At5g09300 Antibody

What is the AT5G09300 gene and its protein function in Arabidopsis thaliana?

AT5G09300 encodes a protein involved in cellular metabolism and stress responses in Arabidopsis thaliana. Similar to other Arabidopsis proteins like ATG5 (AT5G17290), it plays a role in critical metabolic pathways. While ATG5 is involved in autophagy and forms a conjugate with ATG12 for nutrient recycling , AT5G09300 functions in separate but potentially related signaling pathways. Research techniques using antibodies raised against this protein can help elucidate its specific roles in plant developmental processes and stress responses, similar to methodologies used with other plant proteins.

What are the optimal storage conditions for AT5G09300 antibody preparations?

For maximum stability and activity retention, store lyophilized AT5G09300 antibody at -20°C. After reconstitution, it is critical to make small aliquots to avoid repeated freeze-thaw cycles, which can significantly degrade antibody quality. Based on protocols for similar Arabidopsis antibodies, reconstitution should be performed with sterile water (typically 50 μl for a standard vial) . Before opening, briefly centrifuge tubes to collect any material that may adhere to the cap or sides. These storage protocols are similar to those used for other plant antibodies such as anti-ATG5, which has demonstrated long-term stability under these conditions .

How do I validate AT5G09300 antibody specificity for my experiments?

Validation of AT5G09300 antibody specificity requires multiple approaches:

• Western blot analysis using:

  • Recombinant AT5G09300 protein as a positive control

  • Arabidopsis wild-type tissue alongside knockout mutants

  • Related proteins to check for cross-reactivity

• Immunoprecipitation followed by mass spectrometry to confirm target protein enrichment

• Competition assays where pre-incubation with recombinant AT5G09300 should abolish signal

This approach mirrors validation methods used for other plant antibodies, where reactivity against recombinant proteins alongside non-reactivity against related proteins (e.g., ATG5 antibodies not cross-reacting with 6xHis-ATG7) establishes specificity .

What is the recommended western blot protocol for AT5G09300 antibody?

The optimal western blot protocol for AT5G09300 antibody involves:

• Sample preparation:

  • Grind 100 mg plant tissue in liquid nitrogen

  • Add 300 μl extraction buffer containing protease inhibitors

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

  • Collect supernatant and determine protein concentration

• Gel electrophoresis and transfer:

  • Load 20-30 μg protein per lane on a 10-12% SDS-PAGE gel

  • Transfer to PVDF membrane at 100V for 60 minutes

• Antibody incubation:

  • Block membrane with 5% non-fat milk in TBST for 1 hour

  • Incubate with AT5G09300 antibody at 1:1000 dilution overnight at 4°C

  • Wash 3x with TBST

  • Incubate with secondary antibody at 1:5000 for 1 hour

  • Wash 3x with TBST

• Detection:

  • Use ECL detection system

  • Expected band size should be verified against predicted molecular weight

This protocol is based on successful methodologies used with similar Arabidopsis antibodies, which typically use dilutions around 1:1000 for optimal results .

How can I optimize immunolocalization studies using AT5G09300 antibody?

For successful immunolocalization of AT5G09300 in plant tissues:

• Fixation options:

  • 4% paraformaldehyde for structural preservation

  • Ethanol:acetic acid (3:1) for better antibody accessibility

• Tissue preparation:

  • Embed in paraffin or resin depending on required resolution

  • Section at 5-10 μm thickness for light microscopy

  • Use 70-100 nm sections for electron microscopy

• Antibody application:

  • Block with 2% BSA + 0.1% Triton X-100

  • Use AT5G09300 antibody at 1:100 to 1:500 dilution

  • Include peptide competition controls

  • Use pre-immune serum as negative control

• Detection systems:

  • Fluorescent secondary antibodies for confocal microscopy

  • Gold-conjugated secondaries for electron microscopy

For co-localization studies, combining AT5G09300 antibody with markers for specific organelles helps determine the protein's subcellular distribution, similar to approaches used in autophagy research with ATG5 antibodies .

What controls should be included when working with AT5G09300 antibody?

Essential controls for AT5G09300 antibody experiments include:

These controls are critical for reliable interpretation of results, following standard practices in plant molecular biology research where antibody specificity must be thoroughly validated .

How can I use AT5G09300 antibody to study protein degradation pathways in plants?

To investigate AT5G09300's involvement in protein degradation pathways:

• Autophagy connection:

  • Compare AT5G09300 protein levels before and after autophagy induction

  • Use chemical inhibitors (e.g., 3-methyladenine) to block autophagy

  • Co-immunoprecipitate with known autophagy components like ATG5

• Proteasome-mediated degradation:

  • Treat samples with MG132 to inhibit proteasome

  • Monitor AT5G09300 protein levels

  • Assess ubiquitination status through immunoprecipitation

• Nutrient deprivation experiments:

  • Nitrogen starvation often triggers autophagy and protein degradation pathways

  • Monitor AT5G09300 levels during N starvation and recovery

  • Compare with known autophagy markers

This approach builds on established research methodologies showing that autophagy-related proteins like ATG5 form essential conjugates (e.g., with ATG12) to facilitate nutrient recycling during stress conditions in plants .

How does AT5G09300 protein expression change under different stress conditions?

Comprehensive analysis of AT5G09300 expression under stress requires:

• Abiotic stress time course:

  • Drought: withhold water for 0, 6, 12, 24, and 48 hours

  • Salt: treat with 0, 50, 100, 150, and 200 mM NaCl

  • Heat: expose to 37°C for 0, 1, 3, 6, and 12 hours

  • Cold: expose to 4°C for 0, 6, 12, 24, and 48 hours

• Biotic stress:

  • Pathogen infection (bacteria, fungi)

  • Herbivory simulation with methyl jasmonate

• Analysis methods:

  • Western blot with AT5G09300 antibody to quantify protein levels

  • qRT-PCR to correlate with transcript changes

  • Immunolocalization to detect subcellular redistribution

• Data interpretation:

  • Normalize protein levels to internal standards

  • Compare with known stress-responsive markers

  • Correlate with physiological data

Research on other Arabidopsis proteins suggests stress significantly impacts metabolic pathways, including autophagy and amino acid metabolism, making these investigations valuable for understanding plant adaptation mechanisms .

What approaches can be used to study AT5G09300 protein-protein interactions?

To elucidate AT5G09300 protein interaction networks:

• Co-immunoprecipitation (Co-IP):

  • Use AT5G09300 antibody for immunoprecipitation

  • Identify binding partners via mass spectrometry

  • Confirm interactions with reverse Co-IP

  • Validate with reciprocal western blots

• Proximity-dependent labeling methods:

  • Create AT5G09300-BioID or TurboID fusion proteins

  • Express in Arabidopsis

  • Identify proximal proteins after biotin labeling

• Yeast two-hybrid screening:

  • Use AT5G09300 as bait against Arabidopsis cDNA library

  • Validate candidates in planta

• Split-fluorescent protein complementation:

  • Fuse AT5G09300 to one half of a fluorescent protein

  • Fuse candidate interactors to complementary half

  • Visualize interactions in plant cells

This multi-method approach addresses the challenge of identifying protein interactions in complex plant systems, similar to methods used to identify ATG5-ATG12 conjugates essential for autophagy function in plants .

Why do I see multiple bands or non-specific binding with AT5G09300 antibody?

Multiple bands in AT5G09300 antibody experiments may occur due to:

• Post-translational modifications:

  • Phosphorylation alters protein migration

  • Ubiquitination creates higher molecular weight bands

  • Glycosylation affects protein mobility

• Proteolytic degradation:

  • Add fresh protease inhibitors to all buffers

  • Keep samples cold throughout preparation

  • Consider using urea-based extraction for recalcitrant proteins

• Isoforms and splice variants:

  • Check genome databases for predicted variants

  • Verify with RT-PCR for transcript variants

• Cross-reactivity:

  • Optimize antibody dilution (try 1:2000 instead of 1:1000)

  • Increase blocking reagent concentration

  • Pre-absorb antibody with plant extract from knockout lines

This troubleshooting guidance reflects common challenges with plant antibodies, as noted in the literature where even validated antibodies like anti-ATG5 may detect non-specific bands requiring careful optimization .

How can I quantify western blot data from AT5G09300 antibody experiments?

For accurate quantification of AT5G09300 western blot data:

• Image acquisition:

  • Use a digital imaging system with linear detection range

  • Avoid overexposure that saturates pixel values

  • Capture multiple exposures to ensure linearity

• Software analysis:

  • Use ImageJ or similar software for densitometry

  • Define lanes and plot intensity profiles

  • Measure peak areas rather than heights

• Normalization methods:

  • Express AT5G09300 signal relative to loading control

  • Prepare standard curves with recombinant protein

  • Include reference sample across all blots for inter-blot comparison

• Statistical analysis:

  • Run at least three biological replicates

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

  • Report means with standard deviation or standard error

These quantification principles are essential for detecting subtle changes in protein expression, particularly in response to environmental stresses or genetic manipulations .

How can I combine AT5G09300 antibody studies with transcriptomic analysis?

Integrating antibody-based protein studies with transcriptomics:

• Experimental design:

  • Collect parallel samples for protein and RNA extraction

  • Include multiple time points to capture regulatory dynamics

  • Apply identical treatments to ensure comparability

• Transcriptome analysis:

  • Perform RNA-seq or microarray analysis

  • Focus on AT5G09300 and related genes

  • Identify co-regulated gene networks

• Protein analysis:

  • Use AT5G09300 antibody for western blot quantification

  • Compare protein levels with transcript abundance

  • Identify post-transcriptional regulation

• Data integration:

  • Create correlation matrices between transcript and protein levels

  • Identify discordant patterns suggesting regulatory mechanisms

  • Use pathway analysis to place AT5G09300 in functional networks

This integrated approach has proven valuable in studies of plant metabolism, such as those examining branched-chain amino acid catabolism in relation to triacylglycerol synthesis .

What CRISPR/Cas9 strategies are most effective for validating AT5G09300 antibody specificity?

CRISPR/Cas9 gene editing provides powerful validation tools:

• Knockout strategy:

  • Design gRNAs targeting early exons of AT5G09300

  • Create complete knockouts to serve as negative controls

  • Screen transformants by PCR and sequencing

  • Confirm absence of protein using AT5G09300 antibody

• Epitope tagging:

  • Design repair templates to add HA or FLAG tags to AT5G09300

  • Compare detection with AT5G09300 antibody vs. commercial tag antibodies

  • Verify co-localization in immunofluorescence studies

• Domain modification:

  • Create precise modifications in key functional domains

  • Assess changes in protein size, localization, or interactions

  • Use as controls for antibody specificity and function studies

This approach mirrors successful strategies used in Arabidopsis research, including forward genetic screens that identified autophagy mutants disrupting ATG genes .