AHL12 Antibody

What is ALOX12 and what biological processes does it regulate?

ALOX12 (Arachidonate 12-lipoxygenase, 12S-type) is an enzyme that catalyzes the region and stereo-specific incorporation of molecular oxygen into polyunsaturated fatty acids, generating lipid hydroperoxides . It primarily converts arachidonate ((5Z,8Z,11Z,14Z)-eicosatetraenoate) to (12S)-hydroperoxyeicosatetraenoate/(12S)-HPETE, a specific bioactive lipid . Through the production of bioactive lipids like (12S)-HPETE, ALOX12 regulates various biological processes including platelet activation . Additionally, it can catalyze the epoxidation of double bonds of polyunsaturated fatty acids such as (14S)-hydroperoxy-docosahexaenoate . ALOX12 may also participate in sequential oxidations of docosahexaenoate (DHA) to generate specialized pro-resolving mediators (SPMs) like resolvin D5 .

What are the key specifications of commercially available ALOX12 antibodies?

The ALOX12 polyclonal antibody is typically generated from rabbits immunized with a KLH conjugated synthetic peptide corresponding to the C-terminal region (amino acids 618-650) of human ALOX12 . These antibodies demonstrate reactivity with human, mouse, and rat samples, making them versatile for comparative studies across species . The calculated molecular weight of the target protein is approximately 75694 Da . These antibodies are supplied in PBS with 0.09% (W/V) sodium azide and are purified through protein A columns followed by peptide affinity purification .

What experimental applications are ALOX12 antibodies suitable for?

ALOX12 antibodies have been validated for multiple experimental applications in molecular and cellular biology research . They can be effectively used in Western blotting (WB) at a dilution of 1:1000 for detecting ALOX12 protein in cell or tissue lysates . For immunofluorescence (IF) studies, a dilution of 1:100 is recommended to visualize the subcellular localization of ALOX12 . These antibodies are also suitable for flow cytometry (FC) and immunohistochemistry on paraffin-embedded tissues (IHC-P) at dilutions of 1:10-1:50, allowing for quantitative and qualitative analysis of ALOX12 expression in different cell populations and tissue sections . Additionally, they can be used in ELISA applications for detecting and quantifying ALOX12 in solution .

How should ALOX12 antibodies be stored to maintain optimal activity?

For maintaining optimal activity, ALOX12 antibodies should be refrigerated at 2-8°C for short-term storage (up to 2 weeks) . For long-term storage, it is recommended to store the antibody at -20°C in small aliquots to prevent freeze-thaw cycles, which can degrade antibody quality and reduce binding efficiency . Repeated freeze-thaw cycles should be strictly avoided as they can lead to denaturation of the antibody, resulting in decreased specificity and sensitivity in experimental applications . When working with the antibody, it's advisable to keep it on ice and return it to proper storage conditions promptly after use.

How can I validate the specificity of ALOX12 antibody in my experimental system?

Validating antibody specificity is crucial for ensuring reliable experimental results. For ALOX12 antibody, a multi-faceted approach is recommended: First, perform Western blot analysis using positive control lysates from tissues known to express ALOX12 (e.g., platelets) alongside tissues with minimal expression . The presence of a single band at approximately 75 kDa indicates specificity. Second, implement siRNA knockdown or CRISPR knockout of ALOX12 in your experimental cell line, followed by immunoblotting to confirm signal reduction . Third, conduct peptide competition assays using the immunizing peptide to confirm binding specificity . Finally, for immunohistochemistry applications, compare staining patterns with published literature on ALOX12 expression and include isotype control antibodies to assess non-specific binding .

What are the optimal fixation and antigen retrieval methods for ALOX12 immunohistochemistry?

For optimal immunohistochemical detection of ALOX12, tissue fixation and antigen retrieval parameters significantly impact staining quality. Tissues should be fixed in 10% neutral buffered formalin for 24-48 hours depending on sample size . For paraffin-embedded sections, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is generally effective for ALOX12 detection . This typically involves heating sections to 95-98°C for 15-20 minutes followed by gradual cooling to room temperature. In some difficult cases, Tris-EDTA buffer (pH 9.0) may provide superior antigen retrieval. For frozen sections, acetone fixation for 10 minutes at -20°C often yields good results. Optimization experiments comparing different antigen retrieval methods are recommended for each specific tissue type and experimental question .

How can I differentiate between various isoforms of ALOX12 in my research?

ALOX12 exists in multiple isoforms, which presents a challenge for isoform-specific detection. The most definitive approach is to use antibodies specifically raised against unique epitopes in each isoform . When using the C-terminal ALOX12 antibody (targeting amino acids 618-650), it's important to verify which isoforms share this sequence . For more precise isoform discrimination, consider combining antibody-based detection with molecular techniques: RT-PCR using isoform-specific primers can confirm expression at the mRNA level . 2D gel electrophoresis followed by Western blotting can separate isoforms based on both molecular weight and isoelectric point. For functional studies, selective inhibitors or siRNAs targeting specific isoforms can help establish isoform-specific roles in your biological system .

What strategies can minimize cross-reactivity when studying ALOX12 in multi-protein complexes?

When investigating ALOX12 in multi-protein complexes, cross-reactivity can confound results. To minimize this, implement a strategic approach: First, use appropriate blocking solutions containing both serum (5% from the species of the secondary antibody) and BSA (3-5%) to reduce non-specific binding . Second, optimize antibody concentrations through titration experiments to determine the minimum concentration yielding specific signal . Third, incorporate stringent washing steps with buffers containing 0.1-0.3% Tween-20 to remove non-specifically bound antibodies. Fourth, consider using native versus denaturing conditions strategically – native conditions maintain protein-protein interactions while denaturing conditions may expose epitopes but disrupt complexes . Finally, validate findings using reciprocal co-immunoprecipitation with antibodies against suspected interacting partners, and confirm specificity with appropriate controls including isotype antibodies and pre-immune serum .

What are common causes of false positive or negative results when using ALOX12 antibody?

False positive results when using ALOX12 antibody may stem from several sources: cross-reactivity with structurally similar proteins (particularly other lipoxygenases), excessive antibody concentration leading to non-specific binding, inadequate blocking, or contamination of secondary antibodies . Conversely, false negative results might occur due to: insufficient antigen retrieval (particularly in fixed tissues), protein degradation during sample preparation, epitope masking by interacting proteins or post-translational modifications, or antibody degradation from improper storage . To mitigate these issues, always include positive and negative controls, optimize antibody concentration for each application, verify results with an alternative antibody targeting a different epitope of ALOX12, and ensure proper sample preparation and storage conditions .

How should I interpret contradictory results between different detection methods using ALOX12 antibody?

Contradictory results between different detection methods (e.g., Western blot vs. immunohistochemistry) require systematic investigation. First, evaluate the nature of the contradiction: is it presence/absence of signal, intensity differences, or localization discrepancies? Second, consider the fundamental differences between techniques – Western blotting detects denatured proteins separated by size, while immunohistochemistry detects proteins in their native environment with preserved cellular context . Epitope accessibility may differ dramatically between these conditions. Third, examine technical variables: sample preparation methods, antibody concentrations, detection systems, and blocking reagents . Fourth, assess biological variables: different cell types or tissues may express variant forms of ALOX12 or contain different interacting proteins that affect antibody binding . To resolve contradictions, perform additional validation experiments including RNA expression analysis, alternative antibodies, genetic knockdown/knockout controls, and consider consulting recent literature addressing similar discrepancies .

What quality control measures should be implemented when working with newly purchased ALOX12 antibody lots?

Implementing robust quality control for new ALOX12 antibody lots is essential for experimental reproducibility. Upon receiving a new lot, perform a comparative Western blot against a known positive control sample alongside your previous antibody lot to assess consistency in band pattern, molecular weight detection, and signal intensity . For immunofluorescence or immunohistochemistry applications, compare staining patterns and intensity between lots using standardized positive control tissues . Document key parameters including background signal, specific-to-nonspecific signal ratio, and detection threshold. Additionally, verify the antibody certificate of analysis from the manufacturer for lot-specific validation data . If significant lot-to-lot variation is observed, contact the manufacturer for technical support and consider alternative validation methods such as immunoprecipitation followed by mass spectrometry to confirm antibody specificity . Maintain detailed records of lot numbers and corresponding validation results to track performance over time.

How can ALOX12 antibodies be utilized in studying lipid metabolism pathways?

ALOX12 antibodies serve as valuable tools for investigating lipid metabolism pathways through multiple sophisticated approaches. They can be employed in chromatin immunoprecipitation (ChIP) assays to identify transcription factors regulating ALOX12 expression in response to metabolic stimuli . For studying enzyme-substrate interactions, use co-immunoprecipitation with ALOX12 antibodies followed by lipidomic analysis of bound fatty acids . To visualize the subcellular localization of ALOX12 during lipid metabolism, perform immunofluorescence co-staining with organelle markers (e.g., ER, lipid droplets) under various metabolic conditions . For quantitative assessment of ALOX12 enzyme activity correlation with protein levels, combine Western blotting with functional enzyme assays measuring 12-HETE production . Additionally, ALOX12 antibodies can be used in tissue microarray analysis to assess expression patterns across different metabolic disorders, providing insights into pathway dysregulation in disease states .

What considerations are important when designing ALOX12 antibody-based detection systems for high-throughput screening?

Designing high-throughput screening (HTS) systems using ALOX12 antibodies requires careful optimization of several parameters. First, antibody specificity and sensitivity must be rigorously validated in the specific assay format to ensure reliable signal detection with minimal background . Second, standardize sample preparation methods to maintain consistent epitope accessibility across all specimens . Third, optimize antibody concentration through checkerboard titration to determine the minimal concentration yielding maximal signal-to-noise ratio, which is crucial for cost-effective screening . Fourth, select an appropriate detection system (chemiluminescence, fluorescence, colorimetric) based on required sensitivity, equipment availability, and compatibility with other assay components . Fifth, implement robust internal controls including positive controls (known ALOX12-expressing samples), negative controls (ALOX12-knockout samples), and technical controls (standardized samples repeated across plates) to normalize inter-plate variation . Finally, validate the HTS system with a pilot screen of known modulators of ALOX12 expression or activity before scaling to full screening capacity .

How can phosphorylation status of ALOX12 be studied using available antibodies?

Studying ALOX12 phosphorylation requires a strategic combination of techniques and carefully selected antibodies. While general ALOX12 antibodies detect total protein regardless of phosphorylation status , phospho-specific antibodies (if available) should be employed to directly detect specific phosphorylation sites. If phospho-specific ALOX12 antibodies are unavailable, alternative approaches include: First, immunoprecipitation of ALOX12 using the C-terminal antibody followed by Western blotting with general anti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosine antibodies . Second, treatment with phosphatase inhibitors (e.g., sodium orthovanadate, β-glycerophosphate) in parallel samples to preserve phosphorylation status during sample preparation . Third, performing Phos-tag™ SDS-PAGE, which separates phosphorylated from non-phosphorylated proteins based on mobility shift, followed by Western blotting with ALOX12 antibody . Fourth, mass spectrometry analysis of immunoprecipitated ALOX12 to identify specific phosphorylation sites and their stoichiometry under different experimental conditions . These approaches can reveal how phosphorylation regulates ALOX12 activity, stability, or interactions in various physiological and pathological contexts.

What methodological adaptations are needed when studying ALOX12 in different tissue types?

Different tissue types require specific methodological adaptations when studying ALOX12 expression and function. For tissues with high lipid content (brain, adipose tissue), modified extraction buffers containing higher detergent concentrations (1-2% Triton X-100 or NP-40) may be necessary to effectively solubilize ALOX12 . In tissues with abundant proteases (pancreas, intestine), stronger protease inhibitor cocktails should be used during sample preparation . For fibrous tissues (muscle, skin), mechanical homogenization followed by longer extraction times may improve protein recovery . When performing immunohistochemistry, tissue-specific autofluorescence (particularly in tissues containing elastin, collagen, or lipofuscin) can be mitigated using Sudan Black B treatment or spectral unmixing techniques . Antigen retrieval methods should be optimized for each tissue type – for instance, trypsin-based enzymatic retrieval may be superior for some connective tissues, while heat-induced citrate buffer retrieval works better for others . Finally, antibody dilutions and incubation times should be systematically optimized for each tissue type to achieve optimal signal-to-noise ratios .

How can ALOX12 antibodies be used to investigate evolutionary conservation of lipid metabolism pathways?

ALOX12 antibodies offer valuable tools for studying evolutionary conservation of lipid metabolism pathways across species. Researchers can employ these antibodies in comparative Western blot analyses of tissue lysates from different species, from mammals to lower vertebrates, to assess conservation of protein size and expression patterns . For more detailed evolutionary studies, combine immunohistochemistry with phylogenetic analysis of ALOX12 sequences to correlate functional conservation with sequence homology . To examine conservation of protein-protein interactions across species, perform co-immunoprecipitation using ALOX12 antibodies in different organisms, followed by mass spectrometry to identify interacting partners . For functional conservation assessment, compare immunolocalization patterns during development or in response to stimuli across species . Additionally, cross-species comparison of post-translational modifications can be conducted using immunoprecipitation with ALOX12 antibodies followed by modification-specific detection methods . These approaches can reveal how ALOX12-dependent lipid metabolism pathways have evolved and been conserved throughout phylogeny, providing insights into fundamental aspects of lipid biology.

How can ALOX12 antibodies be integrated with mass spectrometry for comprehensive protein analysis?

Integrating ALOX12 antibodies with mass spectrometry creates powerful analytical capabilities for comprehensive protein characterization. Immunoprecipitation using ALOX12 antibodies followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) allows identification of post-translational modifications, interaction partners, and protein variants with high specificity . For analyzing ALOX12 in complex samples, implement sequential window acquisition of all theoretical mass spectra (SWATH-MS) combined with ALOX12 antibody-based verification to achieve both breadth and specificity . To study ALOX12 dynamics in different cellular compartments, perform subcellular fractionation followed by immunoprecipitation and MS analysis of each fraction . For absolute quantification, use stable isotope-labeled standard peptides corresponding to ALOX12 sequences in combination with immunoaffinity enrichment . To detect low-abundance ALOX12 complexes, employ crosslinking immunoprecipitation followed by MS (xIP-MS) using optimized crosslinking conditions . These integrated approaches provide unprecedented insights into ALOX12 structure, function, and interaction networks beyond what either technique alone could achieve.

What approaches can combine ALOX12 antibody-based detection with genomic or transcriptomic data analysis?

Integrating ALOX12 antibody-based detection with genomic and transcriptomic analyses enables multi-omics insights into regulatory mechanisms. Correlative analysis between protein levels (detected by ALOX12 antibodies via Western blot or IHC) and mRNA expression (measured by RNA-seq or qPCR) can reveal post-transcriptional regulation mechanisms . For studying transcription factor binding and epigenetic regulation, combine chromatin immunoprecipitation sequencing (ChIP-seq) for relevant transcription factors with ALOX12 antibody-based protein detection to correlate regulatory events with protein outcomes . To investigate genetic variants affecting ALOX12 expression, correlate genotype data (SNPs) with protein levels detected by quantitative immunoassays . For single-cell resolution studies, integrate single-cell RNA-seq with antibody-based immunofluorescence to examine cell-to-cell variability in ALOX12 expression . Additionally, overlay ALOX12 protein expression data with pathway enrichment analyses from transcriptomic datasets to contextualize ALOX12 function within broader biological networks . These integrated approaches provide comprehensive understanding of ALOX12 regulation from genome to proteome, revealing mechanisms that might be missed by any single analytical method.

How can computational modeling enhance interpretation of ALOX12 antibody-based experimental results?

Computational modeling significantly enhances interpretation of ALOX12 antibody-based experimental results through multiple sophisticated approaches. Structure-based epitope prediction can help understand antibody binding sites and potential cross-reactivity risks, improving experimental design and interpretation . For spatiotemporal studies, image analysis algorithms can quantify ALOX12 localization changes in immunofluorescence data, extracting patterns not visible by manual inspection . Systems biology approaches can integrate ALOX12 protein levels (measured by antibody-based methods) with metabolomic data to model enzymatic flux through lipoxygenase pathways . Network analysis incorporating protein-protein interaction data from co-immunoprecipitation experiments can predict functional relationships and identify novel ALOX12-associated pathways . Additionally, machine learning algorithms can be trained on immunohistochemical data to classify tissue samples based on ALOX12 expression patterns, potentially revealing clinically relevant subtypes . These computational approaches transform antibody-based data from descriptive observations into predictive models that generate testable hypotheses about ALOX12 function and regulation.

How reliable are ALOX12 antibodies for biomarker development in inflammatory and metabolic diseases?

The reliability of ALOX12 antibodies for biomarker development depends on several critical factors that must be systematically addressed. For clinical biomarker applications, antibody performance characteristics including sensitivity, specificity, reproducibility, and lot-to-lot consistency must be rigorously validated using reference standards and clinical specimens . Epitope stability is particularly important – the C-terminal region targeted by many ALOX12 antibodies (amino acids 618-650) should be assessed for potential masking by disease-associated modifications or protein interactions . When developing immunoassays, optimize protocols for relevant clinical sample types (serum, plasma, tissue biopsies) and validate analytical parameters including detection limit, dynamic range, precision, and accuracy . For inflammatory conditions, verify that inflammation-associated post-translational modifications do not interfere with antibody binding . Compare antibody-based detection with orthogonal methods (enzymatic activity, mass spectrometry) to confirm that the antibody accurately reflects ALOX12 status in disease contexts . Additionally, conduct appropriate statistical validation including receiver operating characteristic (ROC) analysis to determine diagnostic performance metrics before clinical application .

What methodological considerations are important when studying post-translational modifications of ALOX12 in disease models?

Studying post-translational modifications (PTMs) of ALOX12 in disease models requires careful methodological planning. First, sample preparation protocols must preserve labile PTMs – flash freezing tissues immediately after collection and including appropriate inhibitors (phosphatase inhibitors for phosphorylation, deacetylase inhibitors for acetylation, etc.) in extraction buffers is essential . Second, select appropriate enrichment strategies – while general ALOX12 antibodies capture all forms of the protein, subsequent analysis with PTM-specific antibodies or mass spectrometry is needed to identify modifications . Third, include appropriate controls – compare disease models with healthy controls processed identically, and include samples treated with modifying or demodifying enzymes as technical controls . Fourth, quantify modification stoichiometry (percentage of ALOX12 molecules carrying specific PTMs) rather than just presence/absence of modifications . Fifth, correlate PTM changes with functional outcomes using activity assays to establish biological significance . Finally, validate key findings across multiple disease models and, when possible, in human patient samples to ensure relevance to human pathophysiology .

How can ALOX12 antibodies facilitate understanding of enzyme regulation in pathological conditions?

ALOX12 antibodies enable multi-faceted investigation of enzyme regulation in pathological conditions. To study altered subcellular localization in disease states, perform immunofluorescence co-localization studies with organelle markers in affected tissues, quantifying redistribution patterns compared to healthy controls . For examining protein-protein interactions unique to pathological conditions, use co-immunoprecipitation with ALOX12 antibodies followed by mass spectrometry or Western blotting for suspected partners . To investigate altered expression in specific cell populations, combine immunohistochemistry with cell-type specific markers in tissue sections from disease models . For studying turnover and stability changes, pulse-chase experiments using metabolic labeling followed by immunoprecipitation can reveal altered protein half-life in disease contexts . To examine potential cleavage products or disease-specific isoforms, use Western blotting with antibodies targeting different epitopes . Additionally, correlate ALOX12 protein levels with enzymatic activity measurements in paired samples to identify post-translational regulation mechanisms . These approaches collectively reveal how ALOX12 regulation is perturbed in pathological conditions, potentially identifying novel therapeutic targets.