At1g63360 Antibody

What is At1g63360 protein and why is it studied in Arabidopsis thaliana?

At1g63360 (Uniprot: Q9SH22) is a protein expressed in Arabidopsis thaliana that has gained attention in plant molecular biology research. The protein is referenced in recombinant antibody production studies and plant protein expression systems . Research applications typically focus on protein expression patterns, localization studies, and functional characterization through various immunological techniques.

How should researchers select between different formats of At1g63360 antibodies?

The selection depends on your experimental goals:

• Polyclonal antibodies (like CSB-PA871060XA01DOA) provide broader epitope recognition and higher sensitivity, making them ideal for detecting native proteins in complex samples. These are generated against recombinant At1g63360 protein and purified through antigen affinity methods .

• Monoclonal antibodies offer higher specificity for particular epitopes, providing consistent results across experiments and batches.
  For initial characterization studies, polyclonal antibodies are often preferred, while targeted analysis of specific protein domains may benefit from monoclonal antibodies.

What validation methods confirm At1g63360 antibody specificity?

Rigorous validation requires multiple approaches:

• Genetic controls: Testing antibody reactivity in At1g63360 knockout/knockdown Arabidopsis lines

• Recombinant protein testing: Using purified At1g63360 protein as a positive control

• Western blot analysis: Confirming single band detection at expected molecular weight (verify against predicted MW)

• Peptide competition assays: Pre-incubating antibody with immunizing peptide to confirm signal reduction

• Cross-reactivity assessment: Testing against related plant species specified in the product datasheet

What are the optimal protein extraction methods for At1g63360 detection?

Successful At1g63360 detection depends significantly on extraction methodology. Based on research with similar plant antibodies, the following protocol is recommended:

• Harvest approximately 100mg of plant material

• Extract in 0.2ml homogenization buffer (0.1M EDTA, 0.12M Tris-HCl pH 6.8, 4% SDS, 10% glycerol)

• Add protease inhibitor cocktail to prevent degradation

• Homogenize tissue thoroughly using mechanical disruption

• Centrifuge at 10,000g for 10 minutes to remove cellular debris

• Collect supernatant for further analysis
  Proper protein extraction significantly impacts detection quality, as demonstrated with other plant antibodies like BAK1 .

How should Western blot conditions be optimized for At1g63360 antibody?

For optimal Western blot results with At1g63360 antibody:

• Sample preparation: Use the extraction method described above

• Protein loading: 10-20μg total protein per lane

• Gel percentage: 10-12% SDS-PAGE for optimal separation

• Transfer conditions: Semi-dry or wet transfer (wet transfer recommended for higher MW proteins)

• Blocking: 5% non-fat milk or BSA in TBST (1 hour at room temperature)

• Primary antibody: Dilute At1g63360 antibody 1:5000 in blocking solution

• Incubation: Overnight at 4°C with gentle agitation

• Secondary antibody: Anti-rabbit HRP conjugate (1:10,000)

• Detection: ECL substrate with exposure time optimization

What controls are essential when using At1g63360 antibody in immunoprecipitation experiments?

For rigorous immunoprecipitation experiments:

• Input control: 5-10% of pre-immunoprecipitated lysate

• IgG control: Non-specific rabbit IgG to identify non-specific binding

• No-antibody control: Beads without antibody to assess direct protein-bead interactions

• Knockout/knockdown control: Lysate from At1g63360-deficient plants

• Antibody amount optimization: Typically 2μl antibody per 50μl of Protein G agarose
  Procedural recommendation: Pre-clear lysates with Protein G agarose before immunoprecipitation to reduce background.

How can At1g63360 antibody be used to study protein-protein interactions?

At1g63360 protein interactions can be studied through several approaches:

• Co-immunoprecipitation (co-IP): Precipitate At1g63360 using the specific antibody (2μl per 50μl Protein G agarose) , then identify interacting partners through:

  • Western blot with antibodies against suspected partners

  • Mass spectrometry for unbiased interaction screening

• Proximity labeling: Combine At1g63360 antibody with techniques like BioID or APEX2 to identify proximal proteins

• Bimolecular Fluorescence Complementation (BiFC): Verify direct interactions identified through co-IP
  The analysis of protein complexes must account for possible transient interactions and conditional associations dependent on developmental stage or stress conditions.

How does recombinant antibody production in Arabidopsis seeds affect transcriptome dynamics?

Studies of recombinant antibody production in Arabidopsis seeds provide insights relevant to At1g63360 antibody research. Transcriptome analysis reveals:

• Limited transcriptome changes: Only a small proportion of genes show significant expression changes (56-103 genes)

• Upregulation of protein processing pathways: Genes involved in protein folding, glycosylation, translocation, and vesicle transport are consistently upregulated

• Endoplasmic reticulum stress response: Antibody production triggers the unfolded protein response (UPR)

• Expression timeline: Antibody accumulation begins around 8 days post anthesis (dpa) and increases to significant levels by 14 dpa
  These findings suggest that when studying At1g63360 in transgenic plants expressing antibodies, researchers should consider potential stress responses that might affect experimental outcomes.

What methodological approaches help distinguish between specific and non-specific signals when using At1g63360 antibody?

Distinguishing specific from non-specific signals requires systematic controls:

• Peptide competition assay: Pre-incubate antibody with excess immunizing peptide

• Knockout/knockdown validation: Compare signal between wild-type and At1g63360-deficient plants

• Secondary antibody-only control: Excludes non-specific secondary antibody binding

• Signal quantification: Use imaging software to measure signal-to-noise ratios

• Multiple detection methods: Confirm findings using orthogonal techniques (e.g., mass spectrometry)

• Cross-reactivity assessment: Test antibody against samples from non-reactive species like Hordeum vulgare or Oryza sativa

Why might Western blot analysis with At1g63360 antibody show multiple bands or high background?

Multiple bands or high background can result from several factors:

• Protein degradation: Add fresh protease inhibitors to extraction buffer

• Cross-reactivity: The antibody may recognize related proteins, especially with polyclonal antibodies

• Post-translational modifications: Different forms of At1g63360 may appear as multiple bands

• Non-specific binding: Optimize blocking conditions (try 5% BSA instead of milk)

• Secondary antibody issues: Increase washing steps (5x 5 minutes with TBST)

• Antibody concentration: Try higher dilution (1:10,000 instead of 1:5,000)

• Sample preparation: Ensure complete protein denaturation before loading
  A systematic approach to troubleshooting involves changing one variable at a time and documenting results.

How can researchers enhance signal detection for low-abundance At1g63360 protein?

For enhanced detection of low-abundance At1g63360:

• Subcellular fractionation: Concentrate the relevant cellular compartment

• Protein precipitation: Use TCA or acetone precipitation to concentrate proteins

• Enhanced chemiluminescence: Use high-sensitivity ECL substrates

• Longer exposure times: Incrementally increase exposure during Western blot imaging

• Signal amplification: Consider using biotin-streptavidin systems or tyramide signal amplification

• Tissue selection: Target tissues with higher At1g63360 expression

• Developmental timing: Sample at developmental stages with peak expression

What strategies help maintain At1g63360 antibody stability and activity during storage?

To maximize antibody stability and performance:

• Proper reconstitution: Add 50μl sterile water to lyophilized antibody

• Aliquoting: Divide reconstituted antibody into single-use aliquots

• Storage temperature: Maintain at -20°C (avoid repeated freeze-thaw cycles)

• Glycerol addition: Add sterile glycerol (final concentration 30-50%) for freeze protection

• Contamination prevention: Use sterile technique when handling

• Spin tubes: Briefly centrifuge before opening to collect material from tube walls

• Stability testing: Periodically test antibody performance using positive controls

How might At1g63360 antibody be used in studying plant stress responses?

At1g63360 antibody can provide insights into stress response mechanisms:

• Expression profiling: Monitor At1g63360 protein levels under various stress conditions (drought, salt, pathogen challenge)

• Subcellular relocalization: Track protein movement between cellular compartments during stress

• Post-translational modifications: Identify stress-induced modifications using phospho-specific or other PTM-specific antibodies alongside At1g63360 antibody

• Protein complex dynamics: Examine how stress affects At1g63360 interaction partners

• Comparative analysis: Study At1g63360 response in stress-tolerant vs. sensitive Arabidopsis ecotypes
  This approach would complement transcriptomic studies of stress responses in Arabidopsis.

What considerations are important when adapting At1g63360 antibody protocols across different plant species?

Cross-species applications require careful optimization:

• Sequence homology analysis: Determine protein sequence conservation between species

• Epitope conservation: Verify if the immunogen peptide sequence is conserved in target species

• Preliminary testing: Validate antibody reactivity in the new species at multiple dilutions

• Extraction optimization: Modify buffers to account for species-specific differences (e.g., higher phenolic compounds)

• Predicted cross-reactivity: Consider manufacturer's predicted reactivity (e.g., Thelungiella halophila)

• Non-reactive species: Be aware of known non-reactive species (Hordeum vulgare, Lactuca sativa, Nicotiana benthamiana, Oryza sativa) The antibody may require different working dilutions or detection methods in different species.