At5g10920 Antibody

What is AT5g10920 and what role does it play in plant metabolism?

AT5g10920 encodes argininosuccinate lyase (AtArgH), an enzyme involved in the arginine biosynthesis pathway in Arabidopsis thaliana. This protein has been identified as a redox-sensitive protein that responds to oxidative stress conditions, particularly hydrogen peroxide (H₂O₂) treatment . In proteomic studies, AT5g10920 has been detected with approximately 8.5% coverage using mass spectrometry techniques .

The enzyme catalyzes the conversion of argininosuccinate to arginine and fumarate in the final step of arginine biosynthesis. This metabolic function places AT5g10920 at a critical junction between nitrogen metabolism and various downstream pathways that utilize arginine, including polyamine synthesis, nitric oxide production, and protein synthesis.

What experimental techniques commonly employ AT5g10920 antibodies?

AT5g10920 antibodies are typically utilized in several standard molecular biology techniques:

• Western blotting (WB) - For detecting AT5g10920 protein expression levels in plant tissue extracts

• Immunoprecipitation (IP) - For pulling down AT5g10920 and its interacting partners

• Immunocytochemistry/Immunofluorescence (ICC/IF) - For visualizing subcellular localization

• Immunohistochemistry (IHC) - For detecting tissue-specific expression patterns

When selecting an antibody for these applications, researchers should verify that the antibody has been validated for the specific technique of interest, ideally with supporting data showing specificity in Arabidopsis samples .

How can I confirm the specificity of an AT5g10920 antibody?

Confirming antibody specificity is essential for reliable research results. For AT5g10920 antibodies, consider these validation approaches:

• Use of knockout/knockdown lines: Compare antibody reactivity between wild-type and AT5g10920 mutant Arabidopsis tissues. A specific antibody should show reduced or absent signal in the mutant .

• Peptide competition assay: Pre-incubate the antibody with excess synthetic peptide corresponding to the immunogen. This should block specific binding sites and eliminate true positive signals.

• Recombinant protein controls: Include purified recombinant AT5g10920 protein as a positive control in Western blots to confirm the correct molecular weight detection.

• Multiple antibody comparison: When possible, use antibodies raised against different epitopes of AT5g10920 and compare detection patterns.

• Mass spectrometry validation: Immunoprecipitate the protein and confirm its identity via mass spectrometry, comparing detected peptides with the expected AT5g10920 sequence.

How does oxidative stress affect AT5g10920 expression and post-translational modifications?

AT5g10920 (argininosuccinate lyase) has been identified as a redox-sensitive protein in Arabidopsis cells responding to hydrogen peroxide treatment . When designing experiments to study these responses, researchers should consider:

• Temporal dynamics: AT5g10920 is among the early-responsive proteins to oxidative stress. Time-course experiments (0-24 hours) with H₂O₂ treatment can reveal the kinetics of expression changes and modifications.

• Post-translational modifications: Redox-sensitive proteins often undergo reversible oxidative modifications on cysteine residues. These can be detected using:

  • Redox proteomics approaches with differential alkylation

  • Biotin-switch techniques to identify S-nitrosylation

  • Phosphoproteomic analysis to identify altered phosphorylation states

• Subcellular redistribution: Oxidative stress may trigger changes in protein localization. Fractionation experiments followed by immunoblotting can track movement between cellular compartments.

The table below summarizes key methodological approaches for studying AT5g10920 redox sensitivity:

What considerations should guide experimental design when studying redox-sensitive properties of AT5g10920?

When investigating AT5g10920's redox-sensitive properties, experimental design should account for several factors:

• Sample preparation conditions: Redox-sensitive proteins are vulnerable to artifactual oxidation during extraction. Use anaerobic buffers containing reducing agents (such as DTT or TCEP) and alkylating agents (iodoacetamide or N-ethylmaleimide) to preserve native redox states.

• Physiologically relevant stress conditions: For H₂O₂ treatments, use concentrations that mirror biological stress responses (typically 0.1-5 mM for exogenous application) rather than extreme concentrations that may cause non-specific effects .

• Kinetic analysis: Redox modifications are often transient. Design time-course experiments (minutes to hours) to capture the dynamics of these changes.

• Integration with metabolic pathways: As an arginine biosynthesis enzyme, AT5g10920's redox regulation may affect nitrogen metabolism. Consider measuring related metabolites (arginine, ornithine, citrulline) alongside protein analysis.

• Comparative analysis with other redox-sensitive proteins: Include known redox-responsive proteins as positive controls, such as those identified in the same proteomic studies as AT5g10920, including 2-Cys peroxiredoxin (2-Cys PrxA) and ascorbate peroxidase (APX1) .

How can the interaction between AT5g10920 and other proteins be effectively studied?

To investigate protein-protein interactions involving AT5g10920:

• Co-immunoprecipitation (Co-IP): Use AT5g10920 antibodies to pull down the protein complex, followed by mass spectrometry to identify interacting partners. For increased specificity, consider:

  • Crosslinking prior to lysis (using DSP or formaldehyde)

  • Sequential IPs with antibodies against suspected partners

  • Comparison between normal and stress conditions to identify condition-specific interactions

• Proximity labeling approaches: Express AT5g10920 fused to BioID or APEX2 in Arabidopsis to biotinylate proximal proteins, enabling their subsequent purification and identification.

• Split-reporter systems: For testing specific hypothesized interactions, use split-GFP, split-luciferase, or yeast two-hybrid assays with AT5g10920 and candidate interactors.

• In vitro binding assays: Express recombinant AT5g10920 and candidate partners to characterize direct interactions and binding parameters using techniques like surface plasmon resonance or isothermal titration calorimetry.

For all interaction studies, include appropriate controls:

• IgG or pre-immune serum for Co-IP experiments

• Expression level controls for fusion proteins

• Free tag/enzyme controls for proximity labeling

• Negative and positive interaction controls for split-reporter systems

What are the optimal protocols for AT5g10920 antibody use in Western blotting?

For optimal Western blotting results with AT5g10920 antibodies:

• Sample preparation:

  • Extract proteins from Arabidopsis tissues using a buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail, and 5 mM DTT

  • For redox studies, include 100 mM iodoacetamide to alkylate free thiols and prevent artificial oxidation

  • Use fresh tissue when possible, or snap-freeze and store at -80°C

• Protein separation:

  • Load 20-40 μg of total protein per lane

  • Use 10-12% SDS-PAGE gels (AT5g10920 is approximately 52 kDa)

  • Include recombinant AT5g10920, if available, as a positive control

• Antibody incubation:

  • Block membrane in 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary AT5g10920 antibody at 1:1000-1:5000 dilution (optimize for each antibody)

  • Incubate overnight at 4°C with gentle rocking

  • Wash 4× with TBST, 5 minutes each

  • Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

• Detection optimization:

  • For low abundance detection, use enhanced chemiluminescence (ECL) substrates with extended exposure times

  • Consider fluorescent secondary antibodies for more quantitative results

  • Document using both short and long exposures to capture the dynamic range

How should AT5g10920 antibodies be validated for cross-reactivity in non-Arabidopsis species?

When validating AT5g10920 antibodies for use in other plant species:

• Sequence homology analysis:

  • Perform sequence alignment of AT5g10920 with homologs from target species

  • Calculate percent identity within the antibody epitope region

  • Predict cross-reactivity based on conservation (typically >70% identity suggests possible cross-reactivity)

• Empirical validation:

  • Run parallel Western blots with Arabidopsis (positive control) and target species samples

  • Compare band patterns and molecular weights

  • Confirm specificity using blocking peptides specific to each species' sequence

• Knockout/knockdown controls:

  • If available, use CRISPR or RNAi lines of the target species with reduced expression

  • Compare signal between wild-type and modified lines

• Recombinant protein validation:

  • Express the homologous protein from the target species

  • Test antibody reactivity against both Arabidopsis and target species recombinant proteins

  • Calculate relative affinity based on signal intensity

What sample preparation techniques maximize AT5g10920 detection in plant tissues?

To optimize AT5g10920 detection in different experimental contexts:

• For Western blotting:

  • Use young, actively growing tissues (AT5g10920 may be most abundant in metabolically active tissues)

  • Grind tissue in liquid nitrogen to prevent protein degradation

  • Include phosphatase inhibitors if studying phosphorylation states

  • Add protease inhibitor cocktail to prevent degradation

• For immunohistochemistry:

  • Fix tissues in 4% paraformaldehyde for 2-4 hours

  • Consider antigen retrieval methods (citrate buffer, pH 6.0, 95°C for 10 minutes)

  • Increase antibody penetration by vacuum infiltration of solutions

  • Use 0.1% Triton X-100 in washing steps to improve permeabilization

• For immunoprecipitation:

  • Increase protein extraction efficiency using optimized buffers (e.g., RIPA for stronger extraction)

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Consider a tandem affinity purification approach for highly specific isolation

• For mass spectrometry:

  • Enrich for AT5g10920 using immunoprecipitation before digestion

  • Consider fractionation techniques to reduce sample complexity

  • Use targeted proteomics (PRM or MRM) for detecting specific AT5g10920 peptides

Why might Western blots with AT5g10920 antibodies show inconsistent results?

Several factors can contribute to inconsistent Western blot results with AT5g10920 antibodies:

• Sample degradation:

  • AT5g10920 may be susceptible to proteolysis during extraction

  • Solution: Use fresh protease inhibitor cocktail and keep samples consistently cold

  • Avoid repeated freeze-thaw cycles of protein extracts

• Antibody quality variations:

  • Different lots of the same antibody may have varying affinities

  • Solution: Validate each new lot against a reference sample

  • Consider creating a large batch of positive control sample to normalize between experiments

• Redox state variability:

  • As a redox-sensitive protein, AT5g10920 may exist in multiple oxidation states

  • Solution: Standardize reducing conditions in sample buffer

  • Add excess DTT (100 mM) to fully reduce all protein forms for consistent migration

• Expression level fluctuations:

  • AT5g10920 expression may vary with growth conditions and developmental stage

  • Solution: Carefully control plant growth parameters

  • Document and standardize tissue harvesting procedures (time of day, plant age, etc.)

• Technical variables:

  • Transfer efficiency can vary between experiments

  • Solution: Use stain-free gels or Ponceau staining to verify transfer

  • Include a loading control from a different molecular weight range than AT5g10920

The following troubleshooting table addresses common Western blot issues:

How can background signal be reduced when using AT5g10920 antibodies in immunofluorescence?

High background in immunofluorescence experiments can obscure specific AT5g10920 signals. Try these optimization strategies:

• Blocking optimization:

  • Extend blocking time to 2-3 hours at room temperature

  • Test different blocking agents (BSA, normal serum, casein, commercial blocking buffers)

  • Use the blocking agent that matches the host of your secondary antibody (e.g., goat serum for goat-derived secondaries)

• Antibody dilution optimization:

  • Test a dilution series (typically starting at 1:100 and extending to 1:1000)

  • Incubate primary antibody for longer periods (overnight at 4°C) at higher dilutions

  • Wash more extensively after antibody incubations (5-6 times, 10 minutes each)

• Tissue preparation improvements:

  • Ensure complete fixation without over-fixation (typically 2-4 hours in 4% paraformaldehyde)

  • Add a permeabilization step with 0.2-0.5% Triton X-100 for 30 minutes

  • Perform antigen retrieval if needed (especially for fixed tissues)

• Controls and validation:

  • Include a secondary-only control to assess non-specific binding

  • Pre-adsorb secondary antibodies with plant tissue powder

  • Consider direct conjugation of primary antibodies to fluorophores to eliminate secondary antibody background

• Imaging optimization:

  • Adjust detector gain to minimize background while preserving specific signal

  • Use spectral unmixing to separate autofluorescence from specific signal

  • Apply consistent background subtraction during image processing

What approaches can improve antibody specificity for studying AT5g10920 in complex plant extracts?

To enhance AT5g10920 antibody specificity in complex samples:

• Epitope-specific purification:

  • Perform affinity purification of antibodies using the immunizing peptide

  • Elute specifically bound antibodies for use in critical experiments

  • This isolates the fraction of antibodies that recognize the intended epitope

• Pre-adsorption against plant extracts:

  • Incubate antibodies with extracts from AT5g10920 knockout/knockdown plants

  • This depletes antibodies that bind to non-target proteins

  • Use the pre-adsorbed antibody preparation for experiments

• Two-dimensional Western blotting:

  • Separate proteins by both isoelectric point and molecular weight

  • This provides higher resolution separation of AT5g10920 from similar proteins

  • Identify the specific spot corresponding to AT5g10920 for more confident analysis

• Targeted sample fractionation:

  • Enrich for the subcellular compartment where AT5g10920 is located

  • Reduce sample complexity before applying antibodies

  • This improves signal-to-noise ratio by reducing non-specific binding opportunities

• Multiplexed detection strategies:

  • Use multiple antibodies targeting different regions of AT5g10920

  • True signals should show co-localization of different antibodies

  • This approach can distinguish true signal from cross-reactivity

How can engineered antibodies improve AT5g10920 detection in specialized applications?

Advances in antibody engineering can significantly enhance AT5g10920 detection capabilities:

• Recombinant antibody development:

  • Single-chain variable fragments (scFvs) offer improved tissue penetration

  • Nanobodies derived from camelid antibodies provide access to epitopes in confined spaces

  • These smaller antibody formats can be expressed in plants, eliminating species cross-reactivity issues

• Affinity maturation:

  • In vitro evolution techniques can improve antibody affinity for AT5g10920

  • Higher affinity allows for more dilute antibody use, reducing background

  • Directed evolution approaches like ribosome or phage display can yield antibodies with sub-nanomolar affinities

• Bi-specific antibody applications:

  • Engineered bi-specific antibodies can simultaneously recognize AT5g10920 and another protein of interest

  • This enables direct visualization of protein-protein interactions

  • Such antibodies require careful design to ensure optimal binding to both targets

• Site-specific conjugation:

  • Engineered antibodies with defined conjugation sites avoid heterogeneous labeling

  • This provides consistent fluorophore or enzyme positioning relative to the binding site

  • Results in more reproducible signal generation across experiments

• Developability considerations:

  • Modern antibody engineering considers expression yield, stability, and solubility

  • These properties are critical for consistent research applications

  • Early screening for these qualities ensures reliable antibody performance over time

What mass spectrometry approaches are most effective for characterizing AT5g10920 post-translational modifications?

Mass spectrometry offers powerful tools for characterizing AT5g10920 modifications:

• Enrichment strategies:

  • Immunoprecipitate AT5g10920 using validated antibodies before MS analysis

  • For phosphorylation studies, use TiO2 or IMAC enrichment methods

  • For redox modifications, use strategies like biotin-switch technique followed by avidin purification

• Bottom-up proteomics workflow:

  • Digest immunoprecipitated AT5g10920 with trypsin

  • Target analysis to known modification sites based on predictive algorithms

  • Compare spectra across different stress conditions to identify treatment-specific modifications

• Parallel reaction monitoring (PRM):

  • Develop targeted assays for specific AT5g10920 peptides and their modified forms

  • This provides higher sensitivity for low-abundance modified peptides

  • Enables absolute quantification of modification stoichiometry

• Intact protein MS:

  • Analyze whole AT5g10920 protein to observe combinations of modifications

  • This preserves information about co-occurring modifications on the same protein molecule

  • Can reveal the presence of unexpected modifications

• Cross-linking MS:

  • Use chemical cross-linkers to capture transient AT5g10920 protein interactions

  • This approach can identify binding partners that may regulate AT5g10920 activity

  • Provides structural information about protein complexes involving AT5g10920

Based on studies of redox-sensitive proteins in Arabidopsis, the following table outlines potential AT5g10920 modifications that could be investigated: