At1g64540 Antibody

What is AT1G64540 and what is its function in Arabidopsis thaliana?

AT1G64540 is a gene in Arabidopsis thaliana that encodes a protein involved in plant development and stress responses. Based on genomic analysis, this gene has been identified as a target for artificial microRNA (amiRNA) studies, indicating its potential significance in plant regulatory networks . The gene is part of the transcriptional landscape that shapes Arabidopsis thaliana's pattern-triggered immunity, which is activated upon recognition of molecular patterns of different biological origins .

How is AT1G64540 expression regulated in different plant tissues?

AT1G64540 expression varies across different tissues and developmental stages. Transcriptome analysis reveals that expression patterns can be tissue-specific and developmentally regulated. Studies using autopolyploid Arabidopsis thaliana have shown that gene expression alterations, including those potentially affecting AT1G64540, depend on ecotype (genome composition) and are developmentally specific . This suggests that experimental design for antibody-based detection of AT1G64540 protein must account for these variables.

What genomic tools are available for studying AT1G64540?

Several genomic resources exist for AT1G64540 research:

• amiRNA clones: The Arabidopsis Biological Resource Center (ABRC) provides amiRNA individual clones in pAmiR vector targeting AT1G64540 (stock number CSHL_023355) .

• Gateway Technology: These clones utilize Gateway Technology, which is available under the Gateway Open Architecture Policy for scientific research without restrictive licensing requirements .

• Transformation systems: The pSoup (stock number CD3-1124) helper plasmid is required for efficient transformation of plants with AT1G64540-targeting constructs .

What strategies improve specificity when developing antibodies against plant proteins like AT1G64540?

Developing highly specific antibodies against plant proteins requires multiple strategic approaches:

• Epitope selection optimization: Select unique peptide sequences that:

  • Have low homology with other Arabidopsis proteins

  • Are predicted to be surface-exposed in the native protein

  • Avoid regions with post-translational modifications that could mask epitopes

• Multi-epitope approach: Developing antibodies against 2-3 distinct regions of AT1G64540 increases confidence in detection specificity, similar to approaches used with G-protein-coupled receptors in other research contexts .

• Expression system considerations: For recombinant AT1G64540 protein production, plant-based expression systems may preserve native folding and post-translational modifications better than bacterial systems, improving antibody recognition of the native protein.

How can antibody specificity for AT1G64540 be validated?

Rigorous validation is essential to ensure antibody specificity. A comprehensive validation protocol includes:

• Knockout/knockdown controls: Testing antibodies on tissues from AT1G64540 knockout plants or those treated with amiRNA targeting AT1G64540 (available through ABRC) .

• Western blot analysis: Comparing signal between wild-type and mutant/RNAi samples, with expected size confirmation. Gradient gels (10-15%) provide better resolution for detailed analysis.

• Immunoprecipitation followed by mass spectrometry: This identifies all proteins captured by the antibody, confirming AT1G64540 enrichment and detecting potential cross-reactivity.

• Competition assays: Pre-incubating antibodies with purified AT1G64540 peptide/protein should abolish specific signals in immunodetection methods.

Similar validation approaches have been successfully employed with other antibodies, such as those against angiotensin II receptor type 1 (AT1R) and endothelin-1 type A receptor (ETAR) .

What are optimal protocols for using AT1G64540 antibodies in Western blotting?

The following optimized protocol is recommended for AT1G64540 Western blotting:

Sample preparation:

• Extract total protein from plant tissues using buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail.

• Sonicate briefly (3 × 10s) to improve extraction.

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

• Quantify protein concentration using Bradford assay.

Western blotting parameters:

• Load 20-50 μg of total protein per lane.

• Use 10-12% SDS-PAGE for optimal separation.

• Transfer to PVDF membrane (0.45 μm) at 100V for 60 minutes.

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

• Incubate with primary AT1G64540 antibody (1:1000 dilution) overnight at 4°C.

• Wash 3 × 10 minutes with TBST.

• Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature.

• Develop using enhanced chemiluminescence.

This protocol incorporates principles from antibody-based detection methodologies that have been successfully used in other research contexts .

How should sample preparation be optimized for immunoprecipitation with AT1G64540 antibodies?

Optimized immunoprecipitation of AT1G64540 requires careful consideration of extraction conditions:

Extraction buffer optimization:

Immunoprecipitation protocol:

• Prepare 500 μg total protein extract in 500 μl IP buffer.

• Pre-clear with 50 μl Protein A/G beads for 1 hour at 4°C.

• Incubate pre-cleared lysate with 5 μg AT1G64540 antibody overnight at 4°C with gentle rotation.

• Add 50 μl fresh Protein A/G beads and incubate for 3 hours at 4°C.

• Wash beads 5 times with IP buffer.

• Elute proteins by boiling in 50 μl 2× SDS sample buffer.

This protocol incorporates principles from immunoprecipitation techniques used for other proteins in research contexts .

What approaches are recommended for studying AT1G64540 protein-protein interactions in vivo?

Several complementary approaches can be employed to study AT1G64540 protein-protein interactions:

• Co-immunoprecipitation with AT1G64540 antibody:

  • Perform immunoprecipitation as described above

  • Analyze co-precipitated proteins by mass spectrometry

  • Validate interactions with Western blotting using antibodies against suspected interacting partners

• Proximity-based labeling:

  • Generate transgenic Arabidopsis expressing AT1G64540 fused to BioID or TurboID

  • Biotin-labeled proximal proteins can be purified and identified by mass spectrometry

  • This approach has advantages for capturing transient or weak interactions

• Split-fluorescent protein complementation:

  • Express AT1G64540 fused to one half of a split fluorescent protein

  • Express candidate interacting proteins fused to the complementary half

  • Reconstitution of fluorescence indicates interaction

  • This method allows visualization of interactions in living plant cells

• Yeast two-hybrid screening:

  • Use AT1G64540 as bait to screen Arabidopsis cDNA libraries

  • Validate positive interactions using the above in planta methods

These approaches are similar to those used in studying protein-protein interactions in other research contexts, such as those employed in CAR T cell engineering studies .

How can ChIP-seq experiments with AT1G64540 antibodies be optimized?

Optimizing ChIP-seq for AT1G64540 requires careful consideration of several parameters:

Chromatin preparation:

• Crosslink Arabidopsis tissue with 1% formaldehyde for 10 minutes under vacuum.

• Quench with 0.125 M glycine for 5 minutes.

• Grind tissue in liquid nitrogen and resuspend in extraction buffer.

• Filter through miracloth and isolate nuclei by centrifugation.

• Resuspend nuclei in sonication buffer and sonicate to generate 200-500 bp fragments.

ChIP protocol optimization:

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

• Incubate 10-15 μg of chromatin with 5 μg AT1G64540 antibody overnight at 4°C.

• Add Protein A/G beads and incubate for 3 hours at 4°C.

• Perform sequential washes with increasing stringency buffers.

• Reverse crosslinks and purify DNA for sequencing.

Controls to include:

• Input chromatin (pre-immunoprecipitation)

• IgG control (non-specific antibody)

• Positive control (antibody against known DNA-binding protein)

• Biological replicates (minimum of 3)

This approach incorporates general ChIP-seq principles that have been applied to study transcriptional landscapes in Arabidopsis thaliana .

How does AT1G64540 expression change in polyploid Arabidopsis compared to diploid?

Studies on autopolyploid Arabidopsis have revealed significant insights about gene expression changes between ploidy levels:

• Ploidy-dependent expression:
  Research indicates that gene expression alterations in Arabidopsis autotetraploids depend on ecotype (genome composition) and are developmentally specific . For AT1G64540, expression patterns may vary between diploid and polyploid plants depending on the genetic background.

• Methylation status and expression:
  DNA methylation state can impact gene expression in polyploids. Analysis of the genomic region around AT1G64540 in different ploidy levels could reveal methylation patterns correlating with expression changes .

• Odd vs. even ploidy effects:
  Research has documented interesting differences between odd-number (triploid) and even-number (tetraploid) chromosome sets. These differences could affect AT1G64540 expression, similar to observations with other genes like MRD1 .

• Quantification methods:
  To accurately measure AT1G64540 expression differences:

  • RT-PCR and qRT-PCR with specific primers

  • Control and test reactions with melting curve analysis

  • Normalization against stably expressed reference genes across ploidy levels

These approaches are based on methodologies used in genetic and transcriptome analysis of autopolyploid Arabidopsis thaliana .

What methodologies are effective for studying post-translational modifications of AT1G64540?

Post-translational modifications (PTMs) of AT1G64540 can be studied using several complementary approaches:

• Mass spectrometry-based PTM mapping:

  • Immunoprecipitate AT1G64540 using specific antibodies

  • Digest with multiple proteases to increase sequence coverage

  • Analyze by LC-MS/MS with PTM-specific detection methods

  • Search for phosphorylation, ubiquitination, SUMOylation, and glycosylation

• PTM-specific antibody development:

  • Generate antibodies against predicted phosphorylation sites

  • Use these alongside general AT1G64540 antibodies to determine modification state

  • Validate with phosphatase treatments

• In vivo PTM dynamics:

  • Express tagged AT1G64540 in Arabidopsis

  • Subject plants to various stresses or developmental cues

  • Monitor changes in PTM status over time using mass spectrometry

  • Correlate with functional outcomes

• PTM-mimetic mutants:

  • Generate phosphomimetic (S/T to D/E) or phosphonull (S/T to A) mutations

  • Express these in at1g64540 knockout backgrounds

  • Assess functional consequences through phenotypic analysis

These methodologies incorporate principles used in studying post-translational modifications in various research contexts .

How can non-specific binding be minimized when using AT1G64540 antibodies?

Reducing non-specific binding requires systematic optimization of several parameters:

• Blocking optimization:

  • Test multiple blocking agents (BSA, non-fat milk, normal serum)

  • Optimize blocking time (1-3 hours) and temperature

  • Consider commercial blocking reagents specifically designed for plant samples

• Antibody dilution optimization:

  • Perform dilution series (1:500 to 1:5000) to determine optimal concentration

  • More dilute antibody solutions often reduce background

• Buffer composition adjustment:

  • Increase detergent concentration (0.1-0.3% Tween-20) in wash buffers

  • Add 0.1-0.5 M NaCl to reduce ionic interactions

  • Include 0.1% BSA in antibody dilution buffer

• Pre-adsorption protocol:

  • Pre-incubate antibody with protein extract from at1g64540 knockout tissue

  • This captures antibodies that bind to non-target proteins

  • Use the pre-adsorbed antibody solution for the actual experiment

These approaches incorporate principles used to minimize non-specific binding in antibody-based detection systems across various research contexts .

What controls are necessary when using AT1G64540 antibodies in immunolocalization studies?

Rigorous controls are essential for reliable immunolocalization results:

• Primary antibody controls:

  • Negative control: Omit primary antibody

  • Peptide competition: Pre-incubate antibody with immunizing peptide

  • Genetic control: Use tissue from at1g64540 knockout/knockdown plants

• Secondary antibody controls:

  • Autofluorescence control: No primary or secondary antibody

  • Non-specific binding control: Secondary antibody only

• Tissue preparation controls:

  • Fixation control: Compare multiple fixation methods

  • Compare membrane permeabilization methods

• Positive controls:

  • Include antibody against known subcellular marker

  • Co-localize with fluorescent protein fusion if available

These control strategies are based on standard practices in immunolocalization studies and have been adapted for plant research contexts .

How can CRISPR/Cas9 technology enhance AT1G64540 antibody research?

CRISPR/Cas9 technology offers several advantages for AT1G64540 antibody research:

• Epitope tagging at endogenous locus:

  • Use CRISPR to introduce small epitope tags (HA, FLAG, Myc) at the N or C terminus

  • This preserves endogenous expression levels and regulation

  • Commercial antibodies against these tags provide reliable detection

• Knockout generation for antibody validation:

  • Create precise at1g64540 knockout lines

  • These serve as negative controls for antibody specificity testing

  • Compare multiple knockout lines with different guide RNAs

• Domain-specific mutations:

  • Introduce point mutations in specific domains

  • Test antibody recognition of mutant proteins

  • Correlate structure-function relationships

• Promoter modifications:

  • Modify endogenous promoter to alter expression

  • Test antibody sensitivity across different expression levels

  • Create inducible systems for temporal control

These approaches leverage CRISPR technology to enhance antibody research, similar to strategies used in other molecular biology applications .

What are the advantages of using nanobodies instead of conventional antibodies for AT1G64540 research?

Nanobodies offer several distinct advantages over conventional antibodies:

• Size advantages:

  • Smaller size (~15 kDa vs ~150 kDa for conventional antibodies)

  • Better tissue penetration for in vivo imaging

  • Access to epitopes in protein complexes that might be inaccessible to larger antibodies

• Production benefits:

  • Can be expressed in bacterial systems

  • More consistent batch-to-batch production

  • Genetic fusion to reporters or tags is straightforward

• Experimental applications:

  • Super-resolution microscopy with minimal linkage error

  • Intrabodies for in vivo protein tracking

  • Affinity purification with reduced background

• Stability advantages:

  • Greater thermal stability than conventional antibodies

  • Resistant to harsh conditions during experimental procedures

  • Longer shelf life

The use of nanobodies represents an advanced approach similar to recent innovations in antibody technology described for other research applications .