Anti-15Lipoxygenase 1 (Abcam, ab244205), anti-Glutathione Peroxidase 4 (GPX4) (Abcam, ab125066), anti-Calnexin (Proteintech, 10427-2-AP), anti-ATG4B (CST, 13507S), anti-ATG4B (Proteintech, 15131-1-AP), anti-LC3B (CST, 2775), anti-GABARAP (Abgent, AP1821a), anti-SQSTM1/p62 (Abcam, ab56416), anti-GFP (Abcam, ab290), anti-FLAG (Sigma, F1804), anti-Tom20 (Proteintech, 11802-1-AP), anti-GAPDH (Fudebio-tech, FD0063), anti-β-actin (Fudebio-tech, FD0060), normal rabbit IgG (Santa Cruz, sc-2027), goat anti-mouse IgG-HRP (Fudebio-tech, FDM007), goat anti-rabbit IgG-HRP (Fudebio-tech, FDR007), 1S, 3R-RSL3 (TargetMol, T3646), Fer-1 (Targetmol, T6500), PD146176 (Sigma, P4620), ML355 (Selleck, S6557), Zileuton (Selleck, S1443), Rapamycin (TargetMol, T1537), Bafilomycin A1 (BaF1, Sigma, B1793) Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific, 15338100), Protein A/G Plus-Agarose Immunoprecipitation Reagent (Santa Cruz, sc-2003), DAPI (Beyotime, C1002), LiperFluo (DOJINDO, L248), Lipoxygenase Inhibitor Screening Assay Kit (Cayman, 760700). PC (1,2-dioleoyl-sn-glycero-3-phosphocholine, Avanti, 850375), PE (1-stearoyl-2-eicasotetraenoyl-sn-glycero-3-phosphatidylethanolamine, Avanti, 850804), PE-OOH (1-stearoyl-2-15-hydroperoxy-eicasotetraenoyl-sn-glycero-3-phosphatidylethanolamine, Cayman, Cay25856-100).
Lipoxygenase inhibitor screening assay kit
Manufactured by Cayman Chemical
Sourced in United States
The Lipoxygenase Inhibitor Screening Assay Kit is a laboratory equipment used to detect and measure the inhibitory activity of compounds against the enzyme lipoxygenase. This kit provides a standardized method to screen potential lipoxygenase inhibitors.
Automatically generated - may contain errors
Lab products found in correlation
Eia kit, by Cayman Chemical (4 mentions)
Gen5 data analysis software, by Agilent Technologies (2 mentions)
Anti sqstm1 p62, by Abcam (1 mentions)
Normal rabbit igg, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Lipofectamine ltx reagent with plus reagent, by Thermo Fisher Scientific (1 mentions)
Protein a g plus agarose immunoprecipitation reagent, by Santa Cruz Biotechnology (1 mentions)
Liperfluo, by Dojindo Laboratories (1 mentions)
Anti glutathione peroxidase 4, by Abcam (1 mentions)
Anti calnexin, by Proteintech (1 mentions)
Anti gabarap, by Abcepta (1 mentions)
Anti tom20, by Proteintech (1 mentions)
Fer 1, by Targetmol (1 mentions)
Pd146176, by Merck Group (1 mentions)
Zileuton, by Selleck Chemicals (1 mentions)
Rapamycin, by Targetmol (1 mentions)
Anti gfp, by Abcam (1 mentions)
Anti atg4b, by Proteintech (1 mentions)
21 protocols using lipoxygenase inhibitor screening assay kit
1
Ferroptosis and Autophagy Assessment
2
Lipidation and Deconjugation of LC3
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Gpx4fl/fl MEFs were treated with 500 nM rapamycin for 14 h. Cells were resuspended in PBS and sonicated to extract total proteins. To obtain the LC3-PE, 3.3 mg proteins were used for IP using anti-LC3 antibody and protein A/G plus agarose beads. Centrifuge the lysates at 3000 g/min for 3 min and discard the supernatant. Glycine solution (0.1 M, pH = 2.5) was added and gently mixed the beads. The solution was centrifuged at 3000 g/min for 3 min to collect the supernatant. The elution can be adjusted to neutral pH with NaOH immediately and used for further analysis. LC3-PE was oxidized by ALOX15 according to the instruction manual of lipoxygenase inhibitor screening assay kit (Cayman, 760700). Nordihydroguaiaretic acid (NDGA, positive inhibitor of ALOX15) was added to abort the reaction. Subsequently, ATG4B (0.5 μg) was added or not into the mixture and reacted at 37 °C for 60 min. Finally, the mixture was boiled in sample buffer for 10 min at 100 °C, and immunoblotted by denaturing SDS-PAGE.
3
Lipoxygenase and Cyclooxygenase Inhibition Assay
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The lipoxygenase (LOX) suppression potential was analysed according to Abdelall et al.13 (link) using a lipoxygenase inhibitor screening assay kit (Cayman Chemical, AnnArbor, MI, USA). Cyclooxygenases (COX-1 and COX-2) were measured by an enzyme immunoassay (EIA) kit (Cayman Chemical, AnnArbor, MI, USA) according to the manufacturer’s instruction. COX-2 selectivity index (SI values) which is defined as IC50 (COX-1)/IC50 (COX-2) was estimated and compared to the reference compounds celecoxib, indomethacin and diclofenac as previously described10 (link).
4
Evaluation of Anti-inflammatory Bioactivity of A. zerumbet
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Inhibition of bovine COX-1 and COX-2 by A. zerumbet extract was determined in vitro using an enzyme immuno assay (EIA) kit (Cayman Chemical, AnnArbor, MI, USA) [28 (link)]. A lipoxygenase inhibitor screening assay kit (Cayman Chemical, AnnArbor, MI, USA) was used to evaluate lipoxygenase inhibition activity [28 (link)].
5
Lipoxygenase Inhibition Screening Assay
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Testing of the effect of the test products on the enzymatic activity of lipoxygenase was tested using the Lipoxygenase Inhibitor Screening Assay Kit (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer's instructions. In brief, purified soybean Lipoxygenase was allowed to react with the substrate arachidonic acid in the absence versus presence of test products. The hydroxyperoxides produced as a result of the Lipoxygenase enzymatic reaction were measured in a colorimetric assay, and absorbance read in a microplate reader (BioTek PowerWave, Winooski, VT, USA) at 490–500 nm absorbance.
6
Lipoxygenase Inhibitor Screening Assay
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The positive control (Nordihydroguaiaretic acid [NDGA], catalog No. 760717; Cayman Chemical) and the organic extracts were subjected to a full inhibition curve versus 15-LO using a commercially available lipoxygenase inhibitor screening assay kit (catalog No. 760700; Cayman Chemical, Ann Arbor, Michigan 48108 USA) according to the manufacturer's instructions. NGDA positive control was used at 100 μM final concentration, the organic extracts were serially dissolved in DMSO to produce a series of logarithmic final concentrations (200, 20, and 2 μg/mL), which were subsequently assayed.
7
Cotoneaster Anti-Lipoxygenase Activity
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Anti-lipoxygenase activity of Cotoneaster extracts was determined using the Lipoxygenase Inhibitor Screening Assay Kit (Cayman Chemical, MI, USA) according to the protocol of the manufacturer. The extracts were tested at different concentrations. The effective concentration (μg/mL) in which lipoxygenase activity is inhibited by 50% (IC50) was estimated graphically. Nordihydroguaiaretic acid (NDGA) was used as a positive control.
8
Assessing 15-LOX Inhibition by TBE
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We tested the 15-lipoxygenase (15-LOX) inhibitory activity of TBE by using a Lipoxygenase Inhibitor Screening Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). First, 90 μL of soybean 15-LOX enzyme solution and 10 μL of TBE solution were mixed in the testing wells. The reaction was initiated by adding 10 μL of arachidonic acid substrate solution to all of the wells. All of the testing wells were then placed on a shaker for 5 min, and 90 μL of chromogen was then added to each well to stop the enzyme catalysis and develop the reaction. The absorbance of hydroperoxides produced by 15-LOX from arachidonic acid was measured at 490 nm using a microplate reader (Bio Tek Instruments, Tokyo, Japan).
9
Evaluating Anti-inflammatory Effects of Rhododendron luteum
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The anti-inflammatory activity of Rhododendron luteum Sweet leaf extracts was determined using the Lipoxygenase Inhibitor Screening Assay Kit by Cayman Chemical according to the manufacturer’s instruction. Briefly, 10 μL of the sample was mixed with 90 μL of a lipoxygenase solution. The samples were tested in three different concentrations (50, 100, and 200 μg of dry extract in the reaction mixture). After 5 min of incubation at room temperature, a solution of arachidonic acid was added and the plate was shaken for 10 min. After this time, 100 μL of chromogen was added, and the mixture was shaken for another 5 min. Absorbance was read at 500 nm. Assay buffer instead of extract was used in the negative control. NDGA (nordihydroguaiaretic acid) was used as a positive control.
10
Lipoxygenase and COX Inhibition Assay
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The capacity of the extract to inhibit lipoxygenase was determined using a lipoxygenase inhibitor screening assay kit (Cayman Chemical, Ann Arbor, MI, United States) according to the manufacturer’s instruction and reported study (Abdelall et al., 2016 (link)). The ability of the extract to inhibit ovine COX-1 and COX-2 was determined by using an enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI, United States) according to the manufacturer’s instruction and reported studies (Abdelall et al., 2016 (link)). The data are expressed as IC50 value, which is the concentration causing 50% enzyme inhibition (IC50). Furthermore, the COX-2 selectivity index (SI values) which is defined as IC50 (COX-1)/IC50 (COX-2) was calculated and compared to that of celecoxib, indomethacin, and diclofenac which were used as reference standards.
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