preabsorption with the immunizing peptide: Demonstrating loss of signal

For researchers working with AT1g57690 antibody, validation is particularly important as plant proteins often belong to gene families with similar sequences. Cross-reactivity testing against closely related proteins should be performed prior to using the antibody in critical experiments .

What are the optimal storage conditions for maintaining AT1g57690 antibody activity?

To maintain optimal activity of AT1g57690 antibody, proper storage conditions are essential. For long-term stability:

• Store freeze-dried solid antibody at 2-8°C in the original container protected from light

• After rehydration with the recommended volume of dH₂O, centrifuge if the solution is not clear

• For short-term storage (up to 6 weeks), store the rehydrated antibody at 2-8°C

• For extended storage after rehydration, add an equal volume of glycerol (ACS grade or better) for a final concentration of 50%, and store at -20°C

What working dilutions are recommended for common applications?

These recommendations serve as starting points; optimal dilutions should be determined experimentally for each specific application and lot of antibody. For particularly sensitive applications, conducting a preliminary titration experiment is highly recommended to determine the optimal antibody concentration that provides maximum specific signal with minimal background .

How can AT1g57690 antibody be optimized for Western blotting in plant tissues?

Western blotting with AT1g57690 antibody in plant tissues requires specific optimization due to the complex nature of plant samples. The following protocol modifications can significantly improve results:

• Sample preparation enhancements:

  • Include plant-specific protease inhibitors (e.g., PMSF, leupeptin, aprotinin) in extraction buffers

  • Add polyvinylpolypyrrolidone (PVPP) at 2-5% (w/v) to remove interfering phenolic compounds

  • Include 1-2% β-mercaptoethanol to reduce oxidation of plant proteins

• Membrane blocking optimization:

  • Use 5% non-fat dry milk in TBS-T (preferred over BSA for plant samples)

  • Alternative: 3% BSA with 0.1% Tween-20 if milk produces high background

  • Consider adding 0.05% Triton X-100 to reduce non-specific binding

• Signal enhancement strategies:

  • Extended transfer times (1-2 hours) for efficient protein migration

  • Use of high-sensitivity detection systems such as ECL-plus

  • Optimize primary antibody incubation time (overnight at 4°C often yields best results)

For difficult-to-detect AT1g57690 protein, enrichment through subcellular fractionation prior to Western blotting can increase detection sensitivity by concentrating the target protein . When using alkaline phosphatase-conjugated secondary antibodies, the signal can be developed using BCIP/NBT substrate, which provides excellent sensitivity for plant proteins while minimizing background issues common with plant extracts.

What is the recommended protocol for immunoprecipitation using AT1g57690 antibody?

Immunoprecipitation with AT1g57690 antibody requires careful optimization to maintain protein-protein interactions while achieving efficient target capture. This comprehensive protocol addresses plant-specific challenges:

Day 1: Sample Preparation and Antibody Binding

• Prepare fresh plant tissue extract in a non-denaturing lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with plant protease inhibitor cocktail)

• Homogenize tissue and centrifuge at 14,000 × g for 15 minutes at 4°C

• Pre-clear lysate with Protein A/G beads for 1 hour at 4°C

• Incubate 500 μg of pre-cleared lysate with 2-5 μg of AT1g57690 antibody overnight at 4°C with gentle rotation

Day 2: Precipitation and Analysis

• Add 40 μl of pre-washed Protein A/G magnetic beads and incubate for 2 hours at 4°C

• Wash beads 5× with wash buffer (lysis buffer with reduced detergent concentration)

• Elute bound proteins by boiling in SDS sample buffer or use a gentle elution buffer for preserving protein interactions

• Analyze by Western blot or mass spectrometry

For challenging plant samples, consider using a chemical crosslinking step (0.5-2 mM DSP) prior to cell lysis to stabilize transient protein interactions. The F(ab')₂ fragment version of the antibody may be preferable when working with samples containing Fc receptors to reduce background . Appropriate controls include IgG isotype control antibodies and, ideally, samples from AT1g57690 knockout lines.

How can AT1g57690 antibody be used for chromatin immunoprecipitation (ChIP) assays?

AT1g57690 antibody can be employed in ChIP assays to investigate potential DNA-binding properties or chromatin associations of the AT1g57690 protein. The following protocol adaptations are essential for successful plant ChIP experiments:

Crosslinking and Nuclear Extraction:

• Crosslink fresh plant tissue with 1% formaldehyde for 10 minutes under vacuum

• Quench with 0.125 M glycine for 5 minutes

• Isolate nuclei using a plant-specific nuclear isolation buffer containing 0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 1% Triton X-100, and plant protease inhibitors

• Sonicate chromatin to achieve fragments of 200-500 bp (verify by agarose gel)

Immunoprecipitation:

• Pre-clear chromatin with protein A/G beads for 1 hour at 4°C

• Incubate pre-cleared chromatin with 3-5 μg AT1g57690 antibody overnight at 4°C

• Add protein A/G beads and incubate for 2-3 hours at 4°C

• Perform sequential washes with increasing stringency buffers

• Elute DNA-protein complexes and reverse crosslinks at 65°C overnight

• Purify DNA using phenol-chloroform extraction or commercial kits

Data Analysis and Validation:

• Analyze enriched DNA by qPCR targeting candidate regions or by next-generation sequencing

• Use IgG controls and input samples for normalization

• Confirm enrichment of known target sequences if available

ChIP-seq using AT1g57690 antibody can reveal genome-wide binding patterns, providing insights into the protein's role in transcriptional regulation or chromatin organization . When designing primers for ChIP-qPCR validation, focus on regions with potential regulatory elements and include negative control regions where binding is not expected.

What troubleshooting approaches can resolve inconsistent AT1g57690 antibody results?

When facing inconsistent results with AT1g57690 antibody, a systematic troubleshooting approach can identify and resolve issues:

Signal Absence or Weakness:

• Verify protein expression: Confirm target protein expression in your sample using alternative methods (e.g., RT-PCR)

• Optimize extraction: For membrane-associated proteins, test different detergents (CHAPS, NP-40, Triton X-100)

• Increase antibody concentration: Perform a titration experiment to determine optimal concentration

• Enhance signal detection: Use amplification systems like biotin-streptavidin or tyramide signal amplification

• Evaluate epitope accessibility: Test different antigen retrieval methods for fixed samples

High Background or Non-specific Binding:

• Optimize blocking: Test different blockers (BSA, casein, normal serum) and concentrations

• Adjust washing: Increase washing stringency by adding higher salt concentrations or detergents

• Pre-adsorb antibody: Incubate with control lysates from organisms not expressing the target

• Use F(ab')₂ fragments: Switch to F(ab')₂ fragments if Fc receptor binding is suspected

Cross-reactivity Issues:

• Validate with knockout controls: Compare results with samples lacking the target protein

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity

• Alternative antibody: Test antibodies targeting different epitopes of the same protein

Data Interpretation Table for Troubleshooting:

Maintaining detailed laboratory records of all experimental conditions is crucial for identifying variables contributing to inconsistency .

How does AT1g57690 antibody perform in plant tissue immunofluorescence?

Immunofluorescence with AT1g57690 antibody in plant tissues requires specialized techniques to overcome plant-specific challenges:

Sample Preparation Optimization:

• Fixation: Use 4% paraformaldehyde in PME buffer (50 mM PIPES, 5 mM MgSO₄, 5 mM EGTA, pH 6.9) for 1-2 hours

• Cell wall digestion: Apply enzyme solution (2% cellulase, 1% pectinase in PME) for improved antibody penetration

• Permeabilization: Use 0.5% Triton X-100 for 30 minutes after fixation

• Antigen retrieval: For certain applications, microwave treatment in citrate buffer may improve epitope accessibility

Blocking and Antibody Incubation:

• Block with 3% BSA, 10% normal serum from secondary antibody host species, and 0.1% Triton X-100

• Apply AT1g57690 primary antibody at 1:100-1:200 dilution and incubate overnight at 4°C

• Use fluorophore-conjugated secondary antibodies with minimal plant autofluorescence interference (far-red dyes like Cy5 or Alexa Fluor 647 recommended)

• Include DAPI (1 μg/ml) for nuclear counterstaining

Reducing Plant-Specific Background:

• Include 0.1% Tween-20 in all washing steps

• Use TBS rather than PBS for reduced autofluorescence

• Apply 0.1 M NH₄Cl for 10 minutes to reduce fixation-induced autofluorescence

• Add 0.01% Toluidine Blue to blocking solution to reduce cell wall autofluorescence

Sample Preparation Strategy:

• Tissue selection: Choose tissues with known AT1g57690 expression (refer to publicly available expression databases)

• Growth conditions: Standardize plant growth conditions to minimize variation in protein expression

• Harvesting timing: Consider diurnal or developmental regulation of AT1g57690 expression

• Subcellular fractionation: Enrich for relevant cellular compartments if protein abundance is low

Control Implementation:

• Genetic controls: Include knockout/knockdown lines whenever possible

• Antibody controls: Use isotype controls at equivalent concentrations

• Technical controls: Include loading controls for Western blots, input controls for IP/ChIP

• Biological replicates: Minimum of three independent biological replicates to account for natural variation

Quantification Approaches:

• Signal normalization: Normalize AT1g57690 signals to appropriate housekeeping proteins

• Dynamic range assessment: Verify that signal falls within the linear range of detection

• Statistical analysis: Apply appropriate statistical tests based on experimental design

• Reporting standards: Include all relevant experimental details in publications

Antibody Validation Plan:

• Knockout validation: Confirm absence of signal in knockout lines

• Orthogonal methods: Verify results using independent techniques (e.g., mass spectrometry)

• Peptide competition: Demonstrate specificity through signal reduction with immunizing peptide

• Positive controls: Include samples with known AT1g57690 expression

When studying protein-protein interactions involving AT1g57690, consider the potential impact of the antibody on protein binding interfaces. For such applications, epitope-tagged versions of AT1g57690 may provide complementary approaches, though care must be taken to ensure that tagging does not interfere with protein function or localization .

How can AT1g57690 antibody be applied to investigate plant stress responses?

AT1g57690 antibody offers valuable tools for investigating plant stress responses, providing insights into how this protein functions under various environmental challenges:

Stress-Induced Expression Analysis:

• Time-course experiments: Monitor AT1g57690 protein levels at different timepoints after stress application using Western blot

• Tissue-specific changes: Compare protein expression across different plant tissues under stress conditions

• Stress specificity: Analyze protein response to different stressors (drought, salt, pathogen, heat)

• Recovery dynamics: Track protein levels during stress recovery periods

Protein Modification Under Stress:

• Post-translational modifications: Use phospho-specific antibodies alongside general AT1g57690 antibody to detect stress-induced modifications

• Protein stability: Measure protein half-life under normal versus stress conditions using cycloheximide chase assays

• Protein complexes: Identify stress-specific protein interaction partners using co-immunoprecipitation with AT1g57690 antibody

Subcellular Localization Changes:

• Translocation analysis: Track protein movement between cellular compartments using immunofluorescence

• Membrane association: Determine whether stress induces membrane recruitment using subcellular fractionation

• Nuclear-cytoplasmic shuttling: Quantify nuclear/cytoplasmic distribution ratios under various conditions

Functional Assessment:

• Chromatin association: Use ChIP-seq to identify stress-responsive genomic regions bound by AT1g57690

• Activity correlation: Correlate biochemical activity with protein abundance/modification patterns

• Comparative studies: Analyze AT1g57690 response in stress-tolerant versus sensitive Arabidopsis ecotypes

When designing stress experiments, standardize stress application methods and include appropriate physiological measurements (e.g., relative water content for drought stress, ion leakage for cold stress) to correlate molecular changes with plant physiological responses . For publication-quality results, quantitative image analysis of immunofluorescence data should be performed using software that can correct for plant tissue autofluorescence.

How can AT1g57690 antibody be integrated with emerging single-cell technologies?

The integration of AT1g57690 antibody with cutting-edge single-cell technologies opens new frontiers for understanding cellular heterogeneity in plant systems:

Single-Cell Protein Analysis Applications:

• Mass cytometry (CyTOF): Conjugate AT1g57690 antibody with metal isotopes for simultaneous measurement of multiple proteins in single plant cells

• Imaging mass cytometry: Combine metal-labeled AT1g57690 antibody with tissue imaging for spatial protein analysis

• Single-cell Western blotting: Apply microfluidic platforms to analyze AT1g57690 expression in individual protoplasts

• Proximity extension assays: Develop oligonucleotide-labeled AT1g57690 antibody pairs for ultrasensitive protein quantification

Technical Adaptations for Plant Systems:

• Protoplast optimization: Develop gentle protoplast isolation protocols that preserve protein epitopes

• Signal amplification: Implement rolling circle amplification for detecting low-abundance AT1g57690 protein

• Multiplex antibody panels: Design compatible antibody panels including AT1g57690 and related signaling proteins

• Microfluidic systems: Adapt plant cell capture approaches for compatibility with existing single-cell platforms

Data Integration Strategies:

• Multi-omics correlation: Combine single-cell proteomics with scRNA-seq data to correlate AT1g57690 protein and transcript levels

• Spatial mapping: Integrate spatial transcriptomics with antibody-based protein detection

• Computational modeling: Develop algorithms to infer protein interaction networks from single-cell data

• Trajectory analysis: Track AT1g57690 expression changes during developmental progressions at single-cell resolution

While single-cell technologies are still being adapted for plant systems, preliminary work can focus on developing and validating AT1g57690 antibody protocols in protoplast systems, ensuring compatibility with downstream single-cell applications . The development of highly specific AT1g57690 antibodies with minimal cross-reactivity is particularly important for single-cell applications where signal specificity is critical.

What quality control metrics should be applied to AT1g57690 antibody experiments?

Implementing rigorous quality control metrics ensures the reliability and reproducibility of experiments using AT1g57690 antibody:

Antibody Validation Metrics:

• Specificity score: Quantitative assessment based on knockout validation, peptide competition, and orthogonal methods

• Sensitivity threshold: Minimum detectable protein concentration under standardized conditions

• Linear dynamic range: Range of protein concentrations over which signal correlates linearly with abundance

• Lot-to-lot consistency: Coefficient of variation when testing multiple antibody lots under identical conditions

Experimental QC Parameters:

• Signal-to-noise ratio: Minimum acceptable ratio for different applications (e.g., >5:1 for Western blot, >3:1 for immunofluorescence)

• Positive control signal: Expected signal intensity range for positive controls

• Negative control background: Maximum acceptable background in negative controls

• Technical replicate variation: Coefficient of variation between technical replicates (target <15%)

Documentation Requirements:

• Antibody metadata: Complete documentation including catalog number, lot number, concentration, and storage conditions

• Protocol parameters: Detailed protocol with all critical parameters explicitly stated

• Image acquisition settings: Full documentation of all microscopy or imaging parameters

• Data processing methods: Complete description of normalization and quantification approaches

Results Interpretation Guidelines:

• Minimum fold change: Threshold for considering protein level changes biologically significant

• Correlation requirements: Minimum correlation coefficients between independent methods

• Reproducibility standards: Number of independent replicates showing consistent results

• Statistical significance criteria: Appropriate statistical tests and significance thresholds

For publication-quality work, researchers should create a study-specific quality control checklist incorporating these metrics and documenting compliance for each experiment. Implementing standardized positive controls across experiments allows for inter-experimental normalization and facilitates meta-analysis of results across multiple studies .