Heme Oxygenase (Decycling) 1 (HMOX1) ELISA Kit

Antigen: Heme Oxygenase (Decycling) 1 (HMOX1)

Synonyme: HO1, HO-1, Hmox, Hemox, Hsp32, D8Wsu38e, Ho1, Heox, Ho-1, HEOXG, hsp32, HMOX1, MGC132176, zgc:65984, ARABIDOPSIS THALIANA HEME OXYGENASE 1, ATHO1, F18A8.4, F18A8_4, GENOMES UNCOUPLED 2, GUN2, HEME OXYGENASE, HEME OXYGENASE 1, HEME OXYGENASE 6, HY6, PLASTID HEME OXYGENASE, REVERSAL OF THE DET PHENOTYPE 4, TED4, Hmox1, DKFZp469G141

Reaktivität: Hamster

Applikation: ELISA

Zertifikate: ISO 9001:2008

Produktnummer: ABIN577614

Produktbeschreibung

Weitere Bezeichnung: heme oxygenase 1 (HO-1)

Proben: Serum, Plasma

Spezifität: This assay recognizes recombinant and natural hamster HO-1. No significant cross-reactivity or interference was observed.

Sensitivität: The minimum detectable dose of hamster HO-1 is typically less than 0.16 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. Detection Range: The minimum detectable dose of hamster HO-1 is typically less than 0.16 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

Synonyme: HO1, HO-1, Hmox, Hemox, Hsp32, D8Wsu38e, Ho1, Heox, Ho-1, HEOXG, hsp32, HMOX1, MGC132176, zgc:65984, ARABIDOPSIS THALIANA HEME OXYGENASE 1, ATHO1, F18A8.4, F18A8_4, GENOMES UNCOUPLED 2, GUN2, HEME OXYGENASE, HEME OXYGENASE 1, HEME OXYGENASE 6, HY6, PLASTID HEME OXYGENASE, REVERSAL OF THE DET PHENOTYPE 4, TED4, Hmox1, DKFZp469G141

Anwendungen

Prinzip: The microtiter plate provided in this kit has been pre-coated with an antibody specific to HO-1. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for HO-1 and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain HO-1, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of HO-1 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protokoll: Reagent Preparation:
Bring all reagents to room temperature before use for 30min. 1. Wash Buffer If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. 2. Biotin-antibody Centrifuge the vial before opening. Dilute to the working concentration using Biotin-antibody Diluent(1:100), respectively. The suggested 100-fold dilution can be achieved by adding 10 uL sample to 990uL of Biotin-antibody Diluent for 1ml working solution. 3. HRP-avidin Centrifuge the vial before opening. Dilute to the working concentration using HRP-avidin Diluent(1:100), respectively. The suggested 100-fold dilution can be achieved by adding 10 uL sample to 990uL of HRP-avidin Diluent for 1ml working solution. 5 4. Standard Centrifuge the standard vial at 6000-10000rpm for 30s. Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 40 ng/ml. Allow the standard to sit for a minimum of 15 minutes with gentle and uniform agitation by pipette with 1ml measuring range prior to making serial dilutions. The undiluted standard serves as the high standard (40 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml). Prepare fresh for each assay. Use within 4 hours and discard after use. Standard S7 S6 S5 S4 S3 S2 S1 Concentration (ng/ml) 40 20 10 5 2.5 1.25 0.625 Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. 6.
Assay Procedure:
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. All the reagents should be added directly to the liquid level in the well. The pipette should avoid contacting the inner wall of the well. 1. Add 100μl of Standard, Blank, or Sample per well. Cover with the adhesive strip. Incubate for 2 hours at 37°C. 2. Remove the liquid of each well, don’t wash. 3. Add 100μl of Biotin-antibody working solution to each well. Incubate for 1 hour at 37°C. Biotin-antibody working solution may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform. 4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash: Fill each well with Wash Buffer (200μl) a nd let it stand for 2 minutes, then remove the liquid by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel. Complete removal of liquid at each step is essential to good performance. 8 5. Add 100μl of HRP-avidin working solution to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C. 6. Repeat the aspiration and wash five times as step 4. 7. Add 90μl of TMB Substrate to each well. Incubate for 10-30 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 8. Add 50μl of Stop Solution to each well when the first four wells containing the highest concentration of standards develop obvious blue color. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 9. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Calculation of Results:
Using the professional soft ",Curve Exert 1.3", to make a standard curve is recommended, which can be downloaded from our web. Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the 9 points on the graph. The data may be linearized by plotting the log of the HO-1 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Limitations of the Procedure:
The kit should not be used beyond the expiration date on the kit label.Do not mix or substitute reagents with those from other lots or sources.It is important that the Standard Diluent selected for the standard curve be consistent with the samples being assayed.If samples generate values higher than the highest standard, dilute the samples with the appropriate Standard Diluent and repeat the assay.Any variation in Standard Diluent, operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding.This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded. 10.
Sample collection and storage:
Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum and assay immediately or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles. Cell Culture Supernates Remove particulates by centrifugation for 5min at 2000 rpm. Assay immediately or 7 aliquot and store samples at -20° C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles. Note: Grossly hemolyzed samples are not suitable for use in this assay.

Testdurchführung: Assay Time: 1-3h
Sample Volume: 50-100ul

Applikationshinweise: Detection Wavelength: 450 nm

Bestandteile: Reagent (Quantity): Biotin-antibody (1 x120µl), HRP-avidin (1 x120µl), Sample Diluent (1x20ml), Biotin-antibody Diluent (1x10ml), HRP-avidin Diluent (1x10ml), Wash Buffer (25×concentrate)(1x20ml), TMB Substrate (1x10ml), Stop Solution (1x10ml)

Benötigtes Material: Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer. An incubator which can provide stable incubation conditions up to 37°C±0.5°C.

Lagerung: Storage Unopened kit Store at 2 - 8° C. Do not use beyond kit expiration date. Opened kit May be stored for up to 1 month at 2 - 8° C. Try to keep it in a sealed aluminum foil bag,and avoid the damp May be stored for up to 1 month at 2 - 8° C. If don’t make recent use, better keep it store at -20 C . May be stored for up to 1 month at 2 - 8° C.

Beschränkungen: Nur für Forschungszwecke einsetzbar