At1g54790 Antibody

What is the At1g54790 protein and why is it important in plant research?

At1g54790 encodes a GDSL esterase/lipase in Arabidopsis thaliana that belongs to a large family of hydrolytic enzymes with multiple biochemical functions. This protein is significant in plant research because GDSL enzymes play important roles in plant development, stress responses, and lipid metabolism. The protein is classified in the protein-coding gene category and has orthologs in other plant species, including potential homologs like "GDSL esterase/lipase At1g54790-like" found in Momordica charantia (bitter gourd) .

What types of antibodies are available for At1g54790 detection?

Researchers have developed both polyclonal and monoclonal antibodies for At1g54790 detection. Similar to other plant protein antibodies, these typically include:

How should I validate the specificity of an At1g54790 antibody?

Proper validation of At1g54790 antibodies should include:

• Genetic controls: Testing the antibody on wild-type tissue versus knockout/knockdown lines (e.g., T-DNA insertion mutants of At1g54790) .

• Biochemical tests: Performing pre-absorption controls with recombinant At1g54790 protein to confirm specificity.

• Multiple technique validation: Cross-validating results using different methods such as western blot, immunohistochemistry, and immunoprecipitation.

• Cross-reactivity testing: Evaluating potential cross-reactivity with other GDSL family members, especially those with high sequence homology .
  The most definitive demonstration of antibody specificity is the lack of staining in tissues where the target protein has been knocked out .

What controls should I include when using At1g54790 antibodies in immunolocalization experiments?

Every immunolocalization experiment using At1g54790 antibodies should include these essential controls:

• Negative controls:

  • Secondary antibody-only control to detect non-specific binding

  • Tissue from At1g54790 knockout/knockdown plants

  • Pre-immune serum control (for polyclonal antibodies)

• Positive controls:

  • Tissues with known expression patterns of At1g54790

  • Recombinant At1g54790 protein (if available)

• Specificity controls:

  • Antibody pre-absorption with recombinant At1g54790

  • Competitive binding assays
    The absence of appropriate controls represents a significant red flag in experimental design and may lead to misinterpretation of results .

What is the optimal protein extraction method for detecting At1g54790 in Western blots?

The method of protein extraction significantly impacts the detection of plant membrane-associated proteins like GDSL lipases. Based on protocols optimized for similar plant proteins:
Recommended protocol:

• Grind approximately 200 mg of plant material in liquid nitrogen

• Add extraction buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

• Homogenize thoroughly and centrifuge at 14,000 rpm at 4°C

• Collect supernatant and mix with SDS loading buffer

• Heat at 70°C for 10 minutes (not 95°C, which may cause aggregation)
  Inadequate extraction methods often result in failure to detect the target protein, as demonstrated with other plant membrane-associated proteins like BAK1 .

How should I determine the optimal working dilution for an At1g54790 antibody?

To determine the optimal antibody concentration:

• Perform a dilution series test:

  • For Western blots: Test 1:1000, 1:2000, 1:5000, and 1:10000 dilutions

  • For immunolocalization: Test 1:100, 1:200, 1:500, and 1:1000 dilutions

• Evaluate signal-to-noise ratio at each concentration:

  • The ideal dilution provides clear specific signal with minimal background

  • Too high concentration increases background signal

  • Too low concentration results in weak or undetectable signal

• Consider tissue-specific optimization:

  • Different tissues may require different antibody concentrations due to varying target abundance and matrix effects
    Titration experiments have shown that antibody concentrations between 0.625-2.5 μg/mL often provide optimal signal-to-noise ratio for plant proteins .

How can I use At1g54790 antibodies in co-immunoprecipitation (co-IP) experiments to identify protein interaction partners?

For successful co-IP experiments with At1g54790 antibodies:

• Sample preparation:

  • Use mild lysis buffers containing 0.5-1% NP-40 or Triton X-100

  • Include protease and phosphatase inhibitors

  • Maintain cold temperatures throughout to preserve protein interactions

• Immunoprecipitation protocol:

  • Pre-clear lysate with Protein A/G beads

  • Incubate with At1g54790 antibody (2-5 μg per 500 μg protein extract)

  • Add Protein A/G beads and incubate 1-2 hours at 4°C

  • Wash thoroughly with progressively stringent buffers

  • Elute bound proteins with SDS buffer or low pH glycine

• Analysis methods:

  • Western blot for known/suspected interactors

  • Mass spectrometry for unbiased discovery of novel interaction partners
    Successful co-IP experiments with plant proteins like HDA9 have identified novel interactors such as POWERDRESS (PWR), demonstrating the value of this approach .

What strategies can I employ when At1g54790 antibody shows high background or non-specific binding?

When facing high background issues:

• Optimization strategies:

  • Increase blocking concentration (5% BSA or 5% non-fat milk)

  • Add 0.1-0.5% Tween-20 to washing buffers

  • Reduce primary antibody concentration

  • Increase washing duration and frequency

• Sample-specific approaches:

  • For tissue sections: Pretreat with hydrogen peroxide to block endogenous peroxidases

  • For Western blots: Try alternative blocking agents (casein, commercial blockers)

  • For ELISA: Consider adding 1-5% normal serum from the species of secondary antibody

• Alternative detection systems:

  • Try different secondary antibodies or detection systems

  • Consider using secondary F(ab')2 fragments instead of whole IgG to reduce Fc-mediated binding
    Studies on oligo-conjugated antibodies have shown that concentration adjustments can dramatically reduce background signal without compromising specific binding .

How can I develop a multi-color immunofluorescence assay that includes At1g54790 detection?

For multi-color detection involving At1g54790:

• Antibody selection criteria:

  • Ensure primary antibodies are from different host species

  • Alternatively, use directly conjugated primary antibodies

  • Verify no cross-reactivity between secondary antibodies

• Staining protocol modifications:

  • Sequential staining may be necessary to avoid cross-reactivity

  • If using directly conjugated antibodies, apply simultaneously

  • Include appropriate controls for each antibody individually

• Advanced considerations:

  • Account for spectral overlap between fluorophores

  • Consider using quantum dots for narrow emission spectra

  • Use linear unmixing algorithms for closely spaced emission spectra
    Secondary antibodies should be carefully selected to avoid cross-reactivity. The optimal specificity for each secondary antibody in multiple labeling is IgG(H+L) with adsorption against immunoglobulins from other species used in the experiment .

How do I properly quantify At1g54790 protein expression levels from Western blot data?

For accurate protein quantification:

• Experimental design requirements:

  • Include a dilution series of recombinant At1g54790 or positive control sample

  • Use appropriate loading controls (e.g., tubulin, actin, or total protein stain)

  • Ensure samples are within the linear detection range of the system

• Analysis procedure:

  • Use densitometry software (ImageJ, Image Lab, etc.)

  • Normalize to loading controls

  • Create a standard curve from the dilution series

  • Plot relative or absolute expression levels

• Statistical considerations:

  • Perform at least three biological replicates

  • Apply appropriate statistical tests (t-test, ANOVA)

  • Report confidence intervals or standard error
    Quantitative Western blot analyses have been successfully applied to measure relative protein levels in Arabidopsis studies, as demonstrated in research on histone modifications .

What experimental approaches can resolve contradictory results between At1g54790 antibody staining and gene expression data?

When antibody detection and transcript data don't align:

• Possible explanations:

  • Post-transcriptional regulation mechanisms

  • Protein stability/degradation differences

  • Antibody detection limitations

  • Technical issues with either method

• Resolution strategies:

  • Employ multiple antibodies targeting different epitopes

  • Use tagged transgenic lines (GFP-At1g54790) as complementary approach

  • Perform protein half-life studies with cycloheximide chase

  • Investigate potential post-translational modifications

  • Examine subcellular localization patterns

• Integrated analysis approach:

  • Combine proteomics, transcriptomics, and antibody-based detection

  • Analyze protein complexes that may mask epitopes

  • Consider developmental timing differences
    Discrepancies between protein detection and transcript levels have been observed in several plant studies and often reveal important regulatory mechanisms .

How can I apply machine learning approaches to improve At1g54790 antibody-based detection systems?

Advanced computational methods can enhance antibody-based research:

• Active learning strategies:

  • Start with small labeled datasets and iteratively expand

  • Use uncertainty sampling to prioritize ambiguous data points

  • Implement cross-validation to assess model performance

• Experimental design optimization:

  • Predict optimal antibody concentrations based on previous experiments

  • Identify critical variables affecting antibody performance

  • Reduce experimental iterations through predictive modeling

• Image analysis enhancements:

  • Apply convolutional neural networks for automated signal detection

  • Implement segmentation algorithms for tissue-specific quantification

  • Use transfer learning to leverage data from related protein studies
    Active learning approaches have demonstrated significant improvements in experimental efficiency for antibody-antigen binding predictions, reducing the number of required variants by up to 35% .

How can At1g54790 antibodies be used for single-cell protein analysis in plant tissues?

Single-cell approaches with At1g54790 antibodies include:

• Methodological considerations:

  • Tissue preparation: Optimize protoplast isolation while preserving protein epitopes

  • Fixation protocols: Use mild fixatives that maintain antibody accessibility

  • Signal amplification: Consider tyramide signal amplification or proximity ligation assays

• Single-cell techniques compatible with At1g54790 antibodies:

  • Mass cytometry (CyTOF) with metal-conjugated antibodies

  • Single-cell Western blotting

  • Microfluidic antibody capture

• Data analysis approaches:

  • Dimension reduction techniques (tSNE, UMAP)

  • Clustering algorithms for cell type identification

  • Trajectory inference for developmental studies
    Multimodal analysis combining antibody detection with transcriptomics has been successfully applied in plant research, offering insights into protein-RNA relationships at single-cell resolution .

What are the considerations for developing new epitope-specific antibodies for detecting specific post-translational modifications of At1g54790?

For post-translational modification (PTM)-specific antibodies:

• Epitope selection strategies:

  • Identify likely modification sites through computational prediction

  • Focus on conserved motifs surrounding the modification site

  • Consider accessibility of the epitope in the native protein

• Validation requirements:

  • Test against both modified and unmodified peptides/proteins

  • Verify with mutants where modification sites are altered

  • Confirm with mass spectrometry analysis

  • Validate across different experimental conditions that affect modification status

• Application considerations:

  • Determine if denaturing conditions are required for epitope access

  • Evaluate effects of sample preparation on modification preservation

  • Develop specific blocking strategies for non-specific interactions
    PTM-specific antibodies have been critical in understanding regulatory mechanisms in plants, particularly for histone modifications research .

What are the challenges and solutions for using At1g54790 antibodies in plant species beyond Arabidopsis?

Cross-species applications present specific challenges:

• Sequence homology considerations:

  • Perform sequence alignment of At1g54790 orthologs across target species

  • Identify conserved epitopes as targets for cross-reactive antibodies

  • Consider generating species-specific antibodies for divergent regions

• Validation approaches for cross-species use:

  • Western blot validation with recombinant proteins from target species

  • Include positive controls from Arabidopsis alongside new species

  • Use epitope-tagged versions in non-model species when antibodies fail

• Optimization strategies for diverse plant materials:

  • Adjust extraction buffers for species with different metabolite profiles

  • Modify fixation protocols for species with different cell wall compositions

  • Test multiple antibody concentrations when transferring protocols
    Antibodies developed against Arabidopsis proteins have variable cross-reactivity patterns in other species, as demonstrated by BAK1 antibodies which react with tomato proteins but not with rice or barley proteins .