At3g13980 Antibody

What is At3g13980 and what is its biological significance?

At3g13980 encodes a SKI/DACH domain protein in Arabidopsis thaliana, an important model organism for plant molecular biology research . The SKI/DACH domain is evolutionarily conserved and typically associated with transcriptional regulation and developmental processes in eukaryotes. Based on its classification, this protein likely functions in gene expression regulation pathways, potentially influencing plant growth and developmental processes. Understanding this protein's function can provide insights into fundamental plant biological processes and potentially translate to agricultural applications .

What are the key specifications of commercially available At3g13980 antibodies?

The commercially available At3g13980 antibody is a rabbit polyclonal antibody purified by antigen affinity techniques. The antibody's immunogen is a recombinant Arabidopsis thaliana At3g13980 protein, conferring specificity to the target. The antibody is supplied in an unconjugated form, making it versatile for various detection methods when paired with appropriate secondary detection systems. Research-grade preparations typically include essential components for validation: 200μg of antigen (serving as a positive control) and 1ml of pre-immune serum (serving as a negative control) alongside the purified antibody preparation .

What are the validated applications for At3g13980 antibody?

The At3g13980 antibody has been validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications according to supplier specifications . For ELISA applications, the antibody can detect native or recombinant At3g13980 protein in solution-phase assays. In Western Blot applications, the antibody can identify denatured At3g13980 protein separated by SDS-PAGE and transferred to appropriate membrane substrates. When designing experiments, researchers should incorporate the supplied positive control (recombinant antigen) to establish detection parameters and the negative control (pre-immune serum) to assess background signal levels .

How should Western Blot protocols be optimized for At3g13980 detection?

Western Blot optimization for At3g13980 detection requires systematic adjustment of multiple parameters. Begin with sample preparation: extract plant tissues in buffer containing protease inhibitors to prevent target degradation. For membrane selection, PVDF membranes often provide better protein retention for plant samples compared to nitrocellulose. Primary antibody concentration should be titrated, starting with 1:1000 dilution and adjusting based on signal-to-noise ratio. Extended primary antibody incubation (overnight at 4°C) typically improves specific binding. For blocking, 5% non-fat milk in TBST is standard, but BSA-based blocking may yield improved results for some plant samples. When troubleshooting, systematically vary one parameter at a time while maintaining others constant. Always run the provided positive control (recombinant antigen) alongside experimental samples to verify detection system functionality .

What strategies can address cross-reactivity concerns with At3g13980 antibody?

Addressing cross-reactivity concerns requires multiple validation approaches. First, perform pre-adsorption tests by incubating the antibody with excess recombinant At3g13980 protein before application to samples—specific signals should be significantly reduced or eliminated. Second, compare detection patterns between wild-type plants and At3g13980 knockout/knockdown mutants if available. Third, employ immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody. Western blot analysis should be performed under high-stringency conditions (higher salt concentration, reduced antibody concentration) to minimize non-specific binding. During data analysis, carefully examine band patterns—At3g13980-specific signal should appear at the expected molecular weight (~50-55 kDa), while bands at unexpected molecular weights may indicate cross-reactivity requiring further validation .

How can immunoprecipitation protocols be optimized for At3g13980 studies?

Optimizing immunoprecipitation (IP) protocols for At3g13980 studies requires careful consideration of extraction conditions and binding parameters. Plant tissues should be extracted in non-denaturing buffers (typically Tris-based, pH 7.4-8.0) containing mild detergents (0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions. Pre-clear lysates with protein A/G beads before antibody addition to reduce non-specific binding. For antibody binding, use 2-5 μg of At3g13980 antibody per 500 μg of total protein, and incubate overnight at 4°C with gentle rotation. After capture with protein A/G beads, implement stringent washing steps (at least 4-5 washes) with increasing salt concentrations to remove non-specifically bound proteins. Elution can be performed using either low pH glycine buffer (pH 2.5-3.0) or by boiling in SDS sample buffer, depending on downstream applications. For co-immunoprecipitation studies specifically, crosslinking with formaldehyde or DSP (dithiobis[succinimidyl propionate]) before extraction may stabilize transient protein-protein interactions .

What control systems are essential when designing experiments with At3g13980 antibody?

Robust experimental design with At3g13980 antibody requires implementation of multiple control systems. Primary controls should include: (1) Positive control using the supplied recombinant At3g13980 protein to validate detection system functionality; (2) Negative control using pre-immune serum to establish background signal levels; (3) Secondary antibody-only control to assess non-specific binding of the detection system; (4) Tissue-specific controls comparing tissues with known differential expression of At3g13980; and (5) Technical replicates to ensure reproducibility. For advanced studies, include biological knockdown/knockout controls if available. When using fluorescent detection systems, include autofluorescence controls for plant tissues, which commonly exhibit background fluorescence. In multiplex detection systems, perform single-antibody controls to verify specificity in the presence of multiple detection reagents. Document all control outcomes thoroughly in research notes and publications to support result validity .

How can researchers validate antibody specificity for At3g13980 in different experimental systems?

Validating At3g13980 antibody specificity across experimental systems requires a multi-faceted approach. Begin with epitope analysis by aligning the immunogen sequence against the proteome of your experimental system to identify potential cross-reactive proteins. Perform peptide competition assays by pre-incubating the antibody with excess immunogenic peptide before application to samples—specific signals should be abolished. For genetic validation, compare antibody reactivity in wild-type versus knockout/knockdown systems. When working with new plant species, perform Western blot analysis first to confirm the antibody detects a protein of the expected molecular weight. For immunohistochemistry applications, include peptide competition controls alongside tissue-specific expression analysis. If available, compare reactivity patterns with orthogonal detection methods such as RNA expression analysis or fluorescent protein tagging. For quantitative applications, establish a standard curve using recombinant protein to determine detection limits and linear response range .

What approaches can resolve inconsistent Western blot results with At3g13980 antibody?

Resolving inconsistent Western blot results requires systematic troubleshooting of sample preparation, transfer efficiency, and detection parameters. For sample preparation issues, ensure complete protein extraction with fresh protease inhibitors and consistent protein quantification. Verify protein integrity by Ponceau S staining of membranes post-transfer. For transfer-related inconsistencies, optimize transfer time and voltage for the expected molecular weight of At3g13980, and consider semi-dry versus wet transfer systems for efficiency comparison. For antibody binding issues, prepare fresh dilutions from stock antibody, and test multiple blocking agents (milk, BSA, commercial blockers) to optimize signal-to-noise ratio. Implement extended washing steps (minimum 4 x 10 minutes) to reduce background. For detection problems, verify secondary antibody functionality with a different primary antibody system, and consider enhanced chemiluminescence versus fluorescent detection methods. Document all protocol adjustments in a laboratory notebook to facilitate reproducibility once optimized conditions are established .

How should researchers interpret At3g13980 detection patterns in different plant tissues?

Interpretation of At3g13980 detection patterns requires careful consideration of tissue-specific expression, protein modification states, and subcellular localization. When analyzing tissue-specific expression, normalize band intensity to appropriate loading controls (e.g., actin, tubulin, or total protein via Ponceau S staining). SKI/DACH domain proteins commonly display tissue-specific expression patterns associated with developmental stages, so correlate detection patterns with developmental markers. Multiple bands may indicate post-translational modifications, alternative splicing variants, or proteolytic processing. Verify band specificity through peptide competition assays. For subcellular fractionation studies, compare enrichment patterns against established compartment markers (e.g., histone H3 for nuclear fractions, RuBisCO for chloroplasts). Quantitative analysis should employ densitometry with appropriate standard curves and statistical analysis. When comparing expression across conditions, ensure identical exposure times and image acquisition parameters. For developmental studies, establish a time-course analysis to map expression changes during specific developmental transitions .

How can At3g13980 antibody be implemented in chromatin immunoprecipitation studies?

Implementing At3g13980 antibody in Chromatin Immunoprecipitation (ChIP) studies requires optimization of crosslinking, sonication, and immunoprecipitation parameters specific to plant tissues. For crosslinking, use 1% formaldehyde for 10-15 minutes under vacuum for efficient penetration into plant tissues, followed by quenching with glycine. Grind tissues thoroughly in liquid nitrogen before adding nuclear isolation buffer to prevent protein degradation. Optimize sonication conditions (typically 10-15 cycles of 30 seconds on/30 seconds off) to generate DNA fragments of 200-500 bp, verifying fragment size by agarose gel electrophoresis. Use 3-5 μg of At3g13980 antibody per ChIP reaction, with IgG controls processed in parallel. For plant tissues, implement additional washing steps with lithium chloride buffer to reduce background. Verify enrichment by qPCR before proceeding to genome-wide analysis methods. Target validation should include analysis of known SKI/DACH target genes from related systems as positive controls. For ChIP-seq applications, verify library quality and sequencing depth requirements (typically 20-30 million reads minimum) before processing .

What methodological approaches enable protein-protein interaction studies with At3g13980?

Protein-protein interaction studies with At3g13980 can employ multiple complementary methodologies. Co-immunoprecipitation using the At3g13980 antibody represents the direct approach—extract plant tissues under non-denaturing conditions, immunoprecipitate with At3g13980 antibody, and identify interacting partners by Western blot or mass spectrometry. For in situ validation of interactions, proximity ligation assay (PLA) combines At3g13980 antibody with antibodies against suspected interaction partners. Bimolecular Fluorescence Complementation (BiFC) provides an orthogonal validation method by expressing At3g13980 and partner proteins as fusion constructs with split fluorescent protein fragments. For quantitative interaction analysis, microscale thermophoresis or surface plasmon resonance using the recombinant proteins can determine binding affinities. Yeast two-hybrid screening can identify novel interaction partners from cDNA libraries. For transient interactions, implement crosslinking before extraction or use protein fragment complementation assays (PCA). Advanced approaches include tandem affinity purification (TAP) tagging of At3g13980 followed by mass spectrometry analysis of purified complexes. Each method provides complementary data addressing different aspects of interaction dynamics .