Mouse/Rat Osteopontin (OPN) Quantikine ELISA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Store the unopened kit at 2-8 °C. -Bring all reagents to room temperature before use. -Reconstitute the Human Cytochrome c Standard with deionized or distilled water. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.	-When mixing or reconstituting protein solutions, always avoid foaming. -Substrate Solution should remain colorless until added to the plate. -Keep Substrate solution protected from light. -Stop Solution should be added to the plate in the same order as the Substrate Solution. -Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.

Upstream tips
-Store the unopened kit at 2-8 °C. -Bring all reagents to room temperature before use. -Reconstitute the Human Cytochrome c Standard with deionized or distilled water. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.
Protocol tips
-When mixing or reconstituting protein solutions, always avoid foaming. -Substrate Solution should remain colorless until added to the plate. -Keep Substrate solution protected from light. -Stop Solution should be added to the plate in the same order as the Substrate Solution. -Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.

Publication protocol

Secreted OPN was evaluated in the media from BMF cells infected or non-infected with Leishmania amazonensis parasites by a quantitative sandwich enzyme immunoassay method using the Quantikine mouse osteopontin ELISA kit (R&D Systems, Cat N° MOST00) as described by the manufacturer. Briefly, a polyclonal mouse OPN specific antibody is pre-coated onto a microplate. Samples, standards and controls are added into the wells and mouse OPN present is bound on the immobilized antibody. Following 2 h incubation at room temperature, unbound substances are washed and an enzyme-linked mouse OPN specific polyclonal antibody is added to the wells and incubated for 2 h at room temperature. After removing any unbound antibody-enzyme reagent by five series of washes, a substrate solution is added to the wells and incubated for 30 min at room temperature in the dark. The enzyme reaction yields a blue color that turns yellow after adding the STOP solution. The color intensity corresponding to the amount of mouse OPN bound in the initial step was read at OD 450 with a correction wavelength set at 570 nm in an absorbance microplate Reader ELx800™. Standard curve was prepared by reconstitution of mouse OPN Standard (2500 pg/ml) following by serial dilutions at 8 concentrations in total, from 2500 pg/ml to 39 pg/ml, and 0 pg/ml. Samples were serially diluted to 1:3, 1:9, 1:27, to 1:81 in the buffer provided in the kit. All buffers used are as described and supplied in the kit (https://www.rndsystems.com/products/mouse-rat-osteopontin-opn-quantikine-elisa-kit_most00).

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Manufacturer protocol

Download the product protocol from R&D Systems for Mouse/Rat Osteopontin (OPN) Quantikine ELISA Kit below.

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