Mouse Arachidonate 15-lipoxygenase B (Alox15b) ELISA Kit
The Mouse Arachidonate 15-Lipoxygenase B (ALOX15B) ELISA Kit from Assay Genie is a powerful tool for detecting and quantifying levels of ALOX15B in mouse serum, plasma, and other biological samples. With its high sensitivity and specificity, this kit provides accurate and reliable results for researchers studying the role of ALOX15B in various physiological and pathological processes.Arachidonate 15-Lipoxygenase B is an enzyme involved in the metabolism of arachidonic acid, a key component of the inflammatory pathway. Dysregulation of ALOX15B has been implicated in various diseases such as inflammation, cancer, and neurodegenerative disorders, making it a valuable target for therapeutic interventions.
By using the Mouse ALOX15B ELISA Kit, researchers can gain insights into the expression levels and activity of ALOX15B in mouse models, paving the way for a better understanding of its role in disease progression and the development of novel treatment strategies. Trust Assay Genie for accurate and reproducible results in your research endeavors.
Product Name:
Mouse Arachidonate 15-lipoxygenase B (Alox15b) ELISA Kit
SKU:
MOEB1203
Size:
96T
Target:
Mouse Arachidonate 15-lipoxygenase B (Alox15b)
Synonyms:
15-lipoxygenase 2, 8S-lipoxygenase, Arachidonate 15-lipoxygenase type II, 15-LOX-2, 8S-LOX, 15-LOX-B, Alox8
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Mouse
Detection Range:
0.312-20ng/mL
Sensitivity:
0.192ng/mL
Intra CV:
Provided with the Kit
Inter CV:
Provided with the Kit
Linearity:
Provided with the Kit
Recovery:
Provided with the Kit
Uniprot:
O35936
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Arachidonate 15-lipoxygenase B
Research Area:
Cardiovascular
Subcellular Location:
Cytoplasm
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
ALOX15B: Converts arachidonic acid exclusively to 15S- hydroperoxyeicosatetraenoic acid, while linoleic acid is less well metabolized. Belongs to the lipoxygenase family. 4 isoforms of the human protein are produced by alternative splicing.Protein type: Motility/polarity/chemotaxis; Cell cycle regulation; EC 1.13.11.33; Lipid Metabolism - arachidonic acid; Oxidoreductase; ApoptosisCellular Component: extrinsic to membrane; cytoskeleton; membrane; cytoplasm; plasma membrane; intracellular; cytosolMolecular Function: oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen; 2-aminophenol 1,6-dioxygenase activity; p-cumate 2,3-dioxygenase activity; 2,3-dihydroxy-p-cumate dioxygenase activity; 3,5-dichlorocatechol 1,2-dioxygenase activity; hydroquinone 1,2-dioxygenase activity; metal ion binding; protocatechuate 3,4-dioxygenase type II activity; 5,6-dihydroxy-3-methyl-2-oxo-1,2-dihydroquinoline dioxygenase activity; calcium ion binding; 1,2-dihydroxyfluorene 1,1-alpha-dioxygenase activity; oxidoreductase activity; styrene dioxygenase activity; dihydroxyfluorene dioxygenase activity; 2,6-dichloro-p-hydroquinone 1,2-dioxygenase activity; 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase activity; catechol dioxygenase activity; 3,4-dihydroxyfluorene 4,4-alpha-dioxygenase activity; lipid binding; phenanthrene dioxygenase activity; 2,3-dihydroxy DDT 1,2-dioxygenase activity; 4-hydroxycatechol 1,2-dioxygenase activity; 2,2',3-trihydroxybiphenyl dioxygenase activity; arachidonate 8-lipoxygenase activity; lipoxygenase activity; chlorocatechol 1,2-dioxygenase activity; 1,2-dihydroxynaphthalene-6-sulfonate 1,8a-dioxygenase activity; 3,4-dihydroxyphenanthrene dioxygenase activity; carbazole 1,9a-dioxygenase activity; dihydroxydibenzothiophene dioxygenase activity; arachidonate 15-lipoxygenase activity; 2,3-dihydroxy-ethylbenzene 1,2-dioxygenase activity; dioxygenase activity; iron ion binding; 1,1-dichloro-2-(dihydroxy-4-chlorophenyl)-(4-chlorophenyl)ethene 1,2-dioxygenase activityBiological Process: negative regulation of cell proliferation; lipoxygenase pathway; linoleic acid metabolic process; arachidonic acid metabolic process; lipid metabolic process; hepoxilin biosynthetic process; negative regulation of cell cycle; negative regulation of growth; positive regulation of keratinocyte differentiation
UniProt Protein Details:
NCBI Summary:
This gene belongs to the lipoxygenase (LOX) gene family whose members encode enzymes that catalyze the addition of molecular oxygen to polyunsaturated fatty acids (PUFAs) to yield fatty acid hydroperoxides. The encoded enzyme preferentially metabolizes arachidonic acid to yield 8-hydroxyeicosatetraenoic acid (8-HETE), while metabolizing linoleic acid less efficiently. The gene may also function as a tumor suppressor. This gene is located in a cluster of related genes that spans approximately 75 kilobases on chromosome 11. [provided by RefSeq, Jan 2013]
arachidonate 8S-lipoxygenase; 15-lipoxygenase 2; arachidonate 15-lipoxygenase B; arachidonate 15-lipoxygenase type II; arachidonate 15-lipoxygenase, second type
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
Step
1.
Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.
Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.
Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.
Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.
Repeat the wash process for five times as conducted in step 3.
6.
Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.
Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.
Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.
After experiment, store all reagents according to the specified storage temperature respectively until their expiry.
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
Sample Type
Protocol
Serum
If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma
Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid
Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant
Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates
Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates
The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates
Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk
Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.
