At1g80960 Antibody

What is the At1g80960 Antibody and what protein does it target in Arabidopsis thaliana?

At1g80960 Antibody (CSB-PA882809XA01DOA) is a research-grade antibody that recognizes and binds to the protein encoded by the At1g80960 gene in Arabidopsis thaliana, also known as Mouse-ear cress. This protein is identified by the UniProt accession number Q9SAG4 . The antibody is typically available in research quantities of 2ml/0.1ml and is designed specifically for research applications in plant molecular biology and biochemistry.

The At1g80960 gene encodes a protein involved in plant cellular processes, and the corresponding antibody is an important tool for studying its expression, localization, and function. When designing experiments with this antibody, researchers should consider the specific nature of the target protein, including its predicted molecular weight, cellular localization, and potential post-translational modifications that might affect antibody recognition.

For optimal experimental outcomes, it's essential to understand that this antibody has been developed against a specific epitope of the target protein. Researchers should therefore carefully evaluate the epitope information provided by the manufacturer to assess potential cross-reactivity with homologous proteins in other plant species or within Arabidopsis itself.

How should I validate the specificity of At1g80960 Antibody before experimental use?

Validation of antibody specificity is a critical first step before conducting any major experiments with At1g80960 Antibody. The most definitive approach is to perform Western blot analysis comparing wild-type Arabidopsis extracts with those from at1g80960 knockout or knockdown mutants. The absence or significant reduction of the target band in the mutant sample provides strong evidence for antibody specificity.

Pre-absorption controls represent another valuable validation method. In this approach, the antibody is pre-incubated with excess purified target antigen or a synthesized peptide corresponding to the epitope. A significant reduction in signal following pre-absorption indicates specificity for the intended target. For advanced validation, researchers can employ immunoprecipitation followed by mass spectrometry to confirm the identity of the pulled-down protein.

Multiple detection techniques should be employed for comprehensive validation. Comparing the results from Western blotting, immunofluorescence, and immunoprecipitation can provide confidence in antibody specificity across different experimental contexts. Documentation of these validation experiments is essential for publication and should include images of full Western blots, detailed descriptions of controls, and quantification of signal specificity.

What positive and negative controls are recommended when working with At1g80960 Antibody?

Effective experimental design with At1g80960 Antibody requires thoughtful selection of both positive and negative controls. For positive controls, researchers should consider using Arabidopsis tissues or developmental stages known to express the At1g80960 gene at high levels, based on transcriptomic data or previous publications. Recombinant expression of the target protein in a heterologous system can also serve as an excellent positive control, particularly when tagged with a detectable marker.

For negative controls, genetic materials are particularly valuable. T-DNA insertion lines, CRISPR-generated knockouts, or RNAi lines targeting At1g80960 provide the most definitive negative controls. Additionally, tissues known to have minimal At1g80960 expression based on published expression atlases can serve as biological negative controls.

Technical negative controls should include omission of primary antibody, substitution with non-immune serum of the same species, and pre-absorption with the target antigen. For immunolocalization studies, competing peptide controls and secondary antibody-only controls are essential to distinguish specific staining from background or autofluorescence, which can be particularly problematic in plant tissues containing chlorophyll and other autofluorescent compounds.

What are the recommended applications for At1g80960 Antibody in plant research?

At1g80960 Antibody can be employed across multiple experimental approaches in plant research. Western blotting represents the most common application, allowing detection and semi-quantitative analysis of the target protein in tissue extracts. For this application, researchers should optimize protein extraction methods to ensure preservation of the epitope recognized by the antibody, particularly considering the potentially challenging nature of plant tissues containing cell walls and interfering compounds.

Immunolocalization techniques, including immunofluorescence and immunogold electron microscopy, can provide valuable insights into the subcellular localization of the At1g80960 protein. These approaches require careful optimization of fixation and permeabilization protocols to maintain both cellular architecture and epitope accessibility. For plant tissues, embedding methods that preserve antigenicity while allowing sectioning through rigid cell walls are particularly important.

More advanced applications include chromatin immunoprecipitation (ChIP) if the protein has DNA-binding properties, co-immunoprecipitation for studying protein-protein interactions, and flow cytometry for quantitative analysis at the single-cell level. Each of these applications requires specific optimization for plant systems, accounting for cell wall digestion, vacuole disruption, and removal of plant-specific compounds that might interfere with antibody binding or detection systems.

How can I optimize protein extraction protocols for Western blotting with At1g80960 Antibody?

Effective protein extraction is fundamental to successful Western blotting with At1g80960 Antibody. Plant tissues present unique challenges due to their rigid cell walls, large vacuoles, and abundance of interfering compounds like polyphenols and polysaccharides. A recommended extraction buffer should contain 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS, supplemented with protease inhibitors freshly added before use.

Several plant-specific considerations can improve extraction quality. Addition of polyvinylpolypyrrolidone (PVPP) at 2-5% can effectively absorb polyphenols, while 2-5 mM EDTA helps inactivate metal-dependent proteases common in plant tissues. Antioxidants such as 10 mM DTT or 5 mM β-mercaptoethanol can prevent oxidation of proteins during extraction. For tissues rich in phenolic compounds, inclusion of 2% PEG-4000 can further reduce interference.

The physical disruption method significantly impacts extraction efficiency. For Arabidopsis seedlings or leaves, grinding in liquid nitrogen with a mortar and pestle typically yields the best results, ensuring the sample remains frozen throughout processing. Alternatively, bead-beating systems with zirconia or steel beads can provide efficient disruption while maintaining cold temperatures. Following homogenization, centrifugation should be performed at 15,000 × g for 15-20 minutes at 4°C to remove insoluble debris while retaining the target protein in the supernatant.

What considerations are important for immunoprecipitation experiments using At1g80960 Antibody?

Immunoprecipitation (IP) with At1g80960 Antibody requires careful consideration of experimental conditions to maintain protein-protein interactions while achieving efficient target pulldown. The lysis buffer composition is critical – a gentle buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, and 1 mM EDTA typically preserves interactions while allowing efficient extraction.

Pre-clearing the lysate with protein A/G beads for 1 hour at 4°C before adding the antibody significantly reduces non-specific binding. For the immunoprecipitation step itself, the antibody-to-lysate ratio requires optimization; typically starting with 2-5 μg of antibody per 500 μg of total protein is recommended. The antibody-lysate mixture should be incubated overnight at 4°C with gentle rotation to allow complete antigen binding while minimizing protein degradation.

Several washing steps are crucial for reducing background. A recommended washing protocol includes three washes with lysis buffer followed by two washes with a stringent buffer (lysis buffer with 300 mM NaCl) and a final wash with PBS. Between washes, centrifugation should be gentle (2,500 × g for 2-3 minutes) to avoid bead compaction. For elution, either SDS sample buffer at 95°C for direct analysis or a gentler approach using excess competing peptide may be employed depending on downstream applications.

Why might I see non-specific bands when using At1g80960 Antibody in Western blotting?

Non-specific bands in Western blotting with At1g80960 Antibody can result from several factors that require systematic troubleshooting. Cross-reactivity with structurally similar proteins represents a common cause, particularly in plants where gene duplication events have created families of related proteins. Analyzing the predicted sizes of potential homologs can help identify whether unexpected bands correspond to related proteins.

Protein degradation during sample preparation frequently contributes to additional bands of lower molecular weight than expected. To address this, ensure protease inhibitor cocktails are fresh and comprehensive, sample processing is performed quickly at 4°C, and samples are never subjected to freeze-thaw cycles. Adding additional protease inhibitors specifically effective against plant proteases, such as pepstatin A (1 μg/ml) and leupeptin (1 μg/ml), can provide enhanced protection.

Optimization of blocking conditions can significantly reduce non-specific binding. While 5% non-fat dry milk in TBST is standard, for some plant extracts, alternative blocking agents such as 5% BSA or 3% gelatin may produce cleaner results. Extending blocking time to 2 hours at room temperature or overnight at 4°C, and diluting primary antibody in fresh blocking solution rather than TBST alone, can further reduce background signals.

How can I improve signal detection when working with low-abundance At1g80960 protein?

Detection of low-abundance proteins presents a significant challenge that requires multiple optimization strategies. Sample enrichment techniques such as subcellular fractionation can concentrate the target protein by isolating the cellular compartment where it predominantly resides. For membrane-associated proteins, membrane fractionation using differential centrifugation can significantly increase detection sensitivity.

Antibody concentration and incubation conditions critically affect detection sensitivity. While standard protocols often recommend a 1:1000 dilution, for low-abundance proteins, concentrations of 1:500 or even 1:250 may be necessary. Extending primary antibody incubation to overnight at 4°C with gentle agitation improves binding efficiency. Similarly, signal amplification systems such as biotin-streptavidin or tyramide signal amplification can enhance detection of minimal amounts of target protein.

Enhanced chemiluminescence (ECL) substrate selection significantly impacts detection limits. Standard ECL may be insufficient for very low abundance proteins; instead, femto-sensitivity ECL substrates can increase detection by 10-100 fold. Digital imaging systems with adjustable exposure times and accumulation modes further improve sensitivity compared to film-based detection, allowing capture of weak signals without background saturation.

What strategies can resolve issues with high background in immunofluorescence using At1g80960 Antibody?

High background in plant immunofluorescence studies often stems from inadequate fixation, autofluorescence, or non-specific antibody binding. Fixation optimization is crucial – while 4% paraformaldehyde is standard, testing alternative fixatives such as methanol-acetone (1:1) or ethanol-acetic acid (3:1) may better preserve the epitope while reducing background. Fixation time should be carefully optimized, as over-fixation can increase background through non-specific protein crosslinking.

Plant-specific autofluorescence requires dedicated approaches. Pre-treatment with 0.1% sodium borohydride for 10 minutes can reduce aldehyde-induced fluorescence from fixation. For chlorophyll autofluorescence, extraction with 80% acetone prior to immunostaining or selecting detection fluorophores with emission spectra distinct from chlorophyll autofluorescence (avoiding 650-700 nm range) can improve signal-to-noise ratio.

Blocking and washing protocols significantly impact background levels. Extended blocking (2-3 hours) with 5% normal serum from the secondary antibody host species, supplemented with 0.3% Triton X-100 and 1% BSA, effectively reduces non-specific binding. After antibody incubations, implementing more stringent washing steps (6-8 washes of 10 minutes each) with 0.1% Tween-20 in PBS can dramatically reduce background without compromising specific signals.

How can I use At1g80960 Antibody for protein-protein interaction studies?

At1g80960 Antibody can be powerfully applied to identify protein interaction partners through several complementary approaches. Co-immunoprecipitation (co-IP) represents the most direct method, wherein the antibody captures the target protein along with its binding partners from native plant extracts. This approach requires gentle lysis conditions that preserve protein-protein interactions, typically using buffers containing 0.5% NP-40 or 1% Triton X-100 with physiological salt concentrations (150 mM NaCl).

Proximity-dependent labeling techniques offer a more comprehensive view of the protein interaction landscape. By conjugating the antibody to enzymes such as BioID or APEX2, researchers can label proteins in close proximity to the target in living cells, enabling identification of transient or context-dependent interactions. This approach is particularly valuable for membrane-associated proteins or those involved in dynamic complexes.

Fluorescence-based interaction studies complement biochemical approaches. Combining the At1g80960 Antibody with antibodies against suspected interaction partners in co-localization studies can provide spatial evidence for interactions. For quantitative assessment, Förster Resonance Energy Transfer (FRET) or Proximity Ligation Assay (PLA) can confirm protein-protein interactions at nanometer-scale distances, offering resolution beyond the diffraction limit of conventional microscopy.

What approaches are recommended for quantitative analysis of At1g80960 protein levels across different conditions?

Quantitative analysis of At1g80960 protein requires carefully controlled experimental design and appropriate normalization strategies. For Western blot-based quantification, digital imaging and densitometry software enable precise measurement of band intensity, which correlates with protein abundance. Multiple technical replicates (at least three) and biological replicates (typically three to five) are essential for statistical validity. Loading standardization using total protein staining methods such as Ponceau S or Stain-Free technology provides more reliable normalization than traditional housekeeping proteins, which may vary under experimental conditions.

Enzyme-linked immunosorbent assay (ELISA) offers greater quantitative precision than Western blotting when properly optimized. Developing a sandwich ELISA with At1g80960 Antibody as the capture or detection antibody, combined with standard curves of recombinant protein, can provide absolute quantification of the target protein. This approach requires significant optimization but offers higher throughput and greater dynamic range than gel-based methods.

Flow cytometry represents an advanced approach for single-cell level quantification, particularly valuable for studying cellular heterogeneity. This method requires successful protoplast preparation from plant tissues, followed by fixation, permeabilization, and antibody staining. While technically challenging, flow cytometry provides quantitative data on protein abundance across thousands of individual cells, revealing population distributions masked by bulk measurements.

How can I use At1g80960 Antibody for chromatin immunoprecipitation (ChIP) studies?

Chromatin immunoprecipitation with At1g80960 Antibody can reveal DNA binding sites if the target protein interacts with chromatin, either directly or as part of a complex. Plant-specific ChIP protocols require optimization of crosslinking conditions to balance efficient protein-DNA fixation with subsequent chromatin shearing. Typically, 1% formaldehyde for 10-15 minutes provides adequate crosslinking for most nuclear proteins, though optimization for specific targets is recommended.

Chromatin extraction and fragmentation present unique challenges in plant tissues. After crosslinking, tissues should be ground in liquid nitrogen, followed by nuclear isolation using sucrose gradient centrifugation to reduce cytoplasmic contamination. Sonication parameters require careful optimization to generate chromatin fragments of 200-500 bp; typically, 15-20 cycles of 30 seconds on/30 seconds off at medium power works well for Arabidopsis tissues.

The immunoprecipitation step is critical for ChIP success. Pre-clearing chromatin with protein A/G beads reduces background, while using 3-5 μg of At1g80960 Antibody per immunoprecipitation typically provides sufficient enrichment. Including negative controls (non-immune IgG) and positive controls (antibody against a known chromatin-associated protein) is essential for validating results. After reverse crosslinking and DNA purification, qPCR analysis of predicted binding regions or genome-wide approaches such as ChIP-seq can identify DNA binding sites of the At1g80960 protein or its associated complexes.

What are the optimal storage conditions for maintaining At1g80960 Antibody activity?

Proper storage of At1g80960 Antibody is essential for maintaining its specificity and sensitivity over time. The antibody should be stored at -20°C for long-term preservation, with aliquoting into single-use volumes highly recommended to avoid repeated freeze-thaw cycles. Each freeze-thaw cycle can reduce antibody activity by approximately 10-20%, with most antibodies tolerating no more than 5-10 cycles before significant deterioration occurs.

Special consideration should be given to antibody concentration and buffer composition. At1g80960 Antibody maintained at higher concentrations (>0.5 mg/ml) generally exhibits better stability than dilute solutions. For antibodies showing signs of aggregation or precipitation, filtration through a 0.22 μm filter followed by addition of stabilizing proteins such as 0.1% BSA can often restore performance. Detailed record-keeping of antibody performance across experiments helps track potential deterioration and informs timely replacement.

How should I prepare and dilute At1g80960 Antibody for various experimental applications?

Proper antibody dilution significantly impacts experimental outcomes and reagent economy. For Western blotting, initial titration experiments testing dilutions from 1:500 to 1:5000 will identify the optimal concentration that balances specific signal intensity with minimal background. Once established, the antibody should be diluted in freshly prepared blocking solution (typically 5% non-fat dry milk or 3-5% BSA in TBST) immediately before use.

For immunofluorescence applications, typical starting dilutions range from 1:100 to 1:500, with optimization recommended for each specific tissue type and fixation method. Dilution in blocking buffer containing 1-3% normal serum from the secondary antibody host species helps reduce non-specific binding. Centrifugation of diluted antibody solution at 10,000 × g for 5 minutes before application can remove potential aggregates that might contribute to speckling or high background.

Antibody handling during dilution and application requires attention to temperature and physical conditions. Allow refrigerated antibody vials to equilibrate to room temperature before opening to prevent condensation that can promote microbial growth and protein denaturation. Use only clean pipette tips and low-protein binding tubes for dilution. For optimal binding kinetics, apply antibody solutions at volumes sufficient to completely cover the sample – typically 50-100 μl per coverslip for immunofluorescence or 10 ml per membrane for Western blotting in standard containers.