At5g39120 Antibody

What is At5g39120 and why is it significant in plant research?

At5g39120 is a gene in Arabidopsis thaliana that encodes an RmlC-like cupins superfamily protein (UniProt ID: Q9FIC9) . This protein family has significant research interest due to its potential roles in plant immunity and stress responses. The protein belongs to a structurally conserved class with a β-barrel core that may participate in diverse biochemical functions including enzymatic activities and protein-protein interactions in plant defense mechanisms .

What are the standard applications for At5g39120 antibodies in plant research?

At5g39120 antibodies are primarily utilized in:

• Western blot analysis for protein expression and post-translational modification studies

• ELISA assays for quantitative protein detection

• Immunoprecipitation for protein-protein interaction studies

• Immunohistochemistry for localization studies in plant tissues

These techniques are fundamental for understanding the protein's expression patterns during plant development and in response to biotic and abiotic stresses.

What samples are most appropriate for At5g39120 antibody validation?

For proper validation, researchers should use:

• Positive control: Wild-type Arabidopsis thaliana tissue samples where At5g39120 is expressed

• Negative control: Knockout or knockdown lines of At5g39120 (if available)

• Reference samples: Recombinant At5g39120 protein for establishing detection limits

Tissue samples from different plant organs (leaves, roots, stems) should be tested to establish expression patterns under various conditions (e.g., pathogen challenge, abiotic stress) .

How should researchers prepare plant samples for optimal At5g39120 antibody detection?

For optimal detection:

• Extract proteins using a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and protease inhibitor cocktail

• Homogenize plant tissue thoroughly at 4°C to prevent protein degradation

• Centrifuge at 12,000 × g for 15 minutes to remove cellular debris

• Quantify protein concentration using Bradford or BCA assay

• Store protein samples at -80°C with 10% glycerol to maintain antibody epitope integrity

What are the common challenges when using At5g39120 antibodies in Western blot applications?

Researchers frequently encounter these challenges:

• Non-specific binding due to the conserved nature of cupins superfamily proteins

• Weak signal strength requiring optimization of antibody concentration

• Background noise from secondary antibody cross-reactivity

To overcome these issues:

• Use blocking solutions with 5% non-fat milk in TBST for 1 hour at room temperature

• Optimize primary antibody dilutions (typically starting at 1:1000)

• Include additional washing steps (5 × 5 minutes with TBST)

• Consider using highly purified secondary antibodies with minimal cross-reactivity to plant proteins

How can researchers verify the specificity of At5g39120 antibodies?

Verification methods include:

• Immunoblotting with recombinant protein: Compare detection of purified recombinant At5g39120 protein alongside plant extracts

• Knockout/knockdown analysis: Compare antibody reactivity in wild-type versus At5g39120 mutant plants

• Peptide competition assay: Pre-incubate antibody with the immunizing peptide before application to samples

• Mass spectrometry validation: Confirm identity of immunoprecipitated proteins by MS analysis

What are the recommended storage and handling practices for maintaining At5g39120 antibody efficacy?

To maintain antibody effectiveness:

• Store antibody aliquots at -20°C or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles (limit to <5 cycles)

• For short-term storage (1-2 weeks), keep at 4°C with 0.02% sodium azide

• When handling, maintain sterile conditions and use low-protein binding tubes

• Before use, centrifuge antibody solution briefly to collect contents at the bottom of the tube

How can At5g39120 antibodies be utilized in plant immunity research?

For immunity research applications:

• Pathogen challenge experiments: Monitor At5g39120 protein levels before and after inoculation with pathogens to determine involvement in immune responses

• Co-immunoprecipitation studies: Identify potential interaction partners during immune activation using At5g39120 antibodies for pull-down assays

• Comparative analysis: Examine At5g39120 expression across resistant and susceptible plant varieties during infection

• Subcellular localization: Track protein redistribution upon immune activation using immunofluorescence with At5g39120 antibodies

Recent studies on plant immunity proteins demonstrate that interactions between proteins like ATG6 and NPR1 enhance Arabidopsis resistance to pathogens such as Pst DC3000/avrRps4 . Similar approaches could reveal whether At5g39120 participates in these or parallel immune pathways.

What methodologies are recommended for studying At5g39120 protein-protein interactions?

For interaction studies:

• Co-immunoprecipitation (Co-IP): Using At5g39120 antibodies conjugated to agarose/magnetic beads

  • Protein extraction in non-denaturing conditions with 0.1% NP-40

  • Overnight incubation with antibody-conjugated beads at 4°C

  • Extensive washing with decreasing detergent concentrations

  • Western blot analysis for interacting proteins

• Proximity ligation assay (PLA): For detecting in situ interactions

  • Fixation of plant tissue sections in 4% paraformaldehyde

  • Dual labeling with At5g39120 antibody and antibody against suspected interaction partner

  • Detection of proximal proteins (<40 nm) using species-specific secondary antibodies

• Bimolecular Fluorescence Complementation (BiFC): For validating interactions identified via Co-IP

  • Cloning At5g39120 and candidate interactors into split-YFP vectors

  • Transient expression in plant protoplasts or N. benthamiana

  • Confocal microscopy to detect reconstituted fluorescence

How can researchers integrate At5g39120 antibody data with transcriptomic analyses?

For integrated multi-omics approaches:

• Expression correlation analysis: Compare At5g39120 protein levels (determined by immunoblotting) with corresponding mRNA levels (from RNA-Seq or qRT-PCR)

• Time-course studies: Track both protein and transcript dynamics during stress responses or developmental transitions

• Pathway enrichment: Integrate At5g39120 protein expression data with transcriptomic datasets to identify coordinated regulatory networks

• Post-translational modification mapping: Combine immunoprecipitation with mass spectrometry to identify modifications not apparent at the transcript level

What approaches can be used to study At5g39120 in the context of plant stress responses?

For stress response studies:

• Comparative stress analysis: Apply multiple stresses (drought, salt, pathogen) and monitor At5g39120 protein levels using quantitative Western blotting

• Cellular localization changes: Track protein redistribution during stress using subcellular fractionation followed by immunoblotting

• Transgenic approaches: Compare stress tolerance in plants with altered At5g39120 expression

• Phosphorylation state analysis: Use phospho-specific antibodies (if available) to monitor activation status during stress

How should researchers interpret inconsistencies between At5g39120 transcript and protein levels?

When transcript and protein levels don't correlate:

• Post-transcriptional regulation: Consider microRNA-mediated silencing or mRNA stability factors

• Translational efficiency: Examine polysome association of At5g39120 transcripts

• Protein stability: Conduct protein half-life studies using cycloheximide chase assays

• Compartmentalization: Assess whether protein localization (rather than absolute levels) is changing

• Technical considerations: Verify antibody specificity under the specific experimental conditions

Research on plant immune proteins has shown that protein abundance can be regulated at multiple levels beyond transcription. For instance, ATG6 has been shown to increase NPR1 protein levels and stability in Arabidopsis, highlighting the importance of protein-level regulation in plant immunity .

What experimental design considerations are critical when using At5g39120 antibodies?

Key experimental design factors:

• Appropriate controls:

  • Technical: Secondary antibody only, pre-immune serum controls

  • Biological: Wild-type vs. gene knockout/knockdown lines

  • Loading: Total protein stains (Ponceau S) rather than single reference proteins

• Replication strategy:

  • Minimum three biological replicates from independent plant populations

  • Technical replicates within each biological sample

  • Randomization of sample processing order

• Quantification methods:

  • Digital image analysis using validated software (ImageJ, Image Lab)

  • Standard curves with recombinant protein for absolute quantification

  • Statistical analysis appropriate for the experimental design

How can researchers effectively compare data between different At5g39120 antibody sources?

For cross-antibody comparisons:

• Conduct side-by-side validation with recombinant At5g39120 protein

• Determine epitope regions for each antibody and assess potential differences in detection

• Perform peptide competition assays to confirm specificity

• Test under identical conditions using the same biological samples

• Consider using a normalization strategy based on recombinant protein standards

How does the presence of plant-specific compounds affect At5g39120 antibody performance?

Plant-specific interfering compounds include:

• Phenolic compounds: Add PVPP (polyvinylpolypyrrolidone, 2% w/v) to extraction buffers

• Secondary metabolites: Include β-mercaptoethanol (5mM) to prevent oxidation

• Polysaccharides: Use extraction buffers with higher detergent concentrations (1.5% Triton X-100)

• Proteases: Add complete protease inhibitor cocktail freshly before extraction

• Chlorophyll: Perform acetone precipitation of proteins or use TCA/acetone extraction methods

What are the recommended protocols for subcellular fractionation when working with At5g39120 antibodies?

For subcellular localization studies:

• Nucleus isolation:

  • Homogenize tissue in nuclear isolation buffer (20mM Tris-HCl pH 7.4, 25% glycerol, 20mM KCl, 2mM EDTA, 2.5mM MgCl₂, 250mM sucrose)

  • Filter through miracloth

  • Pellet nuclei at 1,500 × g for 10 minutes

  • Verify purity using anti-H⁺-ATPase (plasma membrane) and anti-HISTONE H3 (nuclear) antibodies

• Membrane fractionation:

  • Homogenize tissue in extraction buffer (50mM HEPES pH 7.5, 250mM sucrose, 15mM EDTA, 5% glycerol)

  • Remove debris by centrifugation at 2,000 × g for 5 minutes

  • Separate membranes by ultracentrifugation at 100,000 × g for 1 hour

  • Verify fractions using anti-H⁺-ATPase (plasma membrane) and anti-CFBPase (cytosolic) markers

The subcellular localization of At5g39120 can provide insights into its function, particularly if it changes during plant development or stress responses.

What methods are recommended for quantitative analysis of At5g39120 protein levels across different plant tissues?

For quantitative tissue comparisons:

• Sample preparation standardization:

  • Collect tissues at the same developmental stage

  • Use identical fresh weight-to-buffer ratios for extraction

  • Process all samples simultaneously

• Quantification approaches:

  • ELISA with recombinant protein standards for absolute quantification

  • Li-COR infrared fluorescence imaging for wider dynamic range

  • Multiplex Western blotting with tissue-specific loading controls

• Data normalization:

  • Normalize to total protein (determined by Bradford assay)

  • Use multiple housekeeping proteins as references

  • Consider tissue-specific reference proteins rather than global references