The 12-HETE (12-Hydroxyeicosatetraenoic Acid) ELISA Kit is specially designed for the accurate quantification of 12-HETE levels in various sample types, including serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring reliable and reproducible results for a wide range of research applications.12-HETE is a metabolite of arachidonic acid that plays a critical role in inflammation and oxidative stress pathways. Abnormal levels of 12-HETE have been associated with various diseases, including cancer, cardiovascular disorders, and inflammatory conditions.
As such, this ELISA kit serves as a valuable tool for studying the role of 12-HETE in disease development and progression, as well as for developing potential therapeutic interventions.With its user-friendly protocol and high-quality components, the 12-HETE ELISA Kit from Assay Genie is an essential tool for researchers and clinicians interested in investigating the biological functions and clinical implications of 12-HETE in health and disease.
Matrices listed below were spiked with certain level of 12-HETE (12-Hydroxyeicosatetraenoic Acid) and the recovery rates were calculated by comparing the measured value to the expected amount of 12-HETE (12-Hydroxyeicosatetraenoic Acid) in samples.
Matrix
Recovery range(%)
Average(%)
serum(n=5)
86-105
95
EDTA plasma(n=5)
87-98
94
heparin plasma(n=5)
88-101
96
Linearity:
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of 12-HETE (12-Hydroxyeicosatetraenoic Acid) 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)
86-104%
85-101%
94-105%
EDTA plasma(n=5)
83-93%
82-100%
82-101%
UFH plasma(n=5)
80-97%
84-99%
84-96%
Intra-Assay:
CV <8%
Inter-Assay:
CV <10%
Step
Procedure
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.
Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank or Sample per well. The blank well is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody working solution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thorough mixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA plate well, avoid touching plate walls and foaming).
3.
Wash: Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 350µL) using a squirt bottle, multi-channel pipette, manifold dispenser or automated washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.
HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC working solution to each well. Cover with a new Plate sealer. Incubate for 30 minutes at 37°C.
5.
Wash: Repeat the aspiration/wash process for five times.
6.
TMB Substrate: Add 90µL of TMB Substrate to each well. Cover with a new plate sealer. Incubate for about 10-20 minutes at 37°C. Protect from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30 minutes. When apparent gradient appeared in standard wells, you can terminate the reaction.
7.
Stop: Add 50µL of Stop Solution to each well. Color turn to yellow immediately. The adding order of stop solution should be as the same as the substrate solution.
8.
OD Measurement: Determine the optical density (OD Value) of each well at once, using a microplate reader set to 450 nm. You should open the microplate reader ahead, preheat the instrument, and set the testing parameters.
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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.
