At1g28610 Antibody

What is the AT1G28610 gene and its encoded protein?

AT1G28610 (also known as GGL3) encodes a member of the GDSL-motif esterase/acyltransferase/lipase family in Arabidopsis thaliana. The protein belongs to the GDSL-like Lipase/Acylhydrolase superfamily, which is characterized by a conserved GDSL motif near the N-terminus . These enzymes display broad substrate specificity and can catalyze various reactions including acyltransfer and hydrolysis with both lipid and non-lipid substrates . The protein has been assigned the UniProt accession number Q9SHP6, which serves as a unique identifier in protein databases .

What experimental applications is the AT1G28610 antibody suitable for?

The AT1G28610 antibody is primarily used in plant molecular biology research focusing on Arabidopsis thaliana. Common applications include Western blotting (immunoblotting), immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC)/immunofluorescence (IF). When selecting an antibody for a specific application, researchers should review validation data from repositories or search engines that compile experimental evidence for antibody performance . For targeted applications in polyploidy research, the antibody has been used to study gene expression alterations in autopolyploid Arabidopsis, where AT1G28610 showed differential expression patterns .

How should I validate the specificity of the AT1G28610 antibody?

Validation of the AT1G28610 antibody should include multiple approaches to ensure specificity. Begin with a Western blot analysis using wild-type Arabidopsis tissue samples alongside negative controls (knockout or knockdown lines if available). The expected molecular weight for the AT1G28610 protein should be confirmed, and absence or reduction of signal in controls should be observed. Secondary validation methods include immunoprecipitation followed by mass spectrometry to confirm target capture, and immunofluorescence with appropriate co-localization markers to verify subcellular localization patterns . For comprehensive validation, consider using tissue from different developmental stages, as gene expression studies have shown that AT1G28610 expression can vary developmentally in both diploid and polyploid plants .

How can I optimize immunoprecipitation protocols with the AT1G28610 antibody?

For optimal immunoprecipitation (IP) with the AT1G28610 antibody, consider the following methodological approach:

• Sample preparation: Homogenize fresh Arabidopsis tissue in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease inhibitors.

• Pre-clearing: Incubate lysate with protein A/G beads for 1 hour at 4°C to reduce non-specific binding.

• Antibody binding: Add 2-5 μg of AT1G28610 antibody (CSB-PA886681XA01DOA) to 500 μl of pre-cleared lysate and incubate overnight at 4°C with gentle rotation .

• Bead capture: Add fresh protein A/G beads and incubate for 2-4 hours at 4°C.

• Washing: Perform stringent washes (at least 4-5) with decreasing salt concentrations to remove non-specific interactions.
  For challenging samples, consider crosslinking the antibody to beads using dimethyl pimelimidate to prevent antibody co-elution. When investigating protein-protein interactions, milder lysis conditions may preserve complexes, while more stringent conditions may be needed when studying post-translational modifications .

What approaches can improve detection sensitivity in Western blots using AT1G28610 antibody?

To enhance Western blot sensitivity with AT1G28610 antibody:

• Sample enrichment: Consider subcellular fractionation to concentrate the target protein, particularly as GDSL lipases may have specific subcellular localizations.

• Blocking optimization: Test different blocking agents (5% BSA often performs better than milk for phospho-specific epitopes).

• Primary antibody incubation: Use the recommended dilution of AT1G28610 antibody (typically 1:1000) in TBS-T with 5% BSA, and incubate overnight at 4°C.

• Signal amplification: Implement a biotin-streptavidin system or use high-sensitivity ECL substrates for chemiluminescence detection.

• Exposure optimization: Use incremental exposure times to determine the optimal signal-to-noise ratio.
  For difficult-to-detect signals, consider antigen retrieval methods such as heat-mediated or enzymatic approaches, which can expose hidden epitopes, particularly if the protein has complex folding or is embedded in membranes . In cases where AT1G28610 shows differential expression between diploid and polyploid plants, loading controls should be carefully selected to account for potential global changes in protein expression .

How can AT1G28610 antibody be used to study gene expression changes in polyploid plants?

The AT1G28610 antibody serves as a valuable tool for studying gene expression alterations in polyploid Arabidopsis. Research has identified AT1G28610 as differentially expressed in autotetraploid lines compared to diploids, making it a potential marker for monitoring polyploidy . To implement this approach:

• Experimental design: Include diploid controls, newly induced autopolyploids, and established polyploid lines to distinguish immediate versus long-term expression changes.

• Tissue selection: Analyze multiple tissue types (seedlings, leaves, reproductive structures) as expression differences may be tissue-specific .

• Quantitative analysis: Combine Western blotting with densitometry to quantify protein levels across ploidy levels.

• Correlation with transcriptome data: Compare protein levels detected by immunoblotting with transcriptome data from microarray or RNA-seq experiments to assess correlation between transcript and protein abundance .
  This approach has revealed that AT1G28610 expression alterations in autopolyploids are ecotype-dependent and developmentally specific, and may be associated with changes in DNA methylation . These findings suggest that the antibody can be used to investigate epigenetic mechanisms regulating gene expression in polyploids.

What considerations are important when using the AT1G28610 antibody in chromatin immunoprecipitation (ChIP) experiments?

When applying AT1G28610 antibody in ChIP experiments:

• Crosslinking optimization: For plant tissues, longer formaldehyde fixation times (15-20 minutes) may be necessary compared to animal cells, with vacuum infiltration to ensure complete tissue penetration.

• Chromatin fragmentation: Optimize sonication conditions specifically for plant tissues, aiming for DNA fragments of 200-500 bp.

• Antibody selection: Verify that the AT1G28610 antibody (CSB-PA886681XA01DOA) has been validated for ChIP applications, as not all antibodies that work in Western blotting work effectively in ChIP .

• Controls: Include mock IP (no antibody), IgG control, and input sample controls in every experiment.

• qPCR primer design: Design primers for regions with potential lipase/acyltransferase activity regulation, considering both proximal promoter regions and potential enhancers.
  Research has shown that alterations in gene expression in polyploids may be associated with changes in DNA methylation . Therefore, combining ChIP with bisulfite sequencing or methylcytosine antibodies (mCIP) for affinity purification can provide insights into the relationship between chromatin state and AT1G28610 expression in different ploidy contexts .

How do I interpret unexpected molecular weight bands when using AT1G28610 antibody?

When encountering unexpected bands with AT1G28610 antibody in Western blots:

• Higher molecular weight bands may indicate:

  • Post-translational modifications (phosphorylation, glycosylation)

  • Protein dimers or multimers, particularly if bands appear at approximately double the expected weight

  • Protein complexes resistant to denaturation

• Lower molecular weight bands may represent:

  • Proteolytic degradation products (improve sample preparation by adding fresh protease inhibitors)

  • Alternative splice variants (verify against known transcript variants in databases)

  • Cross-reactivity with related GDSL lipase family members (Arabidopsis contains multiple GDSL lipases with structural similarities)
    To distinguish between these possibilities, perform additional validation using techniques such as mass spectrometry to identify the proteins in unexpected bands . For investigating post-translational modifications, consider using phosphatase treatment or deglycosylation enzymes before Western blotting to confirm the nature of modifications. In polyploid studies, verify whether unexpected bands appear consistently across different ploidy levels or are specific to certain genetic backgrounds .

What strategies should I employ when confronted with inconsistent results using AT1G28610 antibody?

When facing inconsistent results with AT1G28610 antibody:

• Antibody validation: Confirm antibody specificity using Western blot on wild-type versus knockout/knockdown samples if available.

• Sample preparation variables:

  • Standardize tissue collection (time of day, growth stage, stress conditions)

  • Optimize protein extraction protocols for plant tissues, considering the potential membrane association of lipases

  • Use fresh antibody aliquots and avoid freeze-thaw cycles

• Technical controls:

  • Include loading controls appropriate for plant samples (e.g., actin, tubulin)

  • Run positive control samples with known AT1G28610 expression

  • Implement internal standards for quantification

• Experimental design improvements:

  • Increase biological replicates to account for natural variation

  • Control for environmental variables that might affect GDSL lipase expression

  • Consider circadian regulation of gene expression
    Research has demonstrated that expression of AT1G28610 can vary between ecotypes and different ploidy levels . Therefore, inconsistent results may reflect genuine biological variation rather than technical issues. Comprehensive documentation of experimental conditions and genetic backgrounds is essential for proper interpretation .

How can AT1G28610 antibody contribute to studying epigenetic regulation in polyploid plants?

The AT1G28610 antibody offers valuable insights into epigenetic regulation mechanisms in polyploid plants:

• Combined ChIP-seq and DNA methylation analysis: Use the antibody in ChIP followed by high-throughput sequencing, combined with whole-genome bisulfite sequencing to correlate protein binding with DNA methylation patterns.

• Investigation of paramutation-like phenomena: Research has shown paramutation-like interactions of epialleles in autotetraploid Arabidopsis . The AT1G28610 antibody can help track protein levels associated with genes undergoing such epigenetic interactions.

• Methylation-specific immunoprecipitation: Combined analysis using the AT1G28610 antibody and methylcytosine antibodies can reveal relationships between gene expression and DNA methylation status .

• Trans-generational studies: Track AT1G28610 protein levels across multiple generations of polyploids to determine the stability of expression changes.
  Research has specifically identified AT1G28610 (MRD1) as being upregulated in Col-0 autopolyploid tissues and has linked this expression pattern to DNA methylation state . The antibody can therefore serve as a tool to investigate how genome doubling triggers specific epigenetic changes and how these are maintained across cell divisions and generations.

What methodological approaches can integrate AT1G28610 antibody data with other -omics datasets in polyploidy research?

Integrating AT1G28610 antibody data with other -omics approaches requires sophisticated methodological strategies:

• Multi-omics integration workflow:

  • Protein-level quantification using AT1G28610 antibody (Western blot, ELISA)

  • Transcriptome analysis (RNA-seq, microarray)

  • Epigenome profiling (methylome, ChIP-seq for histone modifications)

  • Metabolomics focusing on lipid substrates (GC-MS, LC-MS)

• Data normalization and comparative analysis:

  • Standardize data across platforms using appropriate reference genes/proteins

  • Apply statistical methods designed for integrating heterogeneous data types

  • Use dimensionality reduction techniques to identify patterns across datasets

• Functional validation approaches:

  • CRISPR-Cas9 mediated gene editing of AT1G28610

  • Overexpression studies combined with phenotypic analysis

  • Enzymatic activity assays with purified protein

• Biological network construction:

  • Identify co-expressed genes and potential interaction partners

  • Map AT1G28610 function within metabolic pathways

  • Determine regulatory relationships with transcription factors
    Research has already demonstrated that combining protein expression data with transcriptome analysis and methylation studies can reveal complex relationships between ploidy, gene expression, and epigenetic regulation . For example, the overexpression of MRD1 (AT1G28610) in Col-0 tetraploid was found to be inherited in Col-0 tetraploid × Ler-0 tetraploid hybrids, indicating genetic and epigenetic factors controlling its expression .