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(1) Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged. (2) ELISA Wash Concentrate must be diluted to working solution prior use. Please see REAGENTS section for details. (3) Reconstitute all assay standards and controls by adding 0.5 mL of distilled or deminerialized water to each vial. Allow the standards and controls to sit undisturbed for 5 minutes, and then mix well by inversions or gentle vortexing. Make sure that all solid is dissolved completely prior to use. These reconstituted standards and controls must be stored at - 18 C or below. Do not exceed 3 freeze-thaw cycles.
(1) Place a sufficient number of streptavidin-coated microwell strips (Cat. 10040B) in a holder to run human osteocalcin standards, controls and unknown samples in duplicate. The unused strips should be resealed in the bag with a desiccant and stored at 2-8 °C. (2) Test Configuration (3) Prepare working HRP conjugated Osteocalcin Antibody and Biotinylated Osteocalcin Antibody by 1:21 fold dilution of the conjugation antibody with the biotinylated antibody solution. Following is a table that outlines the relationship of strips used and antibody mix prepared. (4) Add 25 µL of standards, controls and patient serum/plasma samples into the designated microwell. (5) Add 200 µL of above antibody mixture to each well (6) Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light. (7) Incubate the plate at room temperature, shaking 350 rpm 100 rpm for 1 hour. (8) Remove the aluminum foil and plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL - 400 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used. (9) Add 200 µL of ELISA HRP Substrate into each of the wells. (10) Cover the plate with one new plate sealer and also with aluminum foil to avoid exposure to light. (11) Incubate plate at room temperature static for 20 minutes. (This incubation period may be reduced to 8 ? 15 min if a lower OD reading is demanded to fit to the plate readers specification.) (12) Remove the aluminum foil and plate sealer. Add 50 µL of ELISA Stop Solution into each of the wells. Mix gently. (13) Read the absorbance at 450 nm within 10 minutes in a microplate reader. NOTE: in case extremely low background is required, one can set the instrument to dual wavelength measurement at 450 nm with background wavelength correction set at 595 nm, 620 nm or 630 nm.