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Mouse 15-LO/LOX(15-lipoxygenase) ELISA Kit

SKU:
MOFI01284
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P39654
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich
Synonyms:
15-LO, LOX
Reactivity:
Mouse
€599
Frequently bought together:

Description

system_update_altDatasheetsystem_update_altMSDS
Product Name:Mouse 15-LO/LOX(15-lipoxygenase) ELISA Kit
Product Code:MOFI01284
Size:96 Assays
Alias:15-LO, LOX
Detection Method:Sandwich ELISA
Application:This immunoassay kit allows for the in vitro quantitative determination of Mouse 15-LO/LOX concentrations in serum plasma and other biological fluids.
Sensitivity:0.094ng/ml
Range:0.156-10ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery: Matrices listed below were spiked with certain level of Mouse 15-LO/LOX and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse 15-LO/LOX in samples. Not Available.
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse 15-LO/LOX and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Not Available.
Intra Assay:CV <8%
Inter Assay:CV <10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8-12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir
UniprotP39654
UniProt Protein Function:ALOX15: Converts arachidonic acid to 15S- hydroperoxyeicosatetraenoic acid. Also acts on C-12 of arachidonate as well as on linoleic acid. Belongs to the lipoxygenase family.
UniProt Protein Details:

Protein type:EC 1.13.11.31; EC 1.13.11.33; Motility/polarity/chemotaxis; Oxidoreductase; Lipid Metabolism - linoleic acid; Lipid Metabolism - arachidonic acid

Cellular Component: extrinsic to internal side of plasma membrane; membrane; cytoplasm; plasma membrane; lipid particle; cytosol

Molecular Function:oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen; phosphatidylinositol-4,5-bisphosphate binding; hepoxilin A3 synthase activity; arachidonate 12-lipoxygenase activity; arachidonate 15-lipoxygenase activity; hepoxilin-epoxide hydrolase activity; dioxygenase activity; iron ion binding; metal ion binding; oxidoreductase activity; lipid binding

Biological Process: ossification; positive regulation of heterotypic cell-cell adhesion; wound healing; lipoxygenase pathway; positive regulation of actin filament polymerization; negative regulation of adaptive immune response; phosphatidylethanolamine biosynthetic process; arachidonic acid metabolic process; hepoxilin biosynthetic process; lipid metabolic process; fatty acid metabolic process; apoptotic cell clearance; bone mineralization

UniProt Code:P39654
NCBI GenInfo Identifier:134948633
NCBI Gene ID:11687
NCBI Accession:NP_033790.3
UniProt Secondary Accession:P39654,Q4FJY9, Q5F2E3, Q6PHB2,
UniProt Related Accession:P39654
Molecular Weight:75,445 Da
NCBI Full Name:arachidonate 15-lipoxygenase
NCBI Synonym Full Names:arachidonate 15-lipoxygenase
NCBI Official Symbol:Alox15  
NCBI Official Synonym Symbols:12-LO; 15-LOX; L-12LO; Alox12l; 12/15-LO  
NCBI Protein Information:arachidonate 15-lipoxygenase
UniProt Protein Name:Arachidonate 15-lipoxygenase
UniProt Synonym Protein Names:12/15-lipoxygenase; 12/15-LO; Arachidonate 12-lipoxygenase, leukocyte-type (EC:1.13.11.31); 12-LOX; L-12LO; Arachidonate omega-6 lipoxygenase
UniProt Gene Name:Alox15  
UniProt Entry Name:LOX15_MOUSE

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3. Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4. Add 0.1 ml of properly diluted sample (Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6. Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7. Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9. Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10. Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11. Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12. Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13. Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.

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