Human Arachidonate-12-Lipoxygenase (ALOX12) ELISA Kit

Principle of the Assay

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Human ALOX12. Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to Human ALOX12. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Human ALOX12, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Human ALOX12 in the samples is then determined by comparing the OD of the samples to the standard curve.

For use with: tissue homogenates, cell lysates, and other biological fluids.

For Research Use Only. Not for use in diagnostic procedures.

Target Information

Catalyzes the regio and stereo-specific incorporation of molecular oxygen into free and esterified polyunsaturated fatty acids generating lipid hydroperoxides that can be further reduced to the corresponding hydroxy species (PubMed:17493578, PubMed:1851637, PubMed:8319693, PubMed:8500694, PubMed:18311922, PubMed:32404334). Mainly converts arachidonate ((5Z,8Z,11Z,14Z)-eicosatetraenoate) to the specific bioactive lipid (12S)-hydroperoxyeicosatetraenoate/(12S)-HPETE (PubMed:17493578, PubMed:22984144, PubMed:24282679, PubMed:8319693, PubMed:8500694). Through the production of bioactive lipids like (12S)-HPETE it regulates different biological processes including platelet activation (PubMed:8319693, PubMed:8500694). It can also catalyze the epoxidation of double bonds of polyunsaturated fatty acids such as (14S)-hydroperoxy-docosahexaenoate/(14S)-HPDHA resulting in the formation of (13S,14S)-epoxy-DHA (PubMed:23504711). Furthermore, it may participate in the sequential oxidations of DHA ((4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoate) to generate specialized pro-resolving mediators (SPMs) like resolvin D5 ((7S,17S)-diHPDHA) and (7S,14S)-diHPDHA, that actively down-regulate the immune response and have anti-aggregation properties with platelets (PubMed:32404334). An additional function involves a multistep process by which it transforms leukotriene A4/LTA4 into the bioactive lipids lipoxin A4/LXA4 and lipoxin B4/LXB4, both are vasoactive and LXA4 may regulate neutrophil function via occupancy of specific recognition sites (PubMed:8250832). Can also peroxidize linoleate ((9Z,12Z)-octadecadienoate) to (13S)-hydroperoxyoctadecadienoate/ (13S-HPODE) (By similarity). Due to its role in regulating both the expression of the vascular endothelial growth factor (VEGF, an angiogenic factor involved in the survival and metastasis of solid tumors) and the expression of integrin beta-1 (known to affect tumor cell migration and proliferation), it can be regarded as protumorigenic (PubMed:9751607, PubMed:16638750, PubMed:22237009). Important for cell survival, as it may play a role not only in proliferation but also in the prevention of apoptosis in vascular smooth muscle cells (PubMed:23578768).

GENE ID	239
SWISS PROT	P18054
SYNONYMS	LOX12; 12-LOX; ALOX-12; 12-LO; 12LPG; LOG12; Lipoxygenase 12; 12S-lipoxygenase; Lipoxin synthase 12-LO; Platelet-type lipoxygenase 12

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-Coated Microplate	48T: 6 strips x 8 wells; 96T: 12 strips x 8 wells
Standard (Lyophilized)	48T: 1 vial; 96T: 2 vials
Biotinylated Antibody (100×)	48T: 60 μL; 96T: 120 μL
Streptavidin-HRP (100×)	48T: 60 μL; 96T: 120 μL
Standard/Sample Diluent Buffer	48T: 10 mL; 96T: 20 mL
Biotinylated Antibody Diluent	48T: 6 mL; 96T: 12 mL
HRP Diluent	48T: 6 mL; 96T: 12 mL
Wash Buffer (25×)	48T: 10 mL; 96T: 20 mL
TMB Substrate Solution	48T: 6 mL; 96T: 10 mL
Stop Reagent	48T: 3 mL; 96T: 6 mL
Plate Covers	48T: 1 piece; 96T: 2 pieces

Storage

Storage

*If the kit is opened, Store the whole kit at 4°C. If the kit is not used up in 1 week. Store the Pre-Coated Microplate, Standard ,Biotinylated Antibody and Streptavidin-HRP at -20°C, the rest reagents at 4°C, please used up within 6 months. *If the kit is not opened, store the whole kit: 4°C(short time storage, valid for 6 months); -20°C (long-term storage, valid for 1 year). Avoid repeated freeze-thaw cycles.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay)：CV% < 8% Three samples of known concentration were tested twenty times on 1 plate to assess intra-assay precision. Inter-assay Precision (precision between assays)：CV% < 10% Three samples of known concentration were tested in forty separate assays to assess inter-assay precision.
SENSITIVITY	The minimum detectable dose was 0.108ng/mL.
ASSAY RANGE	0.32–20ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for detection of Human ALOX12. No significant cross-reactivity or interference between Human ALOX12 and analogues was observed.