LOX1.8 Antibody

What is LOX1.8 and what cellular processes does it regulate in plants?

LOX1.8 (Probable linoleate 9S-lipoxygenase 8) is a plant enzyme that belongs to the lipoxygenase family and plays crucial roles in plant physiology. This enzyme catalyzes the hydroperoxidation of lipids containing a cis,cis-1,4-pentadiene structure .

Plant lipoxygenases like LOX1.8 are implicated in multiple physiological processes including:

• Growth and development regulation

• Resistance to pests and pathogens

• Plant senescence mechanisms

• Wound response pathways

• Stress signaling cascades

LOX1.8 is primarily located in the cytoplasm of plant cells and is particularly well-studied in Solanum tuberosum (potato). The enzyme is part of the oxidative metabolism pathway that generates signaling molecules involved in plant defense responses.

What are the optimal storage conditions for LOX1.8 antibodies to maintain reactivity?

For optimal preservation of LOX1.8 antibody activity, adherence to specific storage protocols is essential:

• Upon receipt: Store at -20°C or -80°C immediately .

• Working stock: Can be stored at 2-8°C for up to 1 month after reconstitution.

• Long-term storage: Return to -20°C or -80°C for periods exceeding one month.

• Avoid repeated freeze-thaw cycles: This significantly reduces antibody performance and shelf-life.

The LOX1.8 antibody is typically supplied in a storage buffer containing:

• 50% Glycerol

• 0.01M PBS at pH 7.4

• 0.03% Proclin 300 as a preservative

This formulation helps maintain antibody stability and prevents microbial contamination during storage periods. If precipitation occurs during storage, centrifugation at 1000×g for 5 minutes is recommended before use.

What validation methods confirm LOX1.8 antibody specificity?

Confirming antibody specificity is crucial for reliable experimental results. For LOX1.8 antibodies, the following validation approaches are recommended:

Primary Validation Techniques:

• Western Blot (WB): Should detect a band at the expected molecular weight for LOX1.8 (approximately 97-102 kDa depending on the plant species) .

• ELISA: Both direct and indirect ELISA can be performed using recombinant LOX1.8 protein as a positive control .

• Immunohistochemistry: Comparison with known expression patterns in plant tissues.

Advanced Validation Approaches:

• Peptide Competition Assay: Pre-incubation of the antibody with the immunizing peptide should abolish specific signals.

• Knockout/Knockdown Controls: Testing on tissues with LOX1.8 knockout or knockdown to confirm signal specificity.

• Cross-reactivity testing: Evaluate reactivity against related lipoxygenase family members to ensure specificity.

When validating, always include proper negative controls (non-immune rabbit IgG) and positive controls (tissues known to express high levels of LOX1.8).

How can LOX1.8 antibodies be optimized for dual immunofluorescence with other plant defense markers?

Optimizing dual immunofluorescence protocols with LOX1.8 antibodies requires careful consideration of several parameters:

Protocol Optimization Strategy:

• Antibody Sequential Application:

  • Primary approach: Apply LOX1.8 antibody first (1:200-1:500 dilution), followed by other markers

  • Alternative approach: Apply simultaneously if antibodies are from different host species

• Fluorophore Selection to Minimize Spectral Overlap:

  Antibody	Recommended Fluorophore	Excitation (nm)	Emission (nm)
  LOX1.8	Alexa Fluor 488	495	519
  Marker 2	Alexa Fluor 594	590	617
  Marker 3	Alexa Fluor 647	650	668

• Tissue-Specific Fixation Optimization:

  • Leaf tissue: 4% paraformaldehyde, 4-6 hours at 4°C

  • Root tissue: 2% paraformaldehyde with 0.1% glutaraldehyde, 2 hours at 4°C

  • Stem sections: 3% paraformaldehyde, overnight at 4°C

• Blocking Solution Composition:

  • 3-5% BSA

  • 0.1% Triton X-100

  • 1% normal serum from secondary antibody host species

  • PBS (pH 7.4)

Cross-validation using complementary techniques (e.g., in situ hybridization) is strongly recommended to confirm co-localization patterns observed with dual immunofluorescence.

What are the key considerations when using LOX1.8 antibodies for studying plant-pathogen interactions?

When investigating plant-pathogen interactions using LOX1.8 antibodies, researchers should consider several critical factors:

Experimental Design Considerations:

• Timing of Sampling:

  • Early response: 0-6 hours post-infection

  • Intermediate response: 12-24 hours post-infection

  • Late response: 48-72+ hours post-infection

  LOX1.8 expression typically peaks during the intermediate response phase.

• Tissue-Specific Expression Patterns:

  • Infected tissue: Direct sampling at infection site

  • Systemic response: Sampling distal tissues to assess systemic acquired resistance

  • Control tissues: Paired uninfected tissues from the same plant

• Pathogen-Specific Response Variations:

  Pathogen Type	Expected LOX1.8 Response	Optimal Detection Method
  Bacterial	Rapid induction (4-8h)	Western blot, qPCR
  Fungal	Sustained induction	IHC, Western blot
  Viral	Variable/pathogen-specific	Western blot, ELISA

• Methodological Approach:

  • Use 1:250-1:500 antibody dilution for immunohistochemistry on plant sections

  • Apply detergent permeabilization (0.1-0.3% Triton X-100) to facilitate antibody penetration

  • Include DAB staining controls to distinguish endogenous peroxidase activity from specific antibody signals

• Data Interpretation Challenges:

  • LOX1.8 induction may vary between compatible and incompatible plant-pathogen interactions

  • Distinguish between direct pathogen effects and wound-response signaling

  • Consider temporal dynamics of expression throughout infection progression

How does sample preparation affect LOX1.8 antibody detection in different plant tissues?

Sample preparation significantly impacts LOX1.8 antibody detection sensitivity and specificity across various plant tissues:

Tissue-Specific Sample Preparation Guidelines:

• Leaf Tissue Processing:

  • Optimal fixation: 4% paraformaldehyde in PBS (pH 7.4) for 4 hours

  • For Western blot: Flash-freeze in liquid nitrogen and grind to fine powder

  • Protein extraction buffer: 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 5 mM EDTA, with protease inhibitor cocktail

  • Note: Avoid excessive heat during homogenization to prevent protein degradation

• Root Tissue Processing:

  • Wash thoroughly to remove soil contaminants

  • Fix in 2-3% paraformaldehyde with 0.1% glutaraldehyde for improved structure preservation

  • For sectioning: Embed in paraffin or OCT compound after dehydration

  • Antigen retrieval: Heat-mediated (citrate buffer pH 6.0) may enhance antibody binding

• Seed Tissue Processing:

  • Requires extended fixation (8-12 hours) due to dense tissue structure

  • Additional permeabilization steps recommended (0.5% Triton X-100, 30 minutes)

  • Higher antibody concentration may be necessary (1:100-1:200 dilution)

• Comparative Recovery Efficiency:

  Tissue Type	Extraction Method	Protein Recovery (%)	LOX1.8 Detection Sensitivity
  Young leaf	TCA-acetone	85-90%	High
  Mature leaf	Phenol extraction	75-80%	Moderate
  Root	TCA-acetone	60-70%	Moderate
  Seed	Phenol extraction	50-60%	Low-Moderate

• Critical Factors Affecting Detection:

  • Presence of phenolic compounds and oxidative enzymes may interfere with antibody binding

  • Addition of 2% PVPP and 5 mM DTT to extraction buffer improves detection

  • Cold temperature maintenance throughout processing preserves enzyme structure

What controls are essential when using LOX1.8 antibodies for Western blot analysis?

Implementing appropriate controls is critical for reliable Western blot analysis with LOX1.8 antibodies:

Essential Controls Framework:

• Positive Controls:

  • Recombinant LOX1.8 protein (5-10 ng per lane)

  • Tissue extracts with confirmed high LOX1.8 expression (potato leaf tissue after wounding)

  • Previously validated samples

• Negative Controls:

  • Primary antibody omission

  • Non-immune rabbit IgG at equivalent concentration

  • Samples from tissues with low/no LOX1.8 expression

• Loading Controls:

  • Plant-specific housekeeping proteins (actin, tubulin, GAPDH)

  • Total protein staining (Ponceau S, SYPRO Ruby)

  Loading Control	Molecular Weight	Recommended Dilution
  Plant Actin	42 kDa	1:1000-1:2000
  Plant GAPDH	37 kDa	1:1000-1:5000
  Plant Tubulin	55 kDa	1:1000-1:2000

• Antibody Validation Controls:

  • Peptide competition assay (pre-incubation with immunizing peptide)

  • Serial dilution test to determine optimal antibody concentration

  • Cross-reactivity assessment with related lipoxygenase family members

• Protocol Controls:

  • Molecular weight markers for accurate size determination

  • Gradient gels (4-15%) for better resolution of high molecular weight proteins

  • Membrane cutting controls to verify transfer efficiency

Implementing this comprehensive control framework helps distinguish specific LOX1.8 signals from non-specific background and validates experimental outcomes.

How can researchers troubleshoot weak or absent signals when using LOX1.8 antibodies?

When encountering weak or absent signals with LOX1.8 antibodies, a systematic troubleshooting approach is recommended:

Hierarchical Troubleshooting Strategy:

• Sample Preparation Issues:

  • Problem: Insufficient protein extraction

  • Solution: Optimize extraction buffer (add 1% SDS, increase detergent concentration)

  • Problem: Protein degradation

  • Solution: Add fresh protease inhibitors, maintain cold temperatures throughout processing

• Antibody-Related Factors:

  • Problem: Antibody degradation

  • Solution: Prepare fresh dilutions, avoid repeated freeze-thaw cycles

  • Problem: Insufficient antibody concentration

  • Solution: Increase primary antibody concentration (try 1:100, 1:200, 1:500 dilutions)

  • Problem: Non-optimal incubation conditions

  • Solution: Extend primary antibody incubation to overnight at 4°C

• Technical Parameters:

  • Problem: Inefficient protein transfer

  • Solution: Optimize transfer conditions (increase time/voltage, use PVDF membrane)

  • Problem: Insufficient blocking

  • Solution: Try alternative blocking agents (5% milk, 3-5% BSA, commercial blocking buffers)

  • Problem: Detection system sensitivity

  • Solution: Switch to more sensitive detection methods (ECL Plus, fluorescent secondary antibodies)

• LOX1.8-Specific Considerations:

  • Problem: Antigen masking

  • Solution: Apply gentle denaturation methods, avoid excessive heat

  • Problem: Post-translational modifications affecting epitope recognition

  • Solution: Test different antibody clones targeting different epitopes

  • Problem: Low endogenous expression

  • Solution: Induce expression through wounding or pathogen treatment

• Optimization Matrix for Signal Enhancement:

  Parameter	Standard Condition	Optimization 1	Optimization 2
  Blocking	5% milk, 1h RT	3% BSA, 2h RT	Commercial blocker, 1h RT
  Primary Ab	1:500, 1h RT	1:200, overnight 4°C	1:100, 2h RT
  Secondary Ab	1:2000, 1h RT	1:1000, 2h RT	1:5000, 1h RT
  Wash Buffer	TBST (0.1% Tween)	PBST (0.1% Tween)	TBST (0.3% Tween)
  Detection	Standard ECL	ECL Plus	Fluorescent detection

What methodological approaches can accurately quantify LOX1.8 expression levels in plant stress response studies?

For accurate quantification of LOX1.8 expression in plant stress response studies, multiple complementary approaches should be considered:

Comprehensive Quantification Framework:

• Protein-Level Quantification:

  a) Western Blot Densitometry:

  • Use digital imaging systems with linear dynamic range

  • Normalize to housekeeping proteins or total protein staining

  • Perform in technical triplicates with multiple biological replicates

  b) ELISA-Based Quantification:

  • Sandwich ELISA with capture antibody specific to LOX1.8

  • Standard curve using recombinant LOX1.8 (range: 0.1-100 ng/mL)

  • Calculate concentration using 4-parameter logistic regression

  c) Immunohistochemistry with Digital Image Analysis:

  • Measure signal intensity across defined tissue regions

  • Use calibration standards for semi-quantitative assessment

  • Apply machine learning algorithms for automated quantification

• Transcript-Level Quantification:

  a) RT-qPCR Analysis:

  • Design primers spanning exon-exon junctions

  • Normalize to multiple reference genes validated for stability under stress conditions

  • Calculate relative expression using 2^-ΔΔCt method

  b) RNA-Seq Approach:

  • Measure transcripts per million (TPM) values

  • Perform differential expression analysis

  • Validate key findings with RT-qPCR

• Integrated Multi-Omics Approach:

  Method	Measurement	Advantages	Limitations
  Western Blot	Protein levels	Direct protein measurement	Semi-quantitative
  ELISA	Protein concentration	High sensitivity	Requires specific antibodies
  RT-qPCR	mRNA expression	High sensitivity, specificity	Doesn't measure protein
  RNA-Seq	Transcriptome-wide expression	Comprehensive, unbiased	Expensive, complex analysis
  Proteomics	Protein abundance	Direct measurement	Equipment intensive

• Experimental Design Considerations:

  • Include appropriate time-course sampling (0, 2, 6, 12, 24, 48, 72 hours)

  • Compare multiple stress treatments (drought, pathogen, wounding, temperature)

  • Include appropriate stress-related positive controls (e.g., pathogenesis-related proteins)

  • Validate findings across different plant tissues and developmental stages

• Data Integration Strategy:

  • Correlate transcript and protein-level measurements

  • Apply statistical methods appropriate for time-series data

  • Consider biological relevance of fold-changes rather than statistical significance alone

This multi-faceted approach provides robust quantification of LOX1.8 expression dynamics during plant stress responses.

How can researchers employ LOX1.8 antibodies to investigate the plant immune response signaling cascade?

LOX1.8 antibodies offer powerful tools for dissecting plant immune response pathways:

Methodological Applications for Immune Response Research:

• Co-Immunoprecipitation (Co-IP) Studies:

  • Use LOX1.8 antibody for pull-down experiments to identify interaction partners

  • Coupled with mass spectrometry for unbiased interactome analysis

  • Verification of known and novel protein-protein interactions in the lipoxygenase pathway

  • Buffer optimization: 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 5% glycerol

• Subcellular Localization Studies:

  • Immunofluorescence microscopy to track LOX1.8 relocalization during immune response

  • Co-localization with known defense signaling compartments

  • Monitor temporal dynamics of LOX1.8 localization after pathogen recognition

  Recommended Confocal Settings:

  Parameter	Setting	Notes
  Excitation	488 nm laser	For Alexa Fluor 488 secondary antibody
  Emission filter	500-550 nm	Adjust based on specific fluorophore
  Pinhole	1 Airy unit	Balance resolution and signal strength
  Z-stack interval	0.5-1.0 μm	For 3D reconstruction

• Chromatin Immunoprecipitation (ChIP) Applications:

  • Investigate LOX1.8 interactions with promoter regions of defense-related genes

  • Identify transcription factors that regulate LOX1.8 expression

  • Map epigenetic modifications of the LOX1.8 gene during immune responses

• Proximity Ligation Assay (PLA):

  • Detect in situ interactions between LOX1.8 and other immune signaling components

  • Visualize protein complexes at specific subcellular locations

  • Quantify interaction dynamics during different phases of immune response

• Functional Assays with Inhibition Approach:

  • Use LOX1.8 antibodies to block enzyme function in intact tissues

  • Measure downstream impacts on signaling molecules (jasmonates, oxylipins)

  • Quantify effects on defense gene expression using reporter constructs

This integrated approach allows researchers to establish causal relationships between LOX1.8 activity and specific immune response outcomes.

What are the critical differences between LOX1.8 antibody applications in monocot versus dicot plant systems?

Understanding the critical differences in LOX1.8 antibody applications between monocot and dicot systems is essential for experimental design:

Comparative Analysis Framework:

• Epitope Conservation and Antibody Cross-Reactivity:

  Plant System	Epitope Conservation	Recommended Antibody Dilution	Expected MW
  Dicots (e.g., potato, tomato)	High (>90%)	1:500 for WB, 1:200 for IHC	95-102 kDa
  Monocots (e.g., rice, wheat)	Moderate (70-85%)	1:250 for WB, 1:100 for IHC	92-98 kDa

  Note: Sequence alignment analyses show greater epitope conservation among closely related dicot species compared to monocots .

• Tissue-Specific Expression Patterns:

  Dicots:

  • Highest expression in leaves and reproductive tissues

  • Rapid induction in vascular tissues upon wounding

  • Strong expression in epidermal cells during pathogen attack

  Monocots:

  • Prominent expression in mesophyll cells

  • Constitutive expression in developing seeds

  • More uniform distribution across tissue types

• Sample Preparation Modifications:

  Dicots:

  • Standard fixation protocols generally effective

  • Protein extraction with RIPA or Tris-based buffers sufficient

  Monocots:

  • Higher silica content requires modified fixation (add 0.1% Tween-20 to fixative)

  • Protein extraction may require stronger buffers with higher detergent concentrations

  • Extended incubation times for antibody penetration (12-18 hours at 4°C)

• Immunohistochemistry Optimization:

  Dicots:

  • Antigen retrieval: citrate buffer (pH 6.0), 95°C for 10 minutes

  • Background control: 5% normal goat serum sufficient

  Monocots:

  • Antigen retrieval: EDTA buffer (pH 8.0), 95°C for 20 minutes

  • Background control: additional blocking with 0.1% glycine and 2% BSA recommended

  • Increased antibody concentration may be necessary (1.5-2× higher than for dicots)

• Developmental Timing Considerations:

  LOX1.8 expression patterns differ significantly between monocots and dicots throughout development, necessitating sampling adjustments based on growth stage rather than chronological age.

How can mass spectrometry complement LOX1.8 antibody-based detection in lipoxygenase pathway research?

Integrating mass spectrometry (MS) with LOX1.8 antibody-based approaches creates a powerful analytical framework:

Complementary MS-Antibody Research Strategy:

• Validation of Antibody Specificity:

  • Immunoprecipitation coupled with MS (IP-MS) to confirm antibody target specificity

  • Identification of LOX1.8 post-translational modifications that may affect antibody recognition

  • Characterization of potential cross-reactive proteins in different plant species

  Typical IP-MS Workflow:

  1. Immunoprecipitate with LOX1.8 antibody from plant extract

  2. Perform on-bead or in-gel digestion with trypsin

  3. Analyze peptides using LC-MS/MS

  4. Compare detected peptides against plant proteome databases

• Quantitative Proteomics Approach:

  Approach	Application	Key Advantages
  SILAC	Differential expression analysis	Accurate relative quantification
  Label-free quantification	Temporal dynamics of LOX1.8 expression	Simplified workflow
  Selected reaction monitoring (SRM)	Absolute quantification of LOX1.8	High sensitivity for targeted analysis
  Data-independent acquisition (DIA)	Comprehensive pathway analysis	Unbiased detection of pathway components

• Substrate and Product Analysis:

  • LC-MS/MS detection of LOX1.8-specific lipid hydroperoxide products

  • Correlation of enzyme activity with protein levels detected by antibodies

  • Characterization of substrate specificity across plant species

  Critical MS Parameters:

  • Ionization mode: Negative ESI for fatty acids, positive for oxylipins

  • Mass resolution: >60,000 FWHM for complex lipid mixtures

  • MS/MS fragmentation: CID or HCD for structural characterization

• Integrated Workflow for Comprehensive Analysis:

  • Use antibody-based detection for protein localization and relative quantification

  • Apply targeted proteomics for absolute quantification of LOX1.8 and pathway components

  • Perform untargeted lipidomics to characterize downstream metabolites

  • Correlate protein abundance with enzymatic products to assess functional activity

• Advanced Applications:

  • Crosslinking MS to map LOX1.8 interaction networks

  • Top-down proteomics to characterize full-length LOX1.8 proteoforms

  • Imaging MS to visualize spatial distribution of LOX1.8 and its products in tissue sections

This integrated approach overcomes the limitations of antibody-only or MS-only methods, providing comprehensive insights into LOX1.8 function in the lipoxygenase pathway.

What emerging technologies are enhancing LOX1.8 antibody applications in plant-microbe interaction studies?

Cutting-edge technologies are revolutionizing how LOX1.8 antibodies can be utilized in plant-microbe interaction research:

Emerging Technological Platforms:

• Single-Cell Immunodetection Systems:

  • Microfluidic devices for single-cell antibody staining

  • Integration with transcriptomics for multi-omics analysis

  • Spatial mapping of LOX1.8 expression heterogeneity within tissues

  • Applications: Identifying specialized cells with elevated LOX1.8 expression during pathogen response

• Advanced Microscopy Techniques:

  Technique	Resolution	Application for LOX1.8 Research
  STORM/PALM	20-30 nm	Nanoscale organization of LOX1.8 in membranes
  Expansion microscopy	70 nm	3D visualization of LOX1.8 distribution
  Lattice light-sheet	300 nm	Live-cell dynamics of LOX1.8 trafficking
  FRET microscopy	10 nm	Direct protein-protein interactions

• Engineered Antibody Formats:

  • Single-domain antibodies (nanobodies) against LOX1.8 for improved tissue penetration

  • Bispecific antibodies to simultaneously detect LOX1.8 and interacting proteins

  • Site-specific conjugation of fluorescent proteins for live-cell imaging

  • Applications: Real-time monitoring of LOX1.8 dynamics during infection processes

• CRISPR-Based Technologies:

  • Epitope tagging of endogenous LOX1.8 for antibody-based detection

  • CUT&Tag for genome-wide profiling of transcription factors regulating LOX1.8

  • Base editing to introduce specific mutations affecting antibody epitopes

  • Applications: Creating reporter lines for live monitoring of LOX1.8 expression

• Artificial Intelligence Integration:

  • Deep learning algorithms for automated quantification of immunostaining patterns

  • Predictive modeling of LOX1.8 expression based on pathogen-associated molecular patterns

  • Computer vision for high-throughput phenotyping of LOX1.8-mediated responses

  • Applications: Large-scale screening of germplasm for LOX1.8-dependent resistance traits

These emerging technologies offer unprecedented resolution and throughput for studying LOX1.8 dynamics in plant-microbe interactions.

How do researchers reconcile contradictory findings when using different LOX1.8 antibodies in parallel studies?

Reconciling contradictory results from different LOX1.8 antibodies requires a systematic analytical approach:

Methodological Framework for Resolving Discrepancies:

This systematic approach transforms contradictory findings into opportunities for deeper understanding of LOX1.8 biology and improved experimental design.

What considerations are important when adapting LOX1.8 antibody protocols from model to non-model plant species?

Adapting LOX1.8 antibody protocols to non-model plant species requires careful consideration of multiple factors:

Systematic Adaptation Framework:

• Sequence Homology Analysis:

  • Perform multiple sequence alignment of LOX1.8 across target species

  • Focus on epitope regions recognized by available antibodies

  • Calculate percent identity and similarity in epitope regions

  • Predict cross-reactivity based on conservation of critical residues

  Estimated Cross-Reactivity Based on Epitope Conservation:

  Epitope Conservation	Expected Cross-Reactivity	Recommended Dilution Adjustment
  >90% identity	Strong	Standard (1:500)
  75-90% identity	Moderate	Increase concentration (1:250)
  50-75% identity	Weak	Significant increase (1:100)
  <50% identity	Unlikely	Consider custom antibody development

• Protocol Optimization Strategy:

  a) Sample Preparation Modifications:

  • Adjust buffer compositions based on tissue-specific biochemistry

  • Optimize protein extraction methods for recalcitrant tissues

  • Modify fixation protocols for species with different cell wall compositions

  b) Detection System Enhancements:

  • Increase antibody concentration incrementally (2-5× higher for distant species)

  • Extend incubation times (overnight at 4°C)

  • Test alternative secondary antibodies with higher sensitivity

  • Consider signal amplification systems (tyramide signal amplification, polymer-based detection)

• Validation in Non-Model Systems:

  • Perform Western blot to confirm correct molecular weight detection

  • Include recombinant LOX1.8 protein as positive control

  • Use tissue-specific expression patterns from model species as reference

  • Apply RNA in situ hybridization as complementary approach

• Common Challenges and Solutions:

  Challenge	Solution Approach
  High autofluorescence	Pre-treatment with sodium borohydride or glycine
  Protein extraction difficulties	Test multiple extraction buffers with increasing stringency
  Non-specific binding	Optimize blocking with species-specific serum
  Low signal intensity	Apply tyramide signal amplification
  High background	Perform additional washing steps with increased detergent

• Evolutionary Considerations:

  • Consider the phylogenetic distance between model and target species

  • Account for potential gene duplication events and paralog detection

  • Recognize that protein function may be conserved despite sequence divergence

  • Validate findings with functional assays specific to the target species