At3g19890 Antibody

What is At3g19890 and why is it studied in Arabidopsis thaliana?

At3g19890 encodes a protein in Arabidopsis thaliana that plays significant roles in plant development and stress responses. The gene is located on chromosome 3 and the protein (Q4PSN8) is involved in cellular processes that are critical for understanding plant biology. Research on At3g19890 is important for several reasons:

• It contributes to our understanding of gene expression regulation in plants.

• The protein functions in developmental pathways that influence plant growth.

• Studies suggest potential roles in response to environmental stressors.

• It serves as a model for understanding similar proteins across plant species.

The At3g19890 antibody is therefore a crucial research tool for detecting, quantifying, and localizing this protein in plant tissues, enabling researchers to elucidate its functions and interactions within cellular networks .

What methodologies are recommended for At3g19890 Antibody validation?

Proper validation is essential before implementing At3g19890 antibody in critical experiments. A comprehensive validation approach should include:

Western Blot Validation Protocol:

• Run protein extracts from wild-type and knockout/knockdown Arabidopsis plants side by side.

• Follow standard western blot procedures using 1:1000 primary antibody dilution.

• Confirm expected band size (~predicted kDa based on protein sequence).

• Verify absence or reduction of signal in negative controls.

Immunoprecipitation Validation:

• Perform IP with At3g19890 antibody on plant extracts.

• Analyze precipitated proteins by mass spectrometry.

• Confirm enrichment of At3g19890 protein and known interacting partners.

Immunofluorescence Controls:

• Compare staining patterns between wild-type and mutant tissues.

• Include secondary-antibody-only controls to assess background.

• Use competing peptide controls to confirm specificity.

Robust validation should demonstrate consistent results across multiple techniques and biological replicates to ensure experimental reliability .

What are the optimal storage and handling conditions for At3g19890 Antibody?

To maintain antibody integrity and performance, follow these methodological guidelines:

Storage Conditions:

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

• For working solutions, store at 4°C for up to 2 weeks.

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

• Consider adding glycerol (50% final) for freezer storage.

Handling Recommendations:

• Centrifuge briefly before opening vials to collect liquid at the bottom.

• Use sterile techniques when handling antibody solutions.

• Prepare working dilutions on the day of the experiment when possible.

• When diluting, use high-quality buffers (PBS with 0.1% BSA or similar).

Quality Control Measures:

• Periodically test antibody performance against a reference sample.

• Document lot numbers and correlate with experimental results.

• Monitor for signs of contamination or precipitation.

Proper storage and handling significantly impact experimental reproducibility and extend the useful life of the antibody preparation .

What protocols are recommended for Western blot analysis using At3g19890 Antibody?

Western Blot Protocol for At3g19890 Detection:

Sample Preparation:

• Extract total protein from Arabidopsis tissues using a plant-specific extraction buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Triton X-100, 1mM EDTA, protease inhibitor cocktail).

• Determine protein concentration using Bradford or BCA assay.

• Prepare samples with 20-30μg total protein per lane.

Gel Electrophoresis and Transfer:

• Separate proteins on 10-12% SDS-PAGE.

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

Antibody Incubation:

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

• Incubate with At3g19890 antibody at 1:1000 dilution in blocking buffer overnight at 4°C.

• Wash 3×10 minutes with TBST.

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

• Wash 3×10 minutes with TBST.

Detection:

• Apply ECL substrate and image using appropriate detection system.

• Expected molecular weight: Confirm against protein database prediction.

This protocol can be optimized based on specific experimental conditions and sample types .

How can At3g19890 Antibody be optimized for chromatin immunoprecipitation (ChIP) assays?

ChIP Protocol Optimization for At3g19890:

Crosslinking and Chromatin Preparation:

• Harvest 3-4g of Arabidopsis tissue and crosslink with 1% formaldehyde for 10 minutes under vacuum.

• Quench with 0.125M glycine for 5 minutes.

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

• Filter through miracloth and isolate nuclei by centrifugation.

• Resuspend chromatin and sonicate to achieve fragments of 200-500bp.

Immunoprecipitation:

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

• Incubate pre-cleared chromatin with 5-10μg At3g19890 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.

Optimization Considerations:

• Test both native and crosslinked ChIP approaches, as protein-DNA interaction types may affect efficiency.

• Determine optimal antibody concentration through titration experiments (2-10μg per reaction).

• Consider dual crosslinking (DSG followed by formaldehyde) for improved capture of protein-protein interactions.

• Include appropriate controls: IgG negative control, input samples, and known target regions if available.

Data Analysis:

• Perform qPCR on ChIP DNA using primers for suspected binding regions.

• Calculate enrichment relative to input and IgG control.

• Consider ChIP-seq for genome-wide binding site identification.

This methodological approach provides a robust framework for investigating protein-DNA interactions involving At3g19890 protein in vivo .

What are effective strategies for troubleshooting inconsistent results with At3g19890 Antibody in immunolocalization studies?

Systematic Troubleshooting Approach:

Sample Preparation Issues:

• Fixation optimization: Test multiple fixatives (4% paraformaldehyde, glutaraldehyde combinations) and fixation times (30min-overnight).

• Embedding and sectioning: Ensure consistent section thickness (5-8μm optimal) and complete infiltration.

• Antigen retrieval: Compare heat-induced (citrate buffer, pH 6.0, 95°C for 10-20min) versus enzymatic methods (proteinase K treatment).

Antibody-Related Factors:

• Titration series: Test dilutions from 1:100 to 1:2000 to determine optimal concentration.

• Incubation conditions: Compare overnight at 4°C versus 2-3 hours at room temperature.

• Signal enhancement: Implement tyramide signal amplification for low-abundance targets.

Controls and Validation:

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to verify signal specificity.

• Genetic controls: Include tissue from knockout/knockdown plants.

• Multi-method confirmation: Validate localization patterns using alternative approaches (e.g., fluorescent protein fusions).

Data Collection and Analysis:

• Implement consistent imaging parameters across experiments.

• Use quantitative image analysis to measure signal intensity and distribution.

• Consider tissue-specific autofluorescence and implement appropriate controls.

Table 2.1: Common Immunolocalization Issues and Solutions for At3g19890 Antibody

This systematic approach helps identify and address specific factors contributing to experimental variability .

How can researchers design dual-labeling experiments using At3g19890 Antibody with other cellular markers?

Dual-Labeling Experimental Design:

Pre-Experimental Planning:

• Antibody compatibility assessment:

  • Confirm host species differences between primary antibodies

  • If using same host, consider directly conjugated antibodies

  • Verify secondary antibody cross-reactivity profiles

• Spectral overlap considerations:

  • Select fluorophores with minimal spectral overlap

  • Plan sequential imaging if overlap cannot be avoided

  • Include single-label controls for spectral unmixing

Optimized Protocol:

• Tissue preparation:

  • Fix samples in 4% paraformaldehyde (4 hours at 4°C)

  • Perform permeabilization with 0.1% Triton X-100 (10 minutes)

  • Block with 3% BSA in PBS (1 hour at room temperature)

• Sequential antibody application:

  • Incubate with At3g19890 antibody (1:500) overnight at 4°C

  • Wash 3×15 minutes in PBS

  • Apply corresponding secondary antibody (2 hours at room temperature)

  • Wash 3×15 minutes in PBS

  • Repeat sequence with second primary/secondary pair

• Counterstaining:

  • DAPI for nuclei (1μg/ml, 10 minutes)

  • Appropriate cell wall stain if needed (Calcofluor White, 10 minutes)

Analysis Considerations:

• Implement colocalization analysis using established coefficients (Pearson's, Manders')

• Perform distance measurements for non-overlapping signals

• Consider 3D reconstruction for complex spatial relationships

Table 2.2: Compatible Marker Combinations with At3g19890 Antibody

This approach provides a framework for investigating protein co-localization and spatial relationships within cellular compartments .

What methodological approaches enable cross-species application of At3g19890 Antibody beyond Arabidopsis?

Cross-Species Implementation Strategy:

Epitope Conservation Analysis:

• Perform sequence alignment of At3g19890 protein with homologs from target species

• Calculate percent identity/similarity for the antibody epitope region

• Predict cross-reactivity based on epitope conservation (>70% identity suggests potential cross-reactivity)

Validation Protocol for Non-Arabidopsis Species:

• Western blot validation:

  • Run protein extracts from both Arabidopsis and target species

  • Compare band patterns and molecular weights

  • Perform peptide competition assays in both species

• Immunoprecipitation confirmation:

  • Perform parallel IPs from both species

  • Analyze precipitated proteins by mass spectrometry

  • Verify target protein identity and interacting partners

• Immunolocalization comparative analysis:

  • Process tissues from both species using identical protocols

  • Compare subcellular localization patterns

  • Correlate with orthologous protein localization data

Optimization Considerations:

• Buffer modifications for species-specific tissue types

• Adjusted antibody concentrations (typically 2-5× higher for cross-species applications)

• Extended incubation times to accommodate lower-affinity binding

• Alternative fixation methods for different tissue architectures

Table 2.3: Cross-Species Reactivity Assessment

This systematic approach provides a framework for extending antibody applications across species while maintaining experimental rigor and reliability .

How can researchers quantitatively analyze protein interactions using At3g19890 Antibody in co-immunoprecipitation experiments?

Quantitative Co-Immunoprecipitation Methodology:

Experimental Design:

• Standardized lysate preparation:

  • Harvest tissues at consistent developmental stages

  • Use quantitative protein extraction protocol with defined tissue:buffer ratio

  • Determine protein concentration using Bradford assay with BSA standard curve

• IP setup with quantitative controls:

  • Use consistent antibody:lysate ratios (5μg antibody per mg total protein)

  • Include spike-in controls for normalization

  • Implement parallel IPs with non-specific IgG

Enhanced Protocol:

• Sample preparation:

  • Extract proteins in IP buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% NP-40, 1mM EDTA, protease inhibitors)

  • Pre-clear lysate with Protein A/G beads (1 hour at 4°C)

  • Quantify protein concentration (aim for 1-2mg total protein per IP)

• Immunoprecipitation:

  • Incubate pre-cleared lysate with 5μg At3g19890 antibody overnight at 4°C

  • Add 30μl Protein A/G beads and incubate 3 hours at 4°C

  • Wash beads 4× with IP buffer and 1× with PBS

  • Elute bound proteins with 2× sample buffer or for LC-MS/MS analysis

• Quantitative analysis options:

  • Western blot with normalization to immunoprecipitated At3g19890

  • SILAC or TMT labeling for mass spectrometry quantification

  • Label-free quantification with appropriate statistical analysis

Data Analysis Framework:

• Calculate enrichment ratios:

  • Target protein/At3g19890 protein ratio

  • Specific IP/IgG control ratio

  • Treatment/control condition ratio

• Statistical validation:

  • Perform minimum of three biological replicates

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

  • Calculate confidence intervals for interaction strengths

Table 2.4: Protein Interaction Quantification Methods

This quantitative approach provides robust data on protein interaction dynamics, enabling meaningful comparisons across experimental conditions and treatments .