At3g20160 Antibody

What is the At3g20160 gene in Arabidopsis thaliana and what protein does it encode?

At3g20160 is a gene locus in Arabidopsis thaliana (Mouse-ear cress) that encodes a specific protein with UniProt accession number Q9LJY2 . The gene is located on chromosome 3 and represents part of the extensive genomic research conducted on this model plant organism. Understanding the function of this gene contributes to broader knowledge of plant molecular biology, particularly in signaling pathways and developmental processes.

How is the quality and specificity of At3g20160 Antibody validated for research applications?

Validation of At3g20160 Antibody involves multiple complementary approaches to ensure both specificity and sensitivity. Proper validation typically includes:

• Western blotting against wild-type and knockout/knockdown plants

• Immunoprecipitation followed by mass spectrometry

• Immunohistochemistry with appropriate positive and negative controls

• Pre-absorption tests with the immunizing peptide/protein
  It's important to note that antibodies successfully tested in applications such as Western Blotting or Immunohistochemistry may not necessarily be suitable for all applications such as Flow Cytometry . Researchers should verify that the antibody has been validated specifically for their intended application.

What controls are essential when using At3g20160 Antibody in immunological experiments?

When designing experiments with At3g20160 Antibody, four types of controls should be incorporated to ensure reliable results:

What are the optimal fixation and permeabilization protocols when detecting At3g20160 protein?

The optimal fixation and permeabilization protocol depends on the cellular localization of At3g20160 and the epitope recognized by the antibody. Consider the following approaches:

• For extracellular epitopes: Cells can be used unfixed or with mild fixation (2-4% paraformaldehyde for 10-15 minutes)

• For intracellular epitopes: Fixation is required, with permeabilization using one of these methods:

  • Organic solvents (methanol/acetone) for cytosolic proteins

  • Mild detergents (0.1-0.5% Triton X-100 or 0.01-0.05% saponin) for membrane-associated proteins

  • Specialized permeabilization for nuclear proteins (higher detergent concentration)
    The choice should be guided by the subcellular localization of At3g20160 and the epitope targeted by the antibody . Preliminary experiments comparing different fixation/permeabilization combinations are recommended to determine optimal conditions.

How should At3g20160 Antibody be applied in flow cytometry experiments with plant cells?

When using At3g20160 Antibody for flow cytometry with plant cells, follow this methodological approach:

• Sample preparation:

  • Isolate protoplasts from plant tissue using enzymatic digestion

  • Filter cell suspension through a 40-70 μm mesh to remove aggregates

  • Maintain cells in appropriate osmotic buffer to prevent lysis

• Staining protocol:

  • Block with 10% normal serum from the same host species as the secondary antibody

  • Incubate with primary At3g20160 Antibody at optimized concentration (typically 1-10 μg/ml)

  • Wash thoroughly to remove unbound antibody

  • Incubate with fluorophore-conjugated secondary antibody

  • Include viability dye to exclude dead cells from analysis

• Instrument settings:

  • Adjust forward and side scatter gates for plant protoplasts

  • Set fluorescence compensation based on single-color controls

  • Use unstained and isotype controls to set negative population gates
    Plant cells often require special consideration due to their cell walls, higher autofluorescence, and different size parameters compared to animal cells.

What are the recommended approaches for using At3g20160 Antibody in immunohistochemistry of plant tissues?

For immunohistochemistry applications with At3g20160 Antibody:

• Tissue preparation:

  • Fix tissue samples in 4% paraformaldehyde or Farmer's fixative

  • Embed in paraffin or prepare frozen sections

  • Perform antigen retrieval if necessary (citrate buffer pH 6.0, 95°C for 10-20 minutes)

• Staining procedure:

  • Block endogenous peroxidases with H₂O₂ if using HRP detection

  • Block non-specific binding with 5-10% serum and 1-3% BSA

  • Incubate with At3g20160 Antibody (starting at 1:100-1:500 dilution)

  • Detect with appropriate secondary antibody system

  • Counterstain to visualize tissue architecture

• Controls:

  • Include tissue from knockout/knockdown plants

  • Perform peptide competition assays

  • Include secondary-only controls
    Plant tissues may require longer incubation times and careful optimization of permeabilization due to cell wall barriers.

How can At3g20160 Antibody be integrated into multi-parameter analyses of plant signaling networks?

At3g20160 Antibody can be effectively incorporated into multi-parameter analyses through several sophisticated approaches:

• Multiplexed immunofluorescence:

  • Combine At3g20160 Antibody with antibodies against related pathway components

  • Use primary antibodies from different host species

  • Apply spectrally distinct fluorophores for simultaneous detection

  • Analyze co-localization or expression correlation patterns

• Proximity ligation assays (PLA):

  • Detect protein-protein interactions involving At3g20160

  • Combine At3g20160 Antibody with antibodies against potential interacting partners

  • Signal amplification allows detection of low-abundance interactions

• Sequential immunoprecipitation:

  • Use At3g20160 Antibody to pull down protein complexes

  • Analyze by mass spectrometry to identify interaction partners

  • Validate key interactions with reciprocal co-immunoprecipitation
    These approaches enable researchers to position At3g20160 within its biological context and understand its functional relationships within signaling networks.

What considerations are important when studying At3g20160 expression across developmental stages or stress conditions?

When investigating At3g20160 expression dynamics:

• Experimental design considerations:

  • Include multiple developmental timepoints or stress intensities/durations

  • Maintain consistent harvest times to control for circadian effects

  • Use biological replicates from independent plant populations

• Quantification approaches:

  • Complement antibody-based detection with transcript analysis

  • Employ relative quantification with reference proteins that maintain stable expression

  • Consider tissue-specific extraction to avoid dilution effects from non-expressing cells

• Data normalization:

  • Normalize to cell number or total protein when comparing different conditions

  • Account for changes in reference protein expression under extreme stress conditions

  • Apply appropriate statistical tests for time-series data (repeated measures ANOVA)
    This comprehensive approach allows for robust analysis of At3g20160 regulation across different biological contexts.

What are common issues encountered when using At3g20160 Antibody and how can they be resolved?

How can epitope accessibility be improved when detecting At3g20160 in fixed tissue samples?

Optimizing epitope accessibility requires methodical testing of different approaches:

• Antigen retrieval methods comparison:

  • Heat-induced epitope retrieval (citrate buffer pH 6.0, Tris-EDTA pH 9.0)

  • Enzymatic digestion (proteinase K, trypsin)

  • Detergent treatment (0.1-0.5% Triton X-100, 0.1-0.5% SDS)

• Fixation optimization:

  • Test different fixatives (4% PFA, glutaraldehyde, methanol/acetone)

  • Vary fixation duration (10 min to overnight)

  • Try post-fixation permeabilization steps

• Signal amplification:

  • Tyramide signal amplification for fluorescence detection

  • Polymer-based detection systems for chromogenic detection

  • Nanobody or Fab fragment secondary reagents for better penetration
    Each of these approaches should be systematically tested and compared to determine the optimal protocol for your specific tissue type and experimental question.

How should quantitative data from At3g20160 immunodetection be analyzed and presented?

Quantitative analysis of At3g20160 immunodetection requires rigorous methodological approaches:

• Image-based quantification:

  • Use consistent acquisition parameters across samples

  • Analyze raw, unprocessed images

  • Apply appropriate background subtraction

  • Normalize to cell count or area

  • Present data as relative fluorescence units or fold-change

• Flow cytometry data:

  • Gate on single, viable cells

  • Report median fluorescence intensity rather than mean

  • Present both percentage of positive cells and signal intensity

  • Use histogram overlays and quantile statistics

• Statistical analysis:

  • Apply normality tests before choosing parametric/non-parametric tests

  • Use ANOVA with post-hoc tests for multiple comparisons

  • Report biological replicates (n) and technical replicates

  • Include appropriate error bars (standard deviation or standard error)
    Proper analysis ensures that subtle changes in At3g20160 expression or localization can be reliably detected and accurately interpreted.

How can conflicting results between antibody-based and transcript-based detection of At3g20160 be reconciled?

Discrepancies between protein and transcript levels require systematic investigation:

• Potential biological explanations:

  • Post-transcriptional regulation (miRNA targeting, RNA stability)

  • Translational control mechanisms

  • Protein stability and degradation pathways

  • Temporal delay between transcription and translation

• Technical verification approaches:

  • Confirm antibody specificity with knockout/knockdown controls

  • Validate RNA detection methods with multiple primer pairs

  • Perform time-course experiments to detect temporal dynamics

  • Use absolute quantification methods for both protein and transcript

• Integrated analysis:

  • Calculate protein-to-mRNA ratios across conditions

  • Apply mathematical modeling to account for synthesis and degradation rates

  • Consider subcellular localization changes that might affect detection These discrepancies often reveal important biological regulatory mechanisms rather than experimental artifacts, and their thorough investigation can lead to novel insights about At3g20160 regulation.