Mouse Lipocalin-2/NGAL DuoSet ELISA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Bring all reagents to room temperature before use. Allow all components to sit for a minimum of 15 minutes with gentle agitation after initial reconstitution. -Working dilutions should be prepared and used immediately.	-A standard curve should be generated for each set of samples assayed. -It is suggested to start Reagent Diluent optimization for serum and plasma samples by using PBS supplemented with 10-50% animal serum. -Do not use buffers with animal serum to reconstitute or dilute the Detection Antibody or Streptavidin-HRP B.

Upstream tips
-Bring all reagents to room temperature before use. Allow all components to sit for a minimum of 15 minutes with gentle agitation after initial reconstitution. -Working dilutions should be prepared and used immediately.
Protocol tips
-A standard curve should be generated for each set of samples assayed. -It is suggested to start Reagent Diluent optimization for serum and plasma samples by using PBS supplemented with 10-50% animal serum. -Do not use buffers with animal serum to reconstitute or dilute the Detection Antibody or Streptavidin-HRP B.

Publication protocol

Ileal and colonic contents reconstituted in 100 mg/mL PBS (Gibco/Life Technologies) containing 0.1% Tween-20 (Sigma-Aldrich Corp.) were vortexed at high speed for 20 minutes at room temperature. Samples were centrifuged for 10 minutes at 13,800g (4°C). Clarified supernatants were collected and analyzed immediately or stored at −20°C. Lipocalin levels from supernatants were measured using Mouse Lipocalin-2/NGAL DuoSet enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN) using undiluted or diluted up to 160-fold in kit-recommended diluent (Gibco/Life Technologies) containing 1% BSA (Sigma-Aldrich Corp.). Plates were read on a Victor3 plate reader (PerkinElmer, Waltham, MA) at 450 nm.

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Manufacturer protocol

Download the product protocol from R&D Systems for Mouse Lipocalin-2/NGAL DuoSet ELISA below.

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