Human ALOXE3/Arachidonate lipoxygenase 3 isoform CRA_a ELISA Kit
The Human ALOXE3 (Arachidonate lipoxygenase 3 isoform CRA_a) ELISA Kit is a powerful tool for the precise quantification of ALOXE3 levels in human samples, including serum, plasma, and cell culture supernatants. This kit boasts exceptional sensitivity and specificity, ensuring accurate and reproducible results that are well-suited for a variety of research applications.ALOXE3, also known as arachidonate lipoxygenase 3 isoform CRA_a, is a key enzyme involved in the oxidation of fatty acids, particularly arachidonic acid.
Its role in inflammation, skin barrier function, and lipid metabolism underscores its importance in various diseases and biological processes. The ALOXE3 ELISA Kit provides researchers with a reliable method for studying the expression and function of this enzyme in the context of conditions such as dermatitis, cancer, and metabolic disorders.
Product Name:
Human ALOXE3/Arachidonate lipoxygenase 3 isoform CRA_a ELISA Kit
Product Code:
HUFI02198
Size:
96 Assays
Alias:
ALOXE3
Detection method:
Sandwich ELISA, Double Antibody
Application:
This immunoassay kit allows for the in vitro quantitative determination of Human ALOXE3 concentrations in serum plasma and other biological fluids.
Sensitivity:
0.188ng/ml
Range:
0.313-20ng/ml
Storage:
4°C for 6 months
Note:
For Research Use Only
Recovery:
Matrices listed below were spiked with certain level of Human ALOXE3 and the recovery rates were calculated by comparing the measured value to the expected amount of Human ALOXE3 in samples.
Matrix
Recovery range(%)
Average(%)
serum(n=5)
85-105
96
EDTA plasma(n=5)
92-103
96
UFH plasma(n=5)
86-102
95
Linearity:
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ALOXE3 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample
1:2
1:4
1:8
serum(n=5)
95-105%
95-105%
93-104%
EDTA plasma(n=5)
86-96%
83-91%
83-101%
UFH plasma(n=5)
80-99%
80-99%
83-98%
CV(%):
Intra-Assay: CV<8% Inter-Assay: CV<10%
Component
Quantity
Storage
ELISA Microplate (Dismountable)
8×12 strips
4°C for 6 months
Lyophilized Standard
2
4°C/-20°C
Sample/Standard Dilution Buffer
20ml
4°C
Biotin-labeled Antibody(Concentrated)
120ul
4°C (Protect from light)
Antibody Dilution Buffer
10ml
4°C
HRP-Streptavidin Conjugate(SABC)
120ul
4°C (Protect from light)
SABC Dilution Buffer
10ml
4°C
TMB Substrate
10ml
4°C (Protect from light)
Stop Solution
10ml
4°C
Wash Buffer(25X)
30ml
4°C
Plate Sealer
5
-
Other materials and equipment required:
Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Non-heme iron-containing lipoxygenase which is atypical in that it displays a prominent hydroperoxide isomerase activity and a reduced dioxygenase activity compared to other lipoxygenases. The hydroperoxide isomerase activity catalyzes the isomerization of hydroperoxides, derived from arachidonic and linoleic acid by ALOX12B, into hepoxilin-type epoxyalcohols. The dioxygenase activity requires a step of activation of the enzyme by molecular oxygen. In presence of oxygen, oxygenates polyunsaturated fatty acids, including arachidonic acid, to produce fatty acid hydroperoxides. In the skin, acts downstream of ALOX12B on the linoleate moiety of esterified omega-hydroxyacyl-sphingosine (EOS) ceramides to produce an epoxy-ketone derivative, a crucial step in the conjugation of omega-hydroxyceramide to membrane proteins. Therefore plays a crucial role in the synthesis of corneocytes lipid envelope and the establishment of the skin barrier to water loss. In parallel, it may have a signaling function in barrier formation through the production of hepoxilins metabolites. Plays also a role in adipocyte differentiation through hepoxilin A3 and hepoxilin B3 production which in turn activate PPARG. Through the production of hepoxilins in the spinal cord, it may regulate inflammatory tactile allodynia.
NCBI Summary:
This gene is a member of the lipoxygenase family, which are catabolized by arachidonic acid-derived compounds. The encoded enzyme is a hydroperoxide isomerase that synthesizes a unique type of epoxy alcohol (8R-hydroxy-11R,12R-epoxyeicosa-5Z,9E,14Z-trienoic acid) from 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE). This epoxy alcohol can activate the the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha), which is implicated in epidermal differentiation. Loss of function of the enzyme encoded by this gene results in ichthyosis, implicating the function of this gene in the differentiation of human skin. This gene is part of a cluster of lipoxygenase genes on 17p13.1. Mutations in this gene result in nonbullous congenital ichthyosiform erythroderma (NCIE). Multiple transcript variants encoding different isoforms have been found for this gene.[provided by RefSeq, Sep 2009]
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
Step
Protocol
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.
