Indications d'application |
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Commentaires |
Information on standard material: |
Volume d'échantillon | 100 μL |
Durée du test | 3 h |
Plaque | Pre-coated |
Protocole | The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Heat Shock Protein 90 kDa Beta 1 (HSP90b1). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Heat Shock Protein 90 kDa Beta 1 (HSP90b1). Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Heat Shock Protein 90 kDa Beta 1 (HSP90b1), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Heat Shock Protein 90 kDa Beta 1 (HSP90b1) in the samples is then determined by comparing the O.D. of the samples to the standard curve. |
Préparation des réactifs |
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Prélèvement de l'échantillon |
Serum: Allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000 × g. Assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles. Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles. Tissue Homogenates: The preparation of tissue homogenates will vary depending upon tissue type. For this assay, rinse tissues in ice-cold PBS (0.02mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weigh before homogenization. Mince the tissues to small pieces and homogenize them in 5-10 mL of PBS with a glass homogenizer on ice (Micro Tissue Grinders work, too). Sonicate the resulting suspension with an ultrasonic cell disrupter or subject it to two freeze-thaw cycles to further break the cell membranes. Centrifugate the homogenates for 5 minutes at 5000 × g. Remove the supernate and assay immediately or aliquot and store at -20°C Biological Fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles. |
Préparation de l'échantillon |
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Procédure de l'essai |
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Calcul des résultats |
Average the duplicate readings for each standard, control, and samples and subtract the average zero standard optical density. Create a standard curve on log-log graph paper, with HSP90b1 concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the standard points and it can be determined by regression analysis. Using some plot software, for instance, curve expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. |
Précision du teste |
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Heat Shock Protein 90kDa Beta 1 (HSP90b1) were tested 20 times on one plate, respectively. |
Restrictions | For Research Use only |
Précaution d'utilisation | The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. |
Conseil sur la manipulation |
The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5 % within the expiration date under appropriate storage condition. To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end. |
Stock | 4 °C |
Stockage commentaire |
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Date de péremption | 6 months |
Images (Fournisseur) |