4-HNE(4-Hydroxynonenal) ELISA Kit

4-HNE(4-Hydroxynonenal) ELISA Kit

Sku Size Price Quantity
EK248919 96 Wells $380
MSDSDatasheet
SKU:EK248919
Category:
Species:General
Assay Principle:Quantitative
Assay Type:Competitive Inhibition
Target Name:4-HNE(4-Hydroxynonenal)
Sample Type:serum, plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids
Sensitivity:0.38 ng/mL
Standard:40 ng/mL
Detection Range:0.63-40 ng/mL
Assay Time:2h
Sample Volume:50 - 100 ul
Detection Wavelength:450 nm
Research Area:Signal transduction;Metabolic pathway;Infection immunity;Hormone metabolism;Dermatology;

Components

Assay plate (12 × 8 coated Microwells) 1
Standard (Lyophilized) 2 vials
Biotinylated-Conjugate (100×) 60 μL
Streptavidin-HRP (100×) 120 μL
Standard/Sample Diluent Buffer 20 ml
Biotinylated-Conjugate Diluent 10 ml
HRP Diluent 12 ml
Wash Buffer (25×) 20 ml
TMB Substrate Solution 10 ml
Stop Reagent 6 ml
Plate Covers 2

Test Principle

This assay uses the quantitative enzyme immunoassay method. The provided microtiter plate in the kit is pre-coated with 4-HNE(4-Hydroxynonenal). Standards or samples are added to the appropriate wells of the microtiter plate, along with a biotin-conjugated antibody specific to 4-HNE(4-Hydroxynonenal) Then, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each well and incubated. Following this, TMB substrate solution is added. The enzyme-substrate reaction is stopped by adding a sulfuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of 4-HNE(4-Hydroxynonenal) in the samples is determined by comparing the optical density (OD) of the samples to the standard curve.

Sample Collection

Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4oC before centrifugation for 20 minutes at approximately 1,000×g. Assay freshly prepared serum immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1,000×g at 2-8oC within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.

Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type.

  1. Tissues were rinsed in ice-cold PBS to remove excess blood thoroughly and weighed before homogenization.
  2. Minced the tissues to small pieces and homogenized them in fresh lysis buffer (Different lysis buffer needs to be chosen based on subcellular location of the target protein) (w:v = 1:20-1:50, e.g. 1mL lysis buffer is added in 20-50mg tissue sample) with a glass homogenizer on ice (Micro Tissue Grinders work, too).
  3. The resulting suspension was sonicated with an ultrasonic cell disrupter till the solution is clarified.
  4. Then, the homogenates were centrifuged for 5 minutes at 10,000×g. Collect the supernates and assay immediately or aliquot and store at ≤-20°C.

Cell Lysates - Cells need to be lysed before assaying according to the following directions.

  1. Adherent cells should be washed by cold PBS gently, and then detached with trypsin, and collected by centrifugation at 1,000×g for 5 minutes (suspension cells can be collected by centrifugation directly).
  2. Wash cells three times in cold PBS.
  3. Resuspend cells in fresh lysis buffer with concentration of 107 cells/mL. If it is necessary, the cells could be subjected to ultrasonication till the solution is clarified.
  4. Centrifuge at 1,500×g for 10 minutes at 2-8oC to remove cellular debris. Assay immediately or aliquot and store at ≤-20oC.

Urine - Collect the first urine of the day (mid-stream) and discharge it directly into a sterile container. Centrifuge to remove particulate matter, assay immediately or aliquot and store at ≤ -20°C. Avoid repeated freeze-thaw cycles.

Saliva - Collect saliva using a collection device or equivalent. Centrifuge samples at 1000 × g at 2-8°Cfor15 minutes. Remove particulates and assay immediately or store samples in aliquot at ≤ -20°C. Avoid repeated freeze-thaw cycles.

Feces - Dry feces were collected as much as possible, weighing more than 50 mg. The feces were washed three times with PBS (w:v = 1:9, e.g. 900 µL lysis buffer is added in 100 mg feces), sonicated(or mashed)and centrifuged at 5000×g for 10 minutes, where the supernatant was collected for testing.

Cell culture supernates and other biological fluids - Centrifuge samples for 20 minutes at 1,000×g. Collect the supernates and assay immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.

Note

  1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and contamination.
  2. Sample hemolysis will influence the result, so hemolytic specimen should not be used.
  3. When performing the assay, bring samples to room temperature.
  4. It is highly recommended to use serum instead of plasma for the detection based on quantity of our in-house data.

Reagent Preparation

  • Ensure all kit components and samples are brought to room temperature (18-25°C) before use. Thoroughly dissolve and mix all components before utilizing the kit.
  • If the entire kit will not be used at once, only remove the necessary strips and reagents for the current experiment. Store the remaining strips and reagents as directed.
  • Dilute the 25× Wash Buffer into 1× Wash Buffer with double distilled water.
Standard Working Solution

First, centrifuge the Standard at 1000 x g for 1 minute. Then, reconstitute the Standard by adding 1.0 ml of Standard Diluent Buffer. Allow the mixture to sit for approximately 10 minutes at room temperature, and gently shake it (avoiding foaming).

Prepare 7 tubes with 0.5 ml of Standard Diluent Buffer in each and create a series of dilutions using the Diluted Standard. Remember to thoroughly mix each tube before transferring the solution to the next tube by pipetting the solution up and down several times. Set up 7 points of Diluted Standard, decreasing the concentration each time, and use a Blank with no standard for comparison. It's important to use a new Standard Solution for each experiment to ensure accurate results. When diluting the Standard from high concentration to low concentration, replace the pipette tip for each dilution. Additionally, make sure not to transfer solution into the Blank tube from the previous one.

Tube 6 5 4 3 2 1 0
ng/mL 40 20 10 5 2.5 1.25 0
  • Before using the stock Biotinylated Antibody and Streptavidin-HRP, give them a quick spin. Dilute them to the working concentration 100-fold with Biotinylated Antibody Diluent and HRP Diluent.
  • When using the TMB Substrate Solution, be sure to aspirate the required amount with sterilized tips and avoid returning any leftover solution to the vial.

Assay Procedure

  1. Prepare the micro ELISA plate by allocating wells for the Diluted Standard, Blank, and Sample. Allocate 7 wells for the Standard and 1 well for the Blank.
  2. Add 50 μL of Standard Working Solution or Sample to each well (Blank will use Standard Diluent). Immediately add 50 μL of Biotinylated-Conjugate (1x) to each well, mix well, cover with the Plate Cover, and incubate for 1 hour at 37°C. When adding solutions, be careful to avoid touching the inside wall and causing foaming.
  3. Pour out the liquid from each well, then wash by adding 200 μL of 1x Wash Solution to each well and letting it sit for 1-2 minutes. After decanting the liquid, remove the remaining liquid from all wells by placing the plate onto absorbent paper. Repeat this process 3 times to ensure complete removal of liquid at each step. After the last wash, invert the plate and blot it against clean paper towels to remove excess liquid.
  4. Add 100 μL of Streptavidin-HRP Working Solution (1x) to each well, cover with the Plate Cover, and incubate at 37°C for 60 minutes.
  5. Repeat the aspiration and wash process a total of 5 times as conducted in step 3.
  6. Add 90 μL of TMB Substrate Solution to each well, cover with a new Plate Cover, and incubate for 20 minutes at 37°C (do not exceed 30 minutes) in the dark. The liquid will turn blue by the addition of TMB Substrate Solution. Preheat the Microplate Reader for about 15 minutes before OD measurement and avoid placing the plate in direct light.
  7. Add 50 μL of Stop Reagent to each well. The liquid will turn yellow by the addition of Stop Reagent. Mix the liquid by tapping the side of the plate. The insertion order of the Stop Reagent should be the same as that of the TMB Substrate Solution.
  8. Run the microplate reader and conduct measurements at 450 nm immediately.

Troubleshoot Common Elisa Issues

Pipetting and washing techniques for ELISA
Pipetting technique
  1. Use the correct pipette that is within the range suggested by manufacturer
  2. Confirm tip is firmly seated on the pipette
  3. Confirm there are no air bubbles while pipetting
  4. Change tips between each standard, sample, or reagent
  5. Use different reservoirs for each reagent
  6. Pipette sample into the side of wells to avoid splashing
  7. Always run samples/standards in replicate
Washing Procedure
  1. Completely aspirate liquid from all wells by gently lowering an aspiration tip into the bottom of each well.
    Note: Take care not to scratch the inside of the well.
  2. Fill the wells with at least 500 µL of diluted wash buffer
  3. Let soak for 25 to 30 seconds
  4. Aspirate wash buffer from wells
  5. Repeat as directed in protocol (usually 4-5 times)
  6. After washing is complete, invert plate and tap (forcefully, if necessary) dry on absorbent tissue. Be sure to remove any residual liquid.
  7. Alternatively, a squirt bottle or automated plate washer may be used. Be sure to follow the above.
Problem: No color plate
Possible Cause Solution
Mixed use of component reagents Please read labels clearly when preparing or using
In the process of plate washing and sample addition, the enzyme marker is contaminated and inactivated, and loses its ability to catalyze the color developing agent Confirm that the container holding the ELISA plate does not contain enzyme inhibitors (such as NaN3, etc.), and confirm that the container for preparing the Wash Solution has been washed.
Missing a reagent or a step Review the manual in detail and strictly follow the operating steps.
Problem: Light Color
Possible Cause Solution
The sample uses NaN3 preservative, which inhibits the reaction of the enzyme Samples cannot use NaN3
The sample to be tested may not contain strong positive samples, so the result may be normal In case of doubt, please test again.
Wrong filter used for absorbance reading When TMB is used as the substrate, the absorbance should be read at 450 nm.
Insufficient incubation time Make sure to follow recommended incubation times.
Insufficient color reaction Usually 15 - 30 minutes
The number of washings increases, and the dilution ratio of the concentrated lotion does not meet the requirements Reduce the impact of washing, dilute the concentrated lotion and washing time according to the manual, and accurately record the washing times and dosage.
Distilled water quality problem The prepared lotion must be tested to see if the pH value is neutral.
In the process of plate washing and sample addition, the enzyme marker is contaminated and inactivated, and loses its ability to catalyze the color developing agent. Confirm that the container holding the ELISA plate does not contain enzyme inhibitors (such as NaN3, etc.), confirm that the container for preparing the Washing Solution has been washed, and confirm that the purified water for preparing the Washing Solution meets the requirements and is not contaminated.
The kit has expired or been improperly stored Please use it within the expiration and store it in accordance with the storage conditions recommended in the manual to avoid contamination.
Reagents and samples are not equilibrated before use All reagents and samples should be equilibrated at room temperature for about 30 minutes.
Insufficient suction volume of the pipette, too fast discharge of pipetting suction, too much liquid hanging on the inner wall of the tip or the inner wall is not clean. To calibrate the pipette, the tips should be matched, each time the tips should fit tightly, the pipetting should not be too fast, and the discharge should be complete. The inner wall of the tips should be clean, and it is best to use it once.
Incubation temperature constant temperature effect is not good Keep the temperature constant to avoid the local temperature being too high or too low
When adding liquid, too much remains on the medial wall of wells When adding liquid, the tip should try to add liquid along the bottom of the medial wall of wells without touching the bottom of the hole.
Reuse of consumables The tips should be replaced when different reagents are drawn, and different storage vessels should be used when configuring different reagent components.
The bottom of the microwell is scratched or there is dirt During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.
Cross-contamination during sample addition Try to avoid cross-contamination when adding samples
Cross-contamination from manual plate washing When washing the plates by hand, the first 3 injections of the lotion should be discarded immediately, and the soaking time should be set for the next few times to reduce cross-contamination.
Cross-contamination when clapping Use a suitable absorbent paper towel when clapping the plate, do not pat irrelevant substances into the well of the plate, and try not to pat in the same position to avoid cross-contamination.
The liquid filling head of the plate washer is blocked, resulting in unsatisfactory liquid addition or large residual amount of liquid suction, resulting in the color of plate is chaotic and irregular. Unblock the liquid addition head, so that each well is filled with washing liquid when washing the plate and the residual amount should be small when aspirating liquid.
Incomplete centrifugation of the sample, resulting in coagulation in the reaction well or interference of sediment or residual cellular components Serum plasma should be fully centrifuged at 3000 rpm for more than 6 minutes
The sample is stored for too long time, resulting in contamination. Samples should be kept fresh or stored at low temperature to prevent contamination
Incorrect preparation of Washing Solution or direct misuse of concentrated Washing Solution Please configure according to the manual
Weak or no signal in ELISA
Possible Cause Solution
Reagents not at room temperature at start of assay It is recommended that all reagents be at room temperature before starting the assay. Allow reagents to sit on bench for 15–20 minutes to reach room temperature.
Incorrect storage of components Double check storage conditions on kit label. Most kits need to be stored at 2–8oC.
Expired reagents Confirm expiration dates on all reagents. Do not use reagents that are past the expiration date.
Reagents added/prepared incorrectly Check protocol, ensure reagents were added in the proper order and prepared to correct dilution.
Incorrect dilutions prepared Check pipetting technique—see below—and double check calculations.
Capture antibody didn’t bind to plate If using a ready-to-use kit: Manufactured kits come with plates pre-coated with capture antibody.
If coating your own plate with an Antibody Pair Kit: Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps.
Wells scratched with pipette or washing tips Use caution when dispensing and aspirating into and out of wells. Automated plate washers may need to be calibrated so tips don’t touch bottom of wells.
Plate read at incorrect wavelength Make sure to use recommended wavelength/filter.
Too much signal in ELISA
Possible Cause Solution
Insufficient washing Use appropriate washing procedure—see below. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Plate sealers not used or reused During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.
Incorrect dilutions prepared Check pipetting technique—see below—and double-check calculations.
Longer incubation times thanrecommended Make sure to follow recommended incubation times.
Problem: High background in ELISA
Possible Cause Solution
Insufficient washing Use appropriate washing procedure—see below. Increasing duration of soak steps may also help. Add 30 seconds each time you let wash buffer soak. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Substrateexposed to light prior to use Ensure substrate isnot exposed to light—store in a dark place. Limit exposure to light whilerunning assay.
The yellowingof the whole plate may be caused by wrong addition of other reagents Check the componentsand lot numbers of the reagents before the experiment, and confirm that allcomponents belong to the corresponding kit. Reagents from differentkits or different lot numbers cannot be mixed.
Streptavidin-HRP contaminates the tip and TMB container orpositive control contaminates the Pre-coated Microplate When absorbingdifferent reagents, the tips should be replaced. When configuring differentreagent components, different storage vessels should be used. Please use apipette during operation.
BiotinylatedAntibody or Streptavidin-HRP concentration too high Checkwhether the concentration calculation is correct or use after furtherdilution.
Color development time is too long Pleasestrictly follow the steps of the manual.
Longerincubation times than recommended Make sure to followrecommended incubation times.
Incorrectstandard curve dilutions prepared Check pipettingtechnique—see below—and double-check calculations.
The wrongfilter was used when the absorbance value was read When TMB is used asthe substrate, the absorbance should be read at 450 nm.
Problem: High background in ELISA
Possible Cause Solution
Incorrect standard curve dilutions prepared Check pipetting technique—see below—and double-check calculations.
Capture antibody didn’t bind to plate Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps.
Problem: Poor replicate data in ELISA
Possible Cause Solution
Insufficient washing Use appropriate washing procedure—see below. Increasing duration of soak steps may also help. Add 30 seconds each time you let wash buffer soak. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Capture antibody didn’t bind to plate Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps.
Plate sealers not used or reused During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.
Problem: Inconsistent results assay-to-assay in ELISA
Possible Cause Solution
Insufficient washing Use appropriate washing procedure—see below. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Inconsistent incubation temperature Manufactured kits have optimized protocols. Make sure to follow recommended incubation temperatures. Be aware of fluctuations in temperature due to environmental conditions.
Plate sealers not used or reused During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.
Incorrect dilutions prepared Check pipetting technique—see below—and double-check calculations.

Product Information

SKU:EK248919
Category:

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