At1g12170 Antibody

What is AT1g12170 and what protein does the antibody target?

AT1g12170 is a gene locus in Arabidopsis thaliana that encodes a protein identified as Q9FWW7. This protein is expressed in the mouse-ear cress (Arabidopsis thaliana) and is part of the plant's cellular machinery. The antibody targeting this protein is designed to bind specifically to epitopes on the AT1g12170 gene product, allowing for its detection and quantification in various experimental applications . Research with this antibody contributes to our understanding of Arabidopsis cellular functions and protein interactions.

What experimental applications are suitable for AT1g12170 antibody?

AT1g12170 antibody is suitable for multiple experimental applications in plant molecular biology research. Based on validation patterns established for similar antibodies in Arabidopsis research, applications likely include Western blotting, immunoprecipitation (IP), chromatin immunoprecipitation (ChIP), and immunofluorescence microscopy . For optimal results, researchers should conduct preliminary validation in their specific experimental systems to establish appropriate working dilutions, as reactivity may vary between 1:100-1:250 for immunofluorescence and 1:3000-1:5000 for Western blotting, based on patterns observed with other plant antibodies .

How should AT1g12170 antibody be stored and reconstituted for maximum stability?

For optimal stability, store lyophilized AT1g12170 antibody at -20°C. Upon receipt, reconstitute by adding sterile water to the specified volume (typically 50 μl for research-grade antibodies) and allow complete dissolution before use. After reconstitution, make small aliquots to avoid repeated freeze-thaw cycles that degrade antibody quality. When handling the antibody, briefly spin tubes before opening to collect material that might adhere to the cap or sides. Reconstituted antibodies should continue to be stored at -20°C, with working aliquots kept at 4°C for short-term use (1-2 weeks) .

How can I validate the specificity of AT1g12170 antibody for my research?

To validate AT1g12170 antibody specificity, implement a multi-step approach:

• Positive and negative controls: Use wild-type Arabidopsis tissue (positive control) and if available, AT1g12170 knockout/knockdown lines (negative control) to verify antibody specificity.

• Western blot analysis: Perform Western blots to confirm a single band of expected molecular weight (~45 kDa based on similar Arabidopsis proteins) .

• Peptide competition assay: Pre-incubate the antibody with excess purified antigen peptide before application to verify that binding is blocked, indicating specificity.

• Multiple detection methods: Validate across different applications (Western blot, immunostaining, ChIP) to ensure consistent target recognition.

• Cross-reactivity assessment: Test against related Arabidopsis proteins to ensure the antibody doesn't recognize homologous proteins .

This rigorous validation is essential as demonstrated by studies showing that commercially available antibodies often recognize proteins of similar molecular weight even in knockout models lacking the target protein .

What controls should I include when using AT1g12170 antibody in ChIP experiments?

When using AT1g12170 antibody in ChIP experiments, include these critical controls:

Additionally, consider including a secondary antibody-only control to rule out non-specific binding. For ChIP-seq experiments, spike-in controls with chromatin from another species can provide improved normalization for quantitative comparisons . As demonstrated in histone modification antibody validations, the use of multiple controls significantly improves result reliability and reproducibility .

How can I determine if the AT1g12170 antibody shows cross-reactivity with other plant species?

To assess cross-reactivity of AT1g12170 antibody with other plant species:

• Sequence homology analysis: First, perform bioinformatic analysis to identify homologous proteins in target plant species with high sequence similarity to the Arabidopsis AT1g12170-encoded protein.

• Western blot analysis: Test the antibody against protein extracts from multiple plant species alongside Arabidopsis (positive control). Compare band patterns and molecular weights to identify potential cross-reactivity.

• Epitope conservation assessment: Analyze the specific epitope sequence recognized by the antibody across different plant species to predict cross-reactivity potential.

• Validation in target species: For each new plant species, perform complete validation including negative controls and peptide competition assays.

• Published literature review: Search for previous cross-reactivity reports with this or similar antibodies.

This methodical approach is necessary because research has shown that antibodies developed against Arabidopsis proteins often show reactivity with homologous proteins in related plant species, particularly within Brassicaceae, but may also recognize functionally conserved proteins in more distant species .

How can AT1g12170 antibody be used to study protein-chromatin interactions?

AT1g12170 antibody can be employed to study protein-chromatin interactions through several advanced methodologies:

• Chromatin Immunoprecipitation (ChIP): Optimize the antibody for ChIP to identify DNA binding sites of the AT1g12170 protein. Follow established protocols for plant ChIP, including appropriate crosslinking conditions (1% formaldehyde for 10-15 minutes) and sonication parameters to generate 200-500 bp DNA fragments.

• ChIP-seq analysis: Combine ChIP with next-generation sequencing to map genome-wide binding profiles. Use specialized analysis pipelines (e.g., MACS for peak calling with p=1e-03) and appropriate bioinformatic tools to identify enriched regions .

• Sequential ChIP (Re-ChIP): To investigate co-occupancy with other factors, perform sequential immunoprecipitations using AT1g12170 antibody followed by antibodies against suspected interaction partners.

• Protein complex identification: Couple immunoprecipitation with mass spectrometry to identify proteins that co-occupy chromatin regions with AT1g12170 protein.

• Integration with transcriptome data: Correlate binding profiles with RNA-seq data to establish functional relevance of binding events.

This comprehensive approach has been successfully implemented for studying chromatin-associated proteins in Arabidopsis, revealing important insights into gene regulation mechanisms .

What are the considerations when using AT1g12170 antibody for co-immunoprecipitation studies?

When conducting co-immunoprecipitation (co-IP) studies with AT1g12170 antibody, consider these critical factors:

• Buffer optimization: Test multiple lysis and binding buffers to preserve protein-protein interactions while minimizing background. For plant proteins, buffers containing 20-50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1-5 mM EDTA, and 0.1-1% non-ionic detergents (NP-40 or Triton X-100) are commonly effective.

• Crosslinking evaluation: Determine whether chemical crosslinking (e.g., DSP, formaldehyde) is needed to capture transient interactions, recognizing that crosslinking may increase background.

• Antibody orientation: Compare results between using AT1g12170 antibody as the primary precipitation antibody versus using antibodies against suspected interaction partners.

• Controls implementation:

  • Input control (5-10% of starting material)

  • IgG control (same species and concentration)

  • Reverse co-IP validation

  • Competition with immunizing peptide

• Elution conditions: Optimize elution to maximize recovery while maintaining antibody integrity, typically using low pH (2.5-3.0) glycine buffers or specific peptide elution.

• Validation methods: Confirm interactions through reciprocal co-IPs, proximity ligation assays, or in vitro binding studies.

This methodical approach has proven effective in studies of protein-protein interactions in plant systems, including those involving chromatin-associated factors .

How can I quantitatively measure AT1g12170 protein expression levels across different plant tissues?

To quantitatively measure AT1g12170 protein expression across different plant tissues, implement this comprehensive approach:

• Sample preparation standardization:

  • Harvest tissues at the same developmental stage

  • Use identical extraction buffers with protease inhibitors

  • Normalize protein concentrations using Bradford or BCA assays

• Western blot quantification:

  • Include recombinant protein standards for absolute quantification

  • Perform technical triplicates with biological replicates (n≥3)

  • Use housekeeping proteins like actin as loading controls

  • Employ fluorescent secondary antibodies for wider linear range

  • Analyze using image quantification software (ImageJ/Fiji)

• ELISA development:

  • Develop sandwich ELISA using AT1g12170 antibody and a secondary detection antibody

  • Generate standard curves with purified recombinant protein

  • Validate assay specificity and sensitivity

• Multiplexed protein assays:

  • Consider using Luminex or similar bead-based assays for higher throughput

  • Couple with analysis of other proteins of interest

• Spatial analysis:

  • Complement quantitative data with immunohistochemistry to determine tissue-specific localization

  • Use confocal microscopy with standardized imaging parameters

This multi-method approach provides robust quantitative assessment while accounting for tissue-specific variations in protein expression .

How can I troubleshoot weak or absent signal when using AT1g12170 antibody in Western blots?

When encountering weak or absent signal with AT1g12170 antibody in Western blots, systematically address these common issues:

• Protein extraction optimization:

  • Use harsher extraction buffers containing SDS or urea

  • Increase detergent concentration (0.5-1% SDS)

  • Add protease inhibitors fresh before extraction

  • Consider tissue-specific extraction protocols for Arabidopsis

• Transfer efficiency verification:

  • Confirm transfer with reversible staining (Ponceau S)

  • Optimize transfer conditions (time, voltage, buffer composition)

  • Consider semi-dry vs. wet transfer systems for plant proteins

• Antibody-related adjustments:

  • Test multiple antibody concentrations (1:1000 to 1:10,000 range)

  • Extend primary antibody incubation (overnight at 4°C)

  • Try different blocking agents (5% BSA vs. milk)

  • Reduce washing stringency by decreasing detergent concentration

• Signal enhancement strategies:

  • Use high-sensitivity ECL substrates

  • Consider signal amplification systems

  • Increase exposure time incrementally

  • Try fluorescent-labeled secondary antibodies

• Sample preparation refinement:

  • Avoid excessive heating of samples (65°C max)

  • Use fresh DTT or β-mercaptoethanol

  • Adjust protein loading (increase up to 50 μg/lane)

This systematic approach has proven effective in optimizing detection of low-abundance plant proteins in multiple studies .

What are the best fixation and permeabilization methods when using AT1g12170 antibody for immunolocalization in plant tissues?

For optimal immunolocalization of AT1g12170 protein in plant tissues, consider these fixation and permeabilization methods:

• Chemical fixation protocols:

  • Paraformaldehyde fixation: 4% paraformaldehyde in PBS for 1-2 hours (preserves structure while maintaining antigenicity)

  • Combination fixation: 4% paraformaldehyde with 0.1-0.5% glutaraldehyde for improved ultrastructure

  • Ethanol-acetic acid (3:1): Alternative for certain applications when aldehyde fixation yields poor results

• Permeabilization strategies:

  • Cell wall digestion: Enzymatic treatment with pectolyase (0.2-1%) and cellulase (1-2%) to facilitate antibody penetration

  • Detergent treatment: 0.1-0.5% Triton X-100 or 0.05-0.2% Tween-20 post-fixation

  • Freeze-thaw cycles: For difficult tissues, rapid freezing in liquid nitrogen followed by thawing can improve accessibility

• Tissue-specific considerations:

  • Leaf tissue: Shorter fixation times (30-60 min) to prevent overfixation

  • Root tissue: Extended enzymatic digestion may be necessary

  • Meristematic regions: Reduced detergent concentration to preserve delicate structures

• Antigen retrieval options:

  • Citrate buffer (pH 6.0) heating if epitope masking occurs

  • Protease treatment (1-5 μg/ml proteinase K) briefly if needed

• Blocking optimization:

  • 2-5% BSA with 5-10% normal serum from secondary antibody host species

  • Include 0.1-0.3% Triton X-100 in blocking solution for improved penetration

These approaches have been successfully implemented in immunolocalization studies of various plant proteins, including those in Arabidopsis .

How should I modify ChIP protocols when using AT1g12170 antibody for difficult plant tissues?

When adapting ChIP protocols for AT1g12170 antibody in challenging plant tissues, implement these specialized modifications:

• Tissue preparation refinements:

  • Flash-freeze tissue in liquid nitrogen immediately after collection

  • Grind thoroughly to fine powder before crosslinking

  • For recalcitrant tissues, perform vacuum infiltration with crosslinking solution

  • Consider double-crosslinking with DSG (2 mM) followed by formaldehyde for protein-protein interactions

• Crosslinking optimizations:

  • Test multiple formaldehyde concentrations (0.75-1.5%)

  • Adjust crosslinking times (10-20 minutes)

  • For woody tissues, extend vacuum infiltration during crosslinking

  • Include penetration enhancers like 0.05% Silwet L-77

• Chromatin extraction improvements:

  • Use specialized plant nuclear isolation buffers containing polyvinylpyrrolidone (PVP) to remove phenolics

  • Include β-mercaptoethanol (5 mM) to counteract oxidative compounds

  • Add protease inhibitor cocktails designed for plant tissues

  • Employ filtration steps through Miracloth to remove debris

• Sonication parameter adjustments:

  • Optimize sonication conditions extensively for each tissue type

  • For fibrous tissues, increase cycle numbers while decreasing power

  • Verify fragment size distribution (aim for 200-500 bp)

• Immunoprecipitation enhancements:

  • Extended incubation times (overnight at 4°C)

  • Increase antibody amount (4-10 μg per reaction)

  • Add BSA (0.1-0.5%) to reduce non-specific binding

  • Consider using protein A/G magnetic beads for improved recovery

These specialized modifications have proven effective in ChIP experiments with various plant tissues, including those with high levels of interfering compounds .

How can I distinguish between specific and non-specific signals when using AT1g12170 antibody?

To distinguish between specific and non-specific signals when using AT1g12170 antibody:

• Implement definitive controls:

  • Use knockout/knockdown lines as gold-standard negative controls

  • Include competing peptide controls to confirm epitope specificity

  • Employ secondary antibody-only controls to assess background

  • Compare multiple antibodies targeting different epitopes of the same protein

• Analyze signal characteristics:

  • Specific signals typically show consistent molecular weight across samples

  • Non-specific binding often shows variable patterns between experiments

  • Examine subcellular localization consistency with predicted protein function

  • Verify signal disappearance under competitive conditions

• Quantitative assessment methods:

  • Calculate signal-to-noise ratios across multiple experiments

  • Set strict threshold criteria based on control experiments

  • Perform statistical analysis to differentiate true signal from background

• Cross-validation approaches:

  • Confirm findings with orthogonal techniques (mass spectrometry)

  • Use tagged protein expression to verify antibody detection patterns

  • Correlate protein detection with mRNA expression data

This comprehensive approach is essential as studies have demonstrated that many commercially available antibodies can recognize non-target proteins of similar molecular weights, even in knockout models lacking the target protein .

What bioinformatic tools are recommended for analyzing ChIP-seq data generated using AT1g12170 antibody?

For analyzing ChIP-seq data generated with AT1g12170 antibody, employ these specialized bioinformatic approaches:

• Primary analysis tools:

  • Quality control: FastQC, MultiQC for read quality assessment

  • Alignment: Bowtie2 (v2.1.0) with optimized parameters for plant genomes

  • Duplicate removal: Picard MarkDuplicates to collapse identical reads

  • Peak calling: MACS (1.4.2) with p-value threshold of 1e-03

• Plant-specific considerations:

  • Genome annotation: Use current TAIR10 annotation for Arabidopsis

  • Repetitive region handling: Implement specialized filtering for plant repetitive elements

  • Reference genome selection: Choose appropriate ecotype references when available

• Advanced analysis approaches:

  • Differential binding analysis: DiffBind or similar tools for comparing conditions

  • Motif discovery: MEME-ChIP, HOMER for identifying enriched sequence motifs

  • Gene ontology: agriGO for plant-specific GO enrichment analysis

  • Data integration: Integrate with RNA-seq using tools like BEDTools (2.17.0)

• Visualization platforms:

  • Genome browsers: IGV, JBrowse with TAIR10 tracks

  • Profile plots: deepTools for generating TSS-centered or metagene plots

  • Heatmaps: ComplexHeatmap for visualizing multivariate data

• Statistical frameworks:

  • R (3.2.3 or later) with Bioconductor packages for comprehensive statistical analysis

  • DESeq2 for differential analysis with appropriate normalization

This comprehensive analysis pipeline, adapted from successful plant epigenomic studies, enables robust interpretation of chromatin binding profiles .

How can AT1g12170 antibody be used to investigate protein-protein interactions in stress response pathways?

AT1g12170 antibody can be leveraged to investigate protein-protein interactions in stress response pathways through these advanced methodological approaches:

• Co-immunoprecipitation under stress conditions:

  • Perform parallel co-IPs in control and stress-treated plants

  • Use gentle extraction conditions to preserve stress-induced interactions

  • Compare interaction partners identified by mass spectrometry between conditions

  • Validate key interactions with reciprocal co-IPs

• Proximity-based interaction methods:

  • Adapt proximity ligation assays (PLA) for plant tissues

  • Deploy BioID or TurboID proximity labeling with AT1g12170 fusion proteins

  • Visualize interaction dynamics in living cells using split fluorescent protein systems

• Dynamic interaction assessment:

  • Implement time-course experiments following stress application

  • Quantify changes in interaction strength using quantitative co-IP

  • Correlate interaction dynamics with physiological responses

• Subcellular localization changes:

  • Track protein relocalization during stress using immunofluorescence

  • Compare protein complex composition in different cellular compartments

  • Assess post-translational modifications affecting interactions

• Functional validation approaches:

  • Disrupt specific interactions through targeted mutations

  • Correlate interaction changes with altered stress phenotypes

  • Implement CRISPR-based tagging for endogenous protein tracking

This integrated approach has proven effective in elucidating stress-responsive protein interaction networks in plants, revealing how protein complexes dynamically assemble and disassemble during environmental challenges .