At5g08100 Antibody

What is the At5g08100 gene and what protein does it encode?

The At5g08100 gene in Arabidopsis thaliana encodes an N-terminal nucleophile aminohydrolases (Ntn hydrolases) superfamily protein. According to TAIR10 annotations, this protein contains the InterPro domain "Peptidase T2, asparaginase 2" (InterPro:IPR000246) . The protein is 315 amino acids in length with a calculated molecular weight of 33029.40 Da and an isoelectric point of 5.18 .

The protein sequence begins with MVGWAIALHG and has structural features characteristic of Ntn hydrolases . Bioinformatic analysis demonstrates that At5g08100 shares homology with 3243 proteins across 873 species, including Archaea (113 hits), Bacteria (1502 hits), Metazoa (516 hits), Fungi (215 hits), Plants (193 hits), and other Eukaryotes (704 hits) . This widespread conservation across diverse organisms suggests the protein likely performs an evolutionarily conserved function. Within Arabidopsis, its closest match is another Ntn hydrolase superfamily protein encoded by AT3G16150.1 .

What is the predicted subcellular localization of At5g08100 protein?

Subcellular localization prediction using SUBAcon indicates that At5g08100 protein is predominantly localized in the cytosol with high confidence (score: 1.000) . This cytosolic localization is important for researchers designing extraction protocols and interpreting experimental results, particularly when performing subcellular fractionation or immunolocalization studies.

The protein's localization suggests it likely participates in cytosolic metabolic processes rather than specialized organelle-specific functions. When designing experiments to study this protein, researchers should focus on cytosolic extraction methods and consider how the protein's activities might be integrated with other cytosolic metabolic pathways.

What is the biological function of the At5g08100 protein?

Based on its classification as an Ntn hydrolase superfamily protein with a peptidase T2/asparaginase 2 domain, the At5g08100 protein likely functions in proteolytic processing or nitrogen metabolism. The peptidase T2/asparaginase domain suggests the protein may catalyze the hydrolysis of asparagine to aspartate and ammonia, which is a crucial process in nitrogen metabolism and mobilization in plants.

While the specific function of At5g08100 isn't directly addressed in the search results, research on nitrogen metabolism in Arabidopsis provides contextual insights. Studies have shown that enzymes involved in nitrogen assimilation and amino acid biosynthesis, such as glutamine synthetases, exhibit altered expression under various conditions . Since asparagine serves as a major nitrogen transport compound in plants, At5g08100 may play roles in nitrogen remobilization during processes like leaf senescence, seed development, or stress responses.

What are the optimal conditions for using At5g08100 Antibody in Western blotting?

For optimal Western blotting results with At5g08100 Antibody (CSB-PA556349XA01DOA) , researchers should implement the following protocol:

Sample preparation:

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

• Quantify protein concentration using Bradford or BCA assay

• Mix samples with Laemmli buffer and heat at 95°C for 5 minutes

Gel electrophoresis and transfer:

• Load 20-30 μg of total protein on a 12% SDS-PAGE gel (appropriate for the ~33 kDa At5g08100 protein)

• Run gel at 120V until the dye front reaches the bottom

• Transfer to PVDF membrane using semi-dry transfer (15V for 30 minutes) or wet transfer (100V for 1 hour)

Antibody incubation and detection:

• Block membrane with 5% non-fat dry milk in TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature

• Dilute At5g08100 Antibody 1:1000 in blocking solution and incubate overnight at 4°C

• Wash 3 times with TBST, 5 minutes each

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

• Wash 3 times with TBST, 5 minutes each

• Develop using enhanced chemiluminescence (ECL) substrate

The expected band size for At5g08100 protein is approximately 33 kDa based on its calculated molecular weight . Always include appropriate positive and negative controls to validate antibody specificity.

How can researchers validate the specificity of the At5g08100 Antibody?

Validating antibody specificity is crucial for generating reliable experimental data. For At5g08100 Antibody, researchers should employ these validation strategies:

Genetic controls:

• Use wild-type Arabidopsis as positive control

• Include at5g08100 knockout or knockdown lines as negative controls

• Test overexpression lines for enhanced signal verification

Peptide competition assay:

• Pre-incubate the antibody with excess synthetic peptide corresponding to the immunogen

• Run parallel Western blots with competed and non-competed antibody

• Specific binding will be significantly reduced or eliminated in the competed sample

Recombinant protein validation:

• Express and purify recombinant At5g08100 protein

• Use as positive control in Western blotting

• Verify antibody detects the protein at the expected molecular weight

Mass spectrometry confirmation:

• Perform immunoprecipitation with At5g08100 Antibody

• Analyze precipitated proteins by mass spectrometry

• Confirm the presence of At5g08100 peptides in the precipitated material

Cross-reactivity assessment:

• Test antibody on protein extracts from related plant species with known sequence differences

• Evaluate signal pattern across species to understand epitope conservation

Thorough validation enhances confidence in experimental results and should be documented in publications to facilitate reproducibility.

What protein extraction methods are most effective for isolating At5g08100 from Arabidopsis tissues?

Since At5g08100 is predicted to be a cytosolic protein , extraction methods should be optimized for soluble proteins. Three effective approaches are:

1. Standard cytosolic extraction:

• Grind tissue in liquid nitrogen to a fine powder

• Add extraction buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM DTT, protease inhibitor cocktail

• Homogenize thoroughly and incubate on ice for 30 minutes with occasional mixing

• Centrifuge at 14,000g for 20 minutes at 4°C

• Collect supernatant containing soluble proteins

2. TCA/acetone precipitation method:

• Add 4 volumes of ice-cold 10% TCA in acetone to tissue powder

• Incubate at -20°C for 1 hour

• Centrifuge at 16,000g for 30 minutes at 4°C

• Wash pellet with ice-cold acetone (3 times)

• Air-dry briefly and resuspend in appropriate buffer

3. Phenol extraction method:

• Mix tissue with extraction buffer containing phenol

• Vortex and incubate at room temperature for 10 minutes

• Centrifuge to separate phases

• Collect phenol phase and precipitate proteins with ammonium acetate/methanol

• This method is particularly effective for tissues with high levels of interfering compounds

The choice of extraction method should be based on specific experimental needs and tissue characteristics. For tissues with high phenolic content or secondary metabolites, the phenol extraction method often yields cleaner preparations, while the standard cytosolic extraction is simpler and suitable for most applications.

How can At5g08100 Antibody be used to investigate protein-protein interactions in nitrogen metabolism pathways?

As a potential component of nitrogen metabolism pathways, At5g08100 protein likely interacts with other proteins in these processes. Several techniques can leverage the At5g08100 Antibody to identify and characterize these interactions:

Co-immunoprecipitation (Co-IP):

• Lyse Arabidopsis tissues under non-denaturing conditions to preserve protein-protein interactions

• Incubate lysate with At5g08100 Antibody and protein A/G beads

• Wash beads thoroughly to remove non-specifically bound proteins

• Elute the immunoprecipitated complex and analyze co-precipitated proteins by:

  • Western blotting with antibodies against suspected interaction partners

  • Mass spectrometry for unbiased identification of interacting proteins

Proximity-dependent labeling:

• Create fusion constructs of At5g08100 with BioID or TurboID biotin ligase

• Express in Arabidopsis via stable transformation

• Proteins in close proximity become biotinylated during in vivo labeling

• Purify biotinylated proteins using streptavidin and identify by mass spectrometry

• Validate interactions with At5g08100 Antibody in reverse Co-IP experiments

Immunolocalization co-staining:

• Perform dual immunofluorescence labeling with At5g08100 Antibody and antibodies against other nitrogen metabolism proteins

• Analyze co-localization using confocal microscopy

• Quantify spatial correlation using appropriate statistical methods

When investigating interaction partners, researchers should focus on other proteins involved in nitrogen assimilation and amino acid biosynthesis, such as glutamine synthetases, which have been shown to have altered expression in nitrogen metabolism mutants . The antibody can help validate interactions initially identified through high-throughput approaches like yeast two-hybrid screening or predicted by bioinformatic methods.

What experimental approaches can detect changes in At5g08100 protein expression under nutrient stress conditions?

Nutrient stress significantly impacts plant metabolism, potentially affecting At5g08100 expression or function. The following approaches can detect such changes:

Quantitative Western blotting:

• Expose plants to nutrient stress conditions (N, P, K, Fe, Cu deficiency)

• Collect tissue samples at multiple time points (0, 6, 12, 24, 48, 72 hours)

• Perform Western blotting with At5g08100 Antibody

• Quantify band intensity using densitometry software

• Normalize to total protein (Ponceau S staining) rather than housekeeping proteins

• Compare protein levels with transcript levels (RT-qPCR) to identify post-transcriptional regulation

Tissue-specific protein accumulation:

• Use immunohistochemistry to examine tissue-specific localization under stress

• Quantify signal intensity across different tissues and stress conditions

• Compare with promoter-reporter studies to distinguish transcriptional vs. post-transcriptional regulation

Protein stability assessment:

• Perform cycloheximide chase experiments to assess protein half-life under stress

• Use At5g08100 Antibody to detect remaining protein at various time points

• Calculate degradation rates under different conditions

Research suggests that nutrient deficiencies like copper and iron can affect gene expression patterns in Arabidopsis . Based on studies by Garcia-Molina et al., alternative splicing in the transcriptome changes under Cu and Fe deficiency , which might also affect At5g08100 expression or post-translational modifications. When designing these experiments, researchers should consider that At5g08100, as a potentially nitrogen-related enzyme, may be particularly responsive to conditions that alter the plant's nitrogen economy.

How can researchers optimize immunohistochemistry protocols for At5g08100 localization in plant tissues?

Immunohistochemistry in plant tissues presents unique challenges requiring specialized approaches:

Tissue preparation and fixation:

• Fix tissues in 4% paraformaldehyde in PBS or PEM buffer for 2-4 hours

• For woody tissues, extend fixation time or use stronger fixatives

• Embed in paraffin for sectioning or prepare fresh frozen sections

• Cut sections 5-10 μm thick for optimal antibody penetration

Cell wall considerations:

• Plant cell walls impede antibody penetration

• Treat sections with cell wall degrading enzymes:

  • 2% cellulase, 1% pectinase, 0.5% macerozyme in PBS (pH 5.8)

  • Incubate at room temperature for 30-60 minutes

• Optimize enzyme concentration and treatment time for specific tissues

Antigen retrieval methods:

• Heat-induced epitope retrieval: 10 mM sodium citrate buffer (pH 6.0), 95°C for 10-20 minutes

• Enzymatic retrieval: Proteinase K (10 μg/ml) for 10-15 minutes at 37°C

• Test multiple retrieval methods to determine optimal conditions

Blocking and antibody incubation:

• Block with 5% normal goat serum, 1% BSA, 0.3% Triton X-100 in PBS for 1-2 hours

• Dilute At5g08100 Antibody 1:100 to 1:500 in blocking buffer

• Incubate sections with primary antibody overnight at 4°C in a humidified chamber

• For detection, use fluorophore-conjugated secondary antibodies with minimal spectral overlap with plant autofluorescence

Autofluorescence management:

• Treat sections with 0.1% Sudan Black B in 70% ethanol for 10-20 minutes

• Include 0.1% Toluidine Blue in washing buffer to reduce chlorophyll autofluorescence

• Use narrow bandpass filters on microscopes to minimize autofluorescence detection

• Apply spectral unmixing algorithms during image analysis

Based on SUBAcon prediction, At5g08100 is predominantly cytosolic , so researchers should expect a diffuse cytoplasmic staining pattern rather than organelle-specific localization. Always include appropriate controls in parallel experiments to validate staining specificity.

What are the most common causes of weak signals when using At5g08100 Antibody, and how can they be addressed?

Weak signals when working with At5g08100 Antibody can result from various factors. Here are systematic approaches to diagnose and resolve these issues:

Protein extraction efficiency:

• Problem: Inadequate extraction of At5g08100 protein

• Solutions:

  • Try multiple extraction methods (see question 2.3)

  • Include fresh protease inhibitors to prevent degradation

  • Extract from tissues with high expression levels

  • Enrich for cytosolic proteins through fractionation

Antibody binding conditions:

• Problem: Suboptimal antibody concentration or incubation conditions

• Solutions:

  • Test concentration gradient (1:100, 1:500, 1:1000, 1:2000)

  • Extend incubation time to overnight at 4°C

  • Try different antibody diluents (TBST, commercial solutions)

  • Ensure gentle agitation during incubation

Detection sensitivity limitations:

• Problem: Detection system not sensitive enough

• Solutions:

  • Switch to more sensitive ECL substrate (Super Signal West Femto)

  • Use fluorescent secondary antibodies with digital imaging

  • Implement signal amplification systems (biotin-streptavidin)

  • Increase exposure time incrementally

Epitope accessibility issues:

• Problem: Epitope masked by protein folding or interactions

• Solutions:

  • Add stronger denaturing agents (8M urea) to sample buffer

  • Heat samples at 95°C for 10 minutes before loading

  • Use fresh reducing agents (DTT or β-mercaptoethanol)

  • Try different antigen retrieval methods for immunohistochemistry

The most effective approach is systematic troubleshooting by modifying one parameter at a time while documenting all changes. This methodical process helps identify the critical factors affecting antibody performance with the specific sample type.

How can researchers reduce non-specific binding and background when using At5g08100 Antibody?

Non-specific binding and high background are common challenges when working with plant samples. The following strategies can improve At5g08100 Antibody specificity:

Enhanced blocking protocols:

• Test different blocking agents (5% milk, 3% BSA, 2% casein)

• Extend blocking time to 2 hours at room temperature

• Use commercial synthetic blocking agents designed for plant samples

• Implement sequential blocking with different agents

Optimized washing procedures:

• Increase washing duration (6 x 10 minutes instead of standard 3 x 5 minutes)

• Use higher stringency wash buffers (TBST with 500 mM NaCl)

• Add low concentrations of SDS (0.05%) to wash buffer

• Maintain continuous agitation during washing steps

Plant-specific interfering compound management:

• Include PVPP (polyvinylpolypyrrolidone) at 2% in extraction buffer

• Add 2% β-mercaptoethanol to reduce phenolics

• Incorporate 5-10 mM ascorbic acid as antioxidant

• Use TCA/acetone precipitation to remove interfering compounds

Antibody pre-adsorption:

• Pre-incubate antibody with acetone powder from unrelated plant tissue

• For recombinant antibodies, include 5% extract from knockout plants

• Use protein A/G beads to pre-clear crude extracts

Secondary antibody optimization:

• Reduce secondary antibody concentration

• Use highly cross-adsorbed secondary antibodies

• Select secondary antibodies with minimal reactivity to plant proteins

For immunohistochemistry applications, additional steps to consider include quenching endogenous peroxidases with 3% hydrogen peroxide before antibody incubation and implementing specific protocols to reduce autofluorescence from chlorophyll and cell walls.

What analytical methods can validate At5g08100 protein detection specificity in complex experimental settings?

To ensure experimental rigor, researchers should validate At5g08100 protein detection using multiple complementary approaches:

Genetic validation:

• Compare signals between wild-type plants and confirmed knockout mutants

• Analyze heterozygous plants for intermediate signal intensity

• Examine overexpression lines for enhanced signal

Immunological validation:

• Perform peptide competition assays at multiple competing peptide concentrations

• Test multiple antibody lots under identical conditions

• Compare commercial antibody results with custom-generated antibodies against different epitopes

Biochemical validation:

• Use size-based fractionation methods (gel filtration) to confirm signal appears at expected molecular weight

• Perform 2D electrophoresis (isoelectric focusing followed by SDS-PAGE) to assess specificity

• Implement orthogonal purification strategies before immunodetection

Mass spectrometry confirmation:

• Excise protein band corresponding to At5g08100 from gel after Western blotting

• Perform in-gel digestion and peptide extraction

• Analyze by LC-MS/MS to confirm protein identity

• Compare detected peptides with theoretical tryptic peptides from At5g08100

Image analysis validation (for microscopy):

• Implement rigorous colocalization analysis with known compartment markers

• Use fluorescence correlation spectroscopy to confirm binding specificity

• Apply deconvolution algorithms to improve signal-to-noise ratio

• Conduct fluorescence recovery after photobleaching (FRAP) experiments to validate protein dynamics

By implementing multiple validation strategies, researchers can establish strong confidence in their experimental findings and distinguish genuine biological signals from technical artifacts.

How might At5g08100 Antibody be used in studies investigating plant responses to environmental stresses?

As climate change intensifies environmental stresses on plants, understanding stress response mechanisms becomes increasingly important. At5g08100 Antibody offers several applications in this context:

Stress-induced protein expression dynamics:

• Compare At5g08100 protein levels across multiple stresses (drought, salt, heat, cold, nutrient deficiency)

• Create time-course profiles of protein accumulation during stress and recovery

• Correlate protein changes with physiological parameters (growth, photosynthesis, metabolite levels)

Post-translational modification analysis:

• Use the antibody to immunoprecipitate At5g08100 from stressed plants

• Analyze for stress-induced PTMs (phosphorylation, ubiquitination, SUMOylation)

• Map modifications to specific domains to infer functional significance

• Develop modification-specific antibodies for high-throughput screening

Protein-protein interaction networks under stress:

• Implement stress-specific interaction proteomics using At5g08100 as bait

• Compare interactomes under normal and stress conditions

• Identify stress-specific protein complexes and regulatory mechanisms

Tissue-specific stress responses:

• Map protein distribution changes during stress progression

• Identify tissues that preferentially accumulate or deplete At5g08100

• Correlate with tissue-specific stress damage or tolerance

The potential involvement of At5g08100 in nitrogen metabolism makes it particularly relevant for investigating plant responses to changing nutrient availability, which is a growing concern in agricultural systems affected by climate change.

What considerations are important when designing experiments to investigate At5g08100 interactions with nitrogen metabolism pathways?

When investigating At5g08100's role in nitrogen metabolism, researchers should consider:

Nitrogen source variation:

• Design experiments with different nitrogen sources (nitrate, ammonium, organic N)

• Vary nitrogen concentration to identify dose-dependent responses

• Include nitrogen starvation followed by resupply to capture dynamic responses

Integration with carbon metabolism:

• Monitor carbon:nitrogen ratio effects on At5g08100 levels

• Investigate interactions between sugar signaling and nitrogen metabolism

• Include experiments varying both carbon and nitrogen availability

Hormone crosstalk:

• Examine effects of hormones known to regulate nitrogen metabolism (cytokinin, ABA)

• Test how hormone inhibitors affect At5g08100 expression and activity

• Investigate At5g08100 in hormone signaling mutant backgrounds

Experimental design considerations:

• Include appropriate time points (immediate responses: 0.5-4h; intermediate: 12-24h; long-term: 3-7 days)

• Sample multiple tissues (roots, leaves of different ages, reproductive structures)

• Consider diurnal variations in nitrogen metabolism

Complementary approaches:

• Couple protein-level studies with transcript analysis

• Incorporate metabolomics to track nitrogen-containing compounds

• Implement enzyme activity assays alongside protein quantification

By designing comprehensive experiments that account for these factors, researchers can generate more robust and physiologically relevant insights into At5g08100's role in plant nitrogen metabolism.

How can researchers integrate At5g08100 Antibody data with other -omics approaches for systems biology analyses?

Modern systems biology requires integration of multiple data types. At5g08100 Antibody data can be integrated with other -omics approaches as follows:

Multi-omics experimental design:

• Collect samples simultaneously for proteomics, transcriptomics, and metabolomics

• Ensure biological replicates and statistical power across all platforms

• Include appropriate time points to capture dynamic processes

Data integration strategies:

• Correlate At5g08100 protein levels with its transcript levels to identify post-transcriptional regulation

• Map protein abundance changes onto metabolic pathway models

• Identify metabolites that correlate positively or negatively with At5g08100 levels

Network analysis approaches:

• Use At5g08100 as a node in protein-protein interaction networks

• Build gene regulatory networks incorporating transcription factors affecting At5g08100

• Implement Bayesian network modeling to infer causal relationships

Visualization and computational tools:

• Utilize specialized visualization tools for multi-omics data (Cytoscape, PathVisio)

• Implement machine learning approaches to identify patterns across datasets

• Apply dimension reduction techniques to identify key variables associated with At5g08100 function

Validation experiments:

• Design targeted experiments to test hypotheses generated from systems-level analyses

• Use genetic approaches (CRISPR/Cas9 editing, overexpression) to manipulate At5g08100

• Measure effects on identified networks using At5g08100 Antibody as a tool

By integrating antibody-based protein detection with other data types, researchers can position At5g08100 within the broader context of plant cellular networks and gain more comprehensive insights into its biological functions.