LOX1.3 Antibody

What is LOX1.3 Antibody and what does it target?

LOX1.3 Antibody is a polyclonal antibody raised in rabbits that specifically targets Solanum tuberosum (potato) lipoxygenase 1.3 protein (Uniprot: Q43189). This antibody recognizes specific epitopes on the potato LOX1.3 protein and can be used to detect and quantify this protein in various experimental applications . Unlike human LOX-1/OLR1 antibodies that target receptors involved in cardiovascular pathology, potato LOX1.3 antibodies target plant lipoxygenases involved in plant defense mechanisms and lipid metabolism.

What are the validated applications for LOX1.3 Antibody?

LOX1.3 Antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western Blot (WB) applications. When using this antibody for Western Blotting, it's critical to ensure proper identification of the antigen by comparing observed molecular weights with the expected size of potato LOX1.3 . Current validation has not extended to immunohistochemistry or immunofluorescence applications for this specific antibody.

What are the optimal storage conditions for LOX1.3 Antibody?

For maximum stability and activity retention, LOX1.3 Antibody should be stored according to the following guidelines:

• Store upon receipt at -20°C or -80°C

• Avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of antibody activity

• The antibody is typically supplied in a liquid formulation containing preservation buffer

Unlike light-sensitive conjugated antibodies (such as Alexa Fluor 488-conjugated antibodies described for human LOX-1 ), the unconjugated LOX1.3 Antibody doesn't require protection from light during storage.

How should LOX1.3 Antibody be validated for specificity in plant research?

Validating antibody specificity is crucial for accurate experimental results. A comprehensive validation approach should include:

• Positive controls: Using purified recombinant Solanum tuberosum LOX1.3 protein

• Negative controls: Testing reactivity against other potato lipoxygenase isoforms (LOX1.5, LOX1.6, LOX1.8)

• Knockout/silenced samples: If available, tissues from LOX1.3 knockout or silenced potato lines

• Cross-reactivity assessment: Testing against lipoxygenases from related Solanaceae species

This approach parallels validation methodologies used for human antibodies, such as those described for human LOX-1 antibodies where multiple antibodies targeting different epitopes were used to confirm specificity .

What are the recommended dilutions and sample preparation methods for Western blotting with LOX1.3 Antibody?

For optimal Western blot results with LOX1.3 Antibody:

Sample Preparation:

• Extract total protein from potato tissue using a buffer containing protease inhibitors

• Determine protein concentration using Bradford or BCA assay

• Denature samples with SDS loading buffer (containing DTT or β-mercaptoethanol)

• Load 20-50 μg of total protein per lane

Antibody Dilution and Protocol:

• Primary antibody (LOX1.3): Start with 1:1000 dilution in blocking buffer

• Secondary antibody: Anti-rabbit HRP-conjugated at 1:5000 dilution

• Optimize dilutions for each new lot of antibody

This methodological approach is based on standard antibody protocols, similar to those described for other plant and human antibodies in the search results .

How can LOX1.3 Antibody be used to investigate plant stress responses and defense mechanisms?

Plant lipoxygenases play crucial roles in stress responses and defense. For advanced research applications:

• Stress-Induced Expression Analysis:

  • Compare LOX1.3 protein levels in control vs. stressed plants (drought, pathogen, wounding)

  • Quantify by Western blot densitometry or ELISA

  • Correlate protein levels with lipoxygenase enzymatic activity measurements

• Subcellular Localization Studies:

  • Fractionate potato cells into organelle components

  • Use LOX1.3 antibody to track protein localization changes during stress

  • Compare with transcript localization using in situ hybridization

• Interaction Studies:

  • Employ LOX1.3 antibody in co-immunoprecipitation experiments to identify protein interaction partners

  • Use proximity ligation assays to confirm in vivo interactions

This methodological approach draws on principles similar to those used in human immunological studies, where antibodies like anti-LOX-1 have been used to investigate protein expression in different cellular contexts .

How can researchers address potential cross-reactivity between different LOX isoforms when using LOX1.3 Antibody?

Potato contains multiple lipoxygenase isoforms (LOX1.3, LOX1.5, LOX1.6, LOX1.8) with sequence similarities. To address cross-reactivity concerns:

• Epitope Mapping:

  • Identify the specific epitopes recognized by the LOX1.3 antibody

  • Compare these sequences across different LOX isoforms

  • Predict potential cross-reactivity based on sequence homology

• Pre-absorption Controls:

  • Pre-incubate LOX1.3 antibody with recombinant LOX1.3 protein prior to immunodetection

  • Compare signal with non-pre-absorbed antibody

  • Diminished signal confirms specificity

• Multi-antibody Approach:

  • Use multiple antibodies targeting different LOX1.3 epitopes

  • Compare detection patterns

  • Consistent results across antibodies increase confidence in specificity

This approach parallels the methodology used in human LOX-1 research, where researchers used six different anti-LOX-1 antibodies to confirm protein detection and rule out non-specific binding .

What strategies can address weak or inconsistent signals when using LOX1.3 Antibody?

When encountering weak or inconsistent signals with LOX1.3 Antibody, consider this systematic troubleshooting approach:

For Western blots specifically, increasing exposure time or using enhanced chemiluminescent substrates may improve detection of low-abundance LOX1.3 protein .

How can researchers distinguish between closely related LOX isoforms when using antibody detection methods?

Distinguishing between closely related LOX isoforms presents a significant challenge. Implement these advanced approaches:

• 2D Gel Electrophoresis Combined with Immunoblotting:

  • Separate proteins by both isoelectric point and molecular weight

  • Perform Western blots on 2D gels

  • Compare observed patterns with theoretical pI/MW values for each isoform

• Immunoprecipitation Followed by Mass Spectrometry:

  • Use LOX1.3 antibody to immunoprecipitate the protein

  • Analyze precipitated proteins by mass spectrometry

  • Identify peptides unique to LOX1.3 versus other isoforms

• Comparative Analysis with Recombinant Standards:

  • Run recombinant versions of each LOX isoform alongside samples

  • Compare migration patterns and reactivity

  • Identify isoform-specific mobility or reactivity differences

This methodological approach is similar to that employed in studies distinguishing between human LOX-1 variants, where researchers used specific antibodies and molecular weight comparisons to differentiate between the 37 kDa LOX-1 and 24 kDa soluble LOX-1 .

How can LOX1.3 Antibody be employed in plant immunity and pathogen response studies?

Plant lipoxygenases play critical roles in immunity and pathogen responses. Advanced research applications include:

• Temporal Expression Profiling:

  • Track LOX1.3 protein expression at different time points after pathogen infection

  • Correlate with defense metabolite production (jasmonates, green leaf volatiles)

  • Compare with transcriptional changes using qRT-PCR

• Tissue-Specific Localization During Infection:

  • Use immunohistochemistry to localize LOX1.3 in infected versus healthy tissues

  • Determine if LOX1.3 accumulates at infection sites

  • Compare with pathogen distribution using dual labeling

• Genotype-Dependent Response Analysis:

  • Compare LOX1.3 protein levels across resistant and susceptible potato cultivars

  • Correlate with resistance phenotypes

  • Identify potential cultivar-specific post-translational modifications

This research approach draws conceptual parallels to immunological studies where human LOX-1 antibodies have been used to investigate immune responses, though in an entirely different biological context .

What considerations should be made when developing quantitative assays using LOX1.3 Antibody?

Developing reliable quantitative assays requires careful standardization:

• ELISA Development Considerations:

  • Determine optimal antibody coating concentration (for direct ELISA)

  • Establish standard curves using recombinant LOX1.3 protein

  • Validate linearity across physiologically relevant concentration ranges

  • Assess potential matrix effects from plant extracts

• Quantitative Western Blot Parameters:

  • Include gradient loading of standards on each blot

  • Use fluorescent secondary antibodies for broader linear range

  • Employ image analysis software with background correction

  • Report results as relative values to internal controls

• Assay Validation Metrics:

  • Determine limit of detection and quantification

  • Assess intra- and inter-assay variability (CV%)

  • Confirm specificity against other potato lipoxygenases

These methodological considerations parallel those used in the development of quantitative assays for human proteins, where researchers must carefully validate antibody-based detection methods for specificity and sensitivity .