At1g27490 Antibody

What is the At1g27490 gene in Arabidopsis thaliana and what protein does this antibody detect?

The At1g27490 gene in Arabidopsis thaliana encodes a protein with UniProt accession number Q9FZI7 . This antibody specifically recognizes epitopes of this protein product. The At1g27490 protein is part of the extensive molecular network in Arabidopsis thaliana, which is widely used as a model organism in plant biology research. When designing experiments using this antibody, researchers should consider the protein's subcellular localization, expression patterns, and potential post-translational modifications that may affect antibody binding.

The antibody is designed to recognize specific epitopes on the protein product, making it valuable for protein detection and quantification in various experimental applications. Understanding the protein's molecular characteristics is essential for interpreting experimental results and designing appropriate controls.

What are the recommended applications for At1g27490 Antibody in plant research?

The At1g27490 Antibody is suitable for multiple experimental applications in plant research, similar to other plant antibodies designed for specific proteins. While the search results don't specify particular applications for this antibody, plant antibodies typically function effectively in Western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence microscopy.

For optimal results in Western blotting, researchers should follow standardized protocols similar to those used with other plant antibodies, which typically include:

These recommendations are based on standard protocols for plant antibodies similar to the actin antibody specifications , and should be optimized for the At1g27490 Antibody through pilot experiments.

How should optimal protein extraction be performed for At1g27490 detection in Arabidopsis tissues?

Protein extraction for At1g27490 detection requires careful methodology to preserve protein integrity while maximizing yield. For Arabidopsis tissues, implement the following extraction protocol:

• Harvest fresh tissue (100-200 mg) and flash-freeze in liquid nitrogen.

• Grind tissue to a fine powder using a pre-chilled mortar and pestle, maintaining frozen conditions throughout.

• Add extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100) supplemented with protease inhibitors (1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin A).

• Use 3-5 volumes of buffer per tissue weight.

• Homogenize thoroughly and incubate with gentle rotation for 30 minutes at 4°C.

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

• Collect the supernatant containing soluble proteins.

• Quantify protein concentration using Bradford or BCA assay.

This methodology ensures optimal extraction of At1g27490 protein while minimizing degradation or modification that could affect antibody recognition. The addition of phosphatase inhibitors may be necessary if studying phosphorylation states of the protein.

What are the optimal Western blotting conditions for detecting At1g27490 protein?

For optimal Western blot detection of At1g27490 protein, follow these methodological steps:

• Resolve 10-30 μg of total protein extract on 10-12% SDS-PAGE gel (exact percentage should be optimized based on the molecular weight of At1g27490).

• Transfer proteins to PVDF or nitrocellulose membrane (100V for 1 hour or 30V overnight at 4°C).

• Block the membrane with 5% non-fat dry milk or BSA in TBS-T for 1 hour at room temperature.

• Incubate with At1g27490 Antibody at 1:3000-1:5000 dilution in blocking buffer overnight at 4°C.

• Wash the membrane 3-4 times with TBS-T, 5 minutes each.

• Incubate with appropriate HRP-conjugated secondary antibody (typically anti-rabbit IgG at 1:5000-1:10000) for 1 hour at room temperature.

• Wash 4-5 times with TBS-T, 5 minutes each.

• Develop using ECL substrate and image using a suitable detection system.

For enhanced sensitivity, consider using a high-sensitivity ECL substrate or implementing signal amplification techniques when detecting low-abundance proteins. This protocol is based on standard approaches used for plant antibodies, similar to the actin antibody methodology .

How can researchers troubleshoot weak or absent signals when using At1g27490 Antibody?

When encountering weak or absent signals with At1g27490 Antibody, systematically evaluate the following potential issues:

• Protein Extraction Efficiency:

  • Ensure complete tissue disruption during extraction

  • Test alternative extraction buffers with different detergent compositions

  • Check protein quantification accuracy

• Antibody Dilution Optimization:

  • Test a range of primary antibody concentrations (1:1000 to 1:5000)

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

  • Optimize secondary antibody concentration

• Sample Preparation:

  • Ensure sample isn't overheated during preparation (can cause protein aggregation)

  • Add fresh protease inhibitors to prevent degradation

  • Check protein loading using a loading control antibody (e.g., anti-actin )

• Detection System:

  • Use fresh ECL reagents

  • Increase exposure time incrementally

  • Consider more sensitive detection methods (enhanced chemiluminescence or fluorescent secondary antibodies)

• Expression Level Verification:

  • Confirm At1g27490 expression in your specific tissues and conditions

  • Consider using tissues with known higher expression of the target protein

Maintaining a detailed log of all troubleshooting steps and observations will help identify the source of the problem and establish reliable detection protocols.

What cross-reactivity considerations should researchers be aware of when using At1g27490 Antibody?

When working with At1g27490 Antibody, researchers must carefully evaluate potential cross-reactivity to ensure experimental validity. Consider these methodological approaches:

• Sequence Homology Analysis:

  • Perform bioinformatic analysis to identify proteins with similar epitopes in your experimental system

  • Check for paralogs or related protein family members in Arabidopsis thaliana

  • Evaluate conservation across species if working with non-Arabidopsis plants

• Experimental Validation:

  • Include negative controls such as knockout/knockdown lines for At1g27490

  • Perform competitive binding assays with recombinant protein

  • Test the antibody on samples with overexpressed At1g27490 protein

• Pre-Absorption Controls:

  • Pre-incubate the antibody with purified recombinant At1g27490 protein before use

  • Compare results between pre-absorbed and regular antibody incubations

• Species Cross-Reactivity:

  • If using in non-Arabidopsis species, perform sequence alignment to determine epitope conservation

  • Validate with recombinant proteins from the test species if available

Thorough cross-reactivity assessment is crucial for meaningful data interpretation, especially when studying protein families with conserved domains or when extending research to other plant species.

How can At1g27490 Antibody be optimized for immunolocalization studies in plant tissues?

Optimizing At1g27490 Antibody for immunolocalization requires specific methodological considerations for plant tissues:

• Tissue Fixation Protocol:

  • For paraffin sections: Fix tissues in 4% paraformaldehyde in PBS for 12-24 hours at 4°C

  • For cryosections: Fix in 4% paraformaldehyde for 1-2 hours, then transfer to 30% sucrose solution

  • Carefully optimize fixation time to preserve antigenicity while maintaining tissue architecture

• Antigen Retrieval Methods:

  • Heat-induced epitope retrieval: Incubate sections in citrate buffer (pH 6.0) at 95°C for 10-20 minutes

  • Enzymatic retrieval: Treat with proteinase K (1-5 μg/ml) for 5-10 minutes at room temperature

  • Test multiple methods as plant tissues often require specific retrieval conditions

• Blocking and Antibody Dilution:

  • Block with 5% normal serum, 3% BSA, and 0.3% Triton X-100 in PBS for 1-2 hours

  • Test At1g27490 Antibody at dilutions between 1:100 and 1:250 for immunofluorescence

  • Incubate sections with primary antibody overnight at 4°C

• Signal Amplification Strategies:

  • Consider using tyramide signal amplification for low-abundance proteins

  • Biotin-streptavidin systems can enhance detection sensitivity

  • Quantum dots can be used for multiplexing with other antibodies

• Controls:

  • Include secondary-only controls

  • Use tissues from knockout plants as negative controls

  • Consider dual labeling with organelle markers to confirm subcellular localization

These optimization steps should be performed sequentially, making one adjustment at a time and documenting results meticulously to establish a reproducible protocol.

What are the key considerations for using At1g27490 Antibody in chromatin immunoprecipitation (ChIP) experiments?

Adapting At1g27490 Antibody for chromatin immunoprecipitation requires careful optimization due to the complex nature of plant chromatin:

• Cross-linking Optimization:

  • Test multiple formaldehyde concentrations (1-3%) and incubation times (10-20 minutes)

  • Consider dual cross-linking with disuccinimidyl glutarate (DSG) followed by formaldehyde for improved efficiency

  • Optimize quenching conditions using glycine (125-250 mM)

• Chromatin Preparation:

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

  • Filter lysates to remove cell debris before immunoprecipitation

  • Pre-clear chromatin with protein A/G beads to reduce background

• Immunoprecipitation Conditions:

  • Test different amounts of At1g27490 Antibody (2-10 μg per reaction)

  • Optimize antibody-chromatin incubation time (4 hours to overnight at 4°C)

  • Include appropriate controls (non-specific IgG, input chromatin)

• Washing Stringency:

  • Implement a series of increasingly stringent washes to reduce non-specific binding

  • Typical wash buffers contain increasing salt concentrations (150-500 mM NaCl)

  • Final washes should include LiCl buffer and TE buffer

• DNA Recovery and Analysis:

  • Reverse cross-links completely (65°C for 4-16 hours)

  • Purify DNA using phenol-chloroform extraction or commercial kits

  • Validate enrichment using qPCR before proceeding to sequencing

The success of ChIP experiments with At1g27490 Antibody will depend largely on the DNA-binding properties of the target protein and the antibody's ability to recognize the protein in its native chromatin context.

How should researchers quantify and statistically analyze Western blot data using At1g27490 Antibody?

Rigorous quantification and statistical analysis of Western blot data using At1g27490 Antibody requires systematic methodology:

• Image Acquisition:

  • Capture images using a dynamic range-appropriate system (CCD camera-based imager)

  • Avoid saturation by taking multiple exposures

  • Use TIFF format with at least 16-bit depth to preserve signal information

• Normalization Strategy:

  • Always include loading controls (actin , tubulin, or GAPDH)

  • Normalize signal intensity of At1g27490 to loading control from the same lane

  • Consider using total protein staining methods (Ponceau S, SYPRO Ruby) as alternative normalization

• Quantification Software:

  • Use specialized software (ImageJ, Image Lab, etc.) for densitometric analysis

  • Define consistent measurement parameters (band boundaries, background subtraction)

  • Create standard curves if performing absolute quantification

• Statistical Analysis Framework:

  • For comparing two groups: Student's t-test or Mann-Whitney U test depending on data distribution

  • For multiple groups: ANOVA with appropriate post-hoc tests (Tukey, Bonferroni)

  • Always perform at least 3-4 biological replicates for statistical validity

• Reporting Results:

  • Present data as mean ± standard deviation or standard error

  • Report exact p-values and statistical tests used

  • Include representative blot images alongside quantification graphs

This systematic approach ensures that subtle changes in At1g27490 protein levels can be accurately quantified and statistically validated across experimental conditions.

How can researchers interpret contradictory results between transcript levels and protein detection using At1g27490 Antibody?

When transcript and protein data for At1g27490 show discrepancies, conduct a methodical analysis following these steps:

• Verify Technical Validity:

  • Confirm antibody specificity using knockout/knockdown controls

  • Check primer specificity for transcript analysis

  • Validate RNA and protein extraction efficiency across samples

• Temporal Consideration Analysis:

  • Implement time-course experiments to track both transcript and protein levels

  • Consider delayed protein synthesis relative to transcription

  • Evaluate whether samples for RNA and protein were collected at equivalent time points

• Post-Transcriptional Regulation Assessment:

  • Investigate microRNA regulation of At1g27490 transcript

  • Evaluate RNA stability using actinomycin D chase experiments

  • Assess translation efficiency using polysome profiling

• Post-Translational Modification Examination:

  • Test for protein degradation rates using cyclohexamide chase assays

  • Investigate potential protein modifications that might affect antibody recognition

  • Consider subcellular localization changes that could affect extraction efficiency

• Experimental Design Matrix:

  Possible Scenario	Experimental Approach	Control/Validation Method
  Rapid protein turnover	Proteasome inhibitor treatment	Monitor protein accumulation
  Inefficient translation	Polysome profiling	Compare transcript association with ribosomes
  Selective tissue expression	Cell/tissue-specific analysis	FACS sorting or laser capture microdissection
  Alternative protein isoforms	Western blot with multiple antibodies	Mass spectrometry confirmation
  Developmental regulation	Developmental time series	Compare transcript/protein across stages

This comprehensive approach helps researchers determine whether discrepancies reflect biological regulation or technical limitations, leading to more accurate interpretations of At1g27490 function.

How can At1g27490 Antibody be validated for use in other plant species beyond Arabidopsis thaliana?

Cross-species validation of At1g27490 Antibody requires systematic methodology to ensure reliable detection in non-Arabidopsis systems:

• Sequence Homology Analysis:

  • Perform BLAST searches using Arabidopsis At1g27490 protein sequence against target species

  • Identify orthologs and calculate percent identity, particularly in the epitope region

  • Create multiple sequence alignments to visualize conservation of the antibody recognition site

• Epitope Conservation Assessment:

  • If epitope information is available, analyze its conservation across species

  • Consider synthetic peptide competition assays to confirm epitope specificity

  • Evaluate potential post-translational modifications that might affect epitope recognition

• Experimental Validation Protocol:

  • Run side-by-side Western blots with both Arabidopsis and target species samples

  • Include recombinant protein (if available) as positive control

  • Test multiple extraction protocols optimized for each species' tissue type

• Cross-Reactivity Controls:

  • Use genetic resources (RNAi, CRISPR knockouts) in the target species when available

  • Perform pre-absorption controls with recombinant proteins or peptides

  • Consider heterologous expression systems to confirm antibody recognition

• Optimization for Non-Arabidopsis Species:

  • Adjust antibody concentration and incubation conditions for each species

  • Modify extraction buffers to account for species-specific differences in cell wall composition

  • Document all modifications required for successful detection

This validation framework ensures that results obtained with At1g27490 Antibody across different plant species are reliable and comparable, enabling evolutionary and comparative studies of protein function.

What experimental design considerations are important when comparing At1g27490 protein levels across different developmental stages or stress conditions?

When designing experiments to compare At1g27490 protein levels across developmental stages or stress conditions, implement these methodological guidelines:

• Standardized Sampling Protocol:

  • Harvest tissues at precisely defined developmental stages using morphological markers

  • Maintain consistent sampling timing relative to stress application

  • Standardize tissue selection (e.g., specific leaf position, root zone)

• Experimental Design Optimization:

  • Use split-plot or randomized complete block designs to minimize environmental variation

  • Include sufficient biological replicates (minimum 3-4) and technical replicates (2-3)

  • Plan time-course experiments to capture dynamic changes

• Control Implementation:

  • Include internal controls for each developmental stage or stress condition

  • Use constitutively expressed proteins as loading controls

  • Consider spike-in controls with recombinant proteins for absolute quantification

• Normalization Strategy:

  • Normalize to total protein content rather than single reference genes when comparing across conditions

  • Verify stability of reference proteins across all experimental conditions

  • Consider multiple normalization methods and compare results for robustness

• Data Analysis Framework:

  • Apply appropriate statistical tests for time-series data (repeated measures ANOVA)

  • Use mixed-effects models when comparing multiple variables

  • Consider normalization to baseline levels when analyzing stress responses

• Interpretation Guidelines:

  • Correlate protein level changes with physiological or phenotypic measurements

  • Consider post-translational modifications that might affect antibody recognition

  • Integrate with transcriptome data for comprehensive understanding

This experimental framework ensures that observed changes in At1g27490 protein levels genuinely reflect biological responses rather than technical variables or sampling artifacts.