ALOX5 Antibody

What is ALOX5 and what is its biological significance?

ALOX5 (Arachidonate 5-Lipoxygenase) is a 78 kDa iron-containing enzyme that catalyzes the first step in leukotriene biosynthesis. It plays a critical role in inflammatory processes by converting arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and further to leukotriene A4, an unstable intermediate. ALOX5 is essential for cysteinyl-leukotriene production, which are critical mediators in inflammatory conditions such as asthma, arthritis, and psoriasis. The enzyme requires Arachidonate 5-lipoxygenase-activating protein (FLAP) for activity and translocates from the cytosol to associate with FLAP at the plasma membrane during activation .

What types of ALOX5 antibodies are available for research?

ALOX5 antibodies are available in several formats:

Different antibodies target various regions of the ALOX5 protein, with immunogens ranging from synthetic peptides to recombinant protein fragments .

Which cell and tissue types normally express ALOX5?

ALOX5 expression has been documented in:

• Bone marrow-derived cells

• B lymphocytes in the mantle zone of lymphoid tissue

• Human B-cell lymphomas (preferentially in mantle cell lymphoma, chronic lymphocytic leukemia, and follicular lymphoma)

• Various cell lines including K562, RT4, DU 145, HL-60, and PC-3

• Human placenta tissue

Interestingly, ALOX5 expression patterns in lymphomas could serve as a potential marker for identifying the cell of origin in B-cell lymphomas .

What are the optimal protocols for Western blot detection of ALOX5?

For optimal Western blot detection of ALOX5:

• Sample preparation:

  • Use fresh tissue lysates or cell extracts

  • Include protease inhibitors to prevent degradation

• Gel electrophoresis:

  • Use 5-20% SDS-PAGE gel

  • Run at 70V (stacking gel)/90V (resolving gel) for 2-3 hours

  • Load 30 μg of protein per lane under reducing conditions

• Transfer and blocking:

  • Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

  • Block with 5% non-fat milk/TBS for 1.5 hours at room temperature

• Antibody incubation:

  • Primary antibody dilution: 1:500-1:2000 (varies by product)

  • Incubate overnight at 4°C

  • Secondary antibody: anti-rabbit/mouse IgG-HRP at 1:500

  • Incubate for 1.5 hours at room temperature

• Detection:

  • Develop using enhanced chemiluminescence (ECL)

  • Expected band size: approximately 78 kDa

Positive controls include human K562 and RT4 whole cell lysates .

How should I optimize immunohistochemistry protocols for ALOX5 detection?

For successful immunohistochemistry detection of ALOX5:

• Tissue preparation:

  • Use formalin-fixed paraffin-embedded (FFPE) specimens

  • Section thickness: 4-6 μm

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Pressure cooker treatment for 2-3 minutes

• Blocking and permeabilization:

  • Block endogenous peroxidase with 3% H₂O₂

  • Permeabilize with 0.1% Triton X-100 if necessary

  • Block with 5% normal serum from the same species as the secondary antibody

• Antibody incubation:

  • Primary antibody dilution: 1:50-1:200

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Secondary antibody incubation: 30-60 minutes at room temperature

• Development and counterstaining:

  • Develop with DAB or other suitable chromogen

  • Counterstain with hematoxylin

  • Mount with permanent mounting medium

ALOX5 immunohistochemistry has proven particularly valuable in characterizing B-cell lymphomas, with distinct expression patterns observed across different lymphoma subtypes .

What are the recommended conditions for immunofluorescence using ALOX5 antibodies?

For immunofluorescence detection of ALOX5:

• Cell preparation:

  • Culture cells on glass coverslips or chamber slides

  • Fix with 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilization and blocking:

  • Permeabilize with 0.1-0.5% Triton X-100 for 10 minutes

  • Block with 5% normal serum or 1% BSA for 30-60 minutes

• Antibody incubation:

  • Primary antibody dilution: 1:10-1:100 (varies by product)

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Secondary antibody (fluorophore-conjugated): 1:200-1:1000

  • Incubate for 1 hour at room temperature protected from light

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI (1:1000) for 5 minutes

  • Mount with anti-fade mounting medium

HeLa cells have been validated as positive controls for ALOX5 immunofluorescence studies .

How can I validate the specificity of my ALOX5 antibody?

Validation strategies for ALOX5 antibodies include:

• Positive and negative controls:

  • Use tissues/cells known to express ALOX5 (e.g., K562 cells, mantle zone B cells)

  • Include tissues/cells lacking ALOX5 expression (e.g., certain DLBCL lines)

• Knockdown/knockout validation:

  • Test antibody reactivity in ALOX5 knockdown or knockout samples

  • Compare with wild-type samples to confirm specificity

• Blocking peptide competition:

  • Pre-incubate antibody with the immunizing peptide

  • Observe elimination of specific signal

• Cross-platform validation:

  • Confirm expression using orthogonal methods (e.g., qPCR, RNAseq)

  • Compare antibody reactivity across different applications (WB, IHC, IF)

• Molecular weight verification:

  • Confirm band appears at expected molecular weight (78 kDa)

Several commercial antibodies have undergone rigorous validation through orthogonal RNAseq, Western blotting with multiple cell lines, and testing across various applications .

What are common pitfalls when using ALOX5 antibodies and how can I address them?

For persistent issues, consider testing alternative ALOX5 antibody clones that target different epitopes of the protein .

What alternatives exist if traditional antibody-based detection methods fail?

When antibody-based detection of ALOX5 proves challenging:

• Activity-based assays:

  • Measure ALOX5 enzymatic activity by quantifying leukotriene production

  • Use HPLC or mass spectrometry to detect 5-HETE and LTA4 metabolites

• Genetic approaches:

  • Use in situ hybridization to detect ALOX5 mRNA

  • Employ GFP-tagged ALOX5 in recombinant systems

• Chemical biology methods:

  • Use activity-based protein profiling with ALOX5-specific probes

  • Apply proximity ligation assays to detect protein interactions

• Proteomics:

  • Use targeted mass spectrometry for ALOX5 protein detection

  • Apply CRISPR-based tagging for endogenous visualization

• Single-cell approaches:

  • Implement single-cell RNA-seq to measure ALOX5 transcript levels

  • Use digital spatial profiling for tissue-based expression analysis

These alternatives can complement antibody-based methods and provide orthogonal validation.

How can ALOX5 antibodies be used to study B-cell lymphoma subtypes?

ALOX5 antibodies have revealed distinctive expression patterns across B-cell lymphoma subtypes:

These findings suggest ALOX5 may serve as a novel marker for identifying the cell of origin in B-cell lymphomas. Immunohistochemistry with ALOX5 antibodies provides valuable information for lymphoma classification and potentially for guiding therapeutic approaches .

How does ALOX5 phosphorylation affect its function and how can this be detected?

ALOX5 activity is regulated by phosphorylation, particularly at Ser523:

• Functional impact:

  • Phosphorylation at Ser523 inhibits ALOX5 activity

  • Controls nuclear export and subcellular localization

  • Regulates interaction with FLAP and other binding partners

• Detection methods:

  • Phospho-specific antibodies (e.g., anti-ALOX5 pSer523)

  • Phosphatase treatment controls

  • Phos-tag SDS-PAGE for mobility shift assays

  • Mass spectrometry for comprehensive phosphosite mapping

• Experimental design:

  • Compare phosphorylation status under different inflammatory stimuli

  • Use kinase inhibitors to manipulate phosphorylation state

  • Correlate phosphorylation with subcellular localization and activity

Phospho-specific antibodies enable researchers to distinguish between active and inactive forms of ALOX5, providing insight into regulatory mechanisms in inflammatory processes .

What considerations are important when using ALOX5 antibodies in flow cytometry?

For successful flow cytometry with ALOX5 antibodies:

• Cell preparation:

  • Gentle fixation (2-4% paraformaldehyde)

  • Thorough permeabilization for intracellular target (0.1% saponin or 0.3% Triton X-100)

  • Maintain single-cell suspension throughout protocol

• Antibody selection and titration:

  • Use antibodies validated for flow cytometry (e.g., clone AECE-1)

  • Titrate antibody to determine optimal concentration

  • Typical starting dilution: 1:50

  • Perform time-course experiments to determine optimal incubation time

• Controls and gating:

  • Include isotype controls to establish background

  • Use known positive and negative cell populations

  • Consider fluorescence-minus-one (FMO) controls for multi-color panels

  • Establish gates based on control populations

• Multi-parametric analysis:

  • Combine with surface markers to identify specific cell subsets

  • Include viability dye to exclude dead cells

  • Consider dual staining with other inflammatory markers

Flow cytometry allows quantitative assessment of ALOX5 expression across different cell populations and under various treatment conditions .

How might ALOX5 antibodies contribute to cancer research beyond lymphoma?

ALOX5 antibodies are increasingly important in broader cancer research:

• Potential applications:

  • Identifying ALOX5 expression in various cancer types

  • Correlating expression with clinical outcomes

  • Monitoring treatment response to ALOX5 inhibitors

  • Developing companion diagnostics

• Recent findings:

  • ALOX5 expression has been detected in prostate cancer cell lines (DU 145, PC-3)

  • Expression patterns may correlate with cancer progression or inflammatory tumor microenvironment

  • Could serve as a biomarker for stratifying patients for targeted therapies

• Methodological approaches:

  • Tissue microarray analysis of large patient cohorts

  • Correlation with inflammatory markers

  • Sequential biopsies during treatment

  • Single-cell analyses of tumor heterogeneity

As mutations in the ALOX5 promoter region have been associated with atherosclerosis and several cancers, immunohistochemical analysis of ALOX5 expression may provide valuable prognostic and predictive information .

What novel techniques are being developed for studying ALOX5 localization and dynamics?

Emerging techniques for ALOX5 localization and dynamics include:

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize subcellular localization

  • Live-cell imaging with fluorescently tagged ALOX5

  • FRET/FLIM to study protein-protein interactions with FLAP

  • Correlative light and electron microscopy for ultrastructural context

• Spatiotemporal analysis:

  • Optogenetic control of ALOX5 activation

  • Photoactivatable or photoswitchable ALOX5 constructs

  • Real-time visualization of translocation during activation

• In situ approaches:

  • Proximity ligation assays to detect ALOX5-FLAP interactions

  • Spatial transcriptomics combined with protein detection

  • Mass cytometry for high-dimensional analysis at single-cell level

• Computational modeling:

  • Simulation of ALOX5 trafficking and activation dynamics

  • Integration of imaging and -omics data

  • Prediction of inhibitor effects on localization and activity

These emerging techniques promise to reveal new insights into ALOX5 regulation and function in inflammatory processes and disease states.

How can ALOX5 antibodies contribute to drug development and personalized medicine?

ALOX5 antibodies have significant potential in drug development and personalized medicine:

• Drug discovery applications:

  • High-throughput screening assays for ALOX5 inhibitor development

  • Target engagement studies to confirm compound binding

  • Pharmacodynamic biomarker development

  • Mechanism-of-action studies for leukotriene pathway modulators

• Personalized medicine approaches:

  • Patient stratification based on ALOX5 expression levels

  • Prediction of response to anti-leukotriene therapies

  • Monitoring treatment efficacy through changes in expression or localization

  • Identification of resistance mechanisms

• Research strategies:

  • Multiplex IHC to correlate ALOX5 with other inflammatory markers

  • Liquid biopsy approaches for non-invasive monitoring

  • Integration with genomic data on ALOX5 promoter variants

  • Development of companion diagnostic assays