Human GDNF 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

For GDNF secretion, 10,000/well h7H-primary pituitary acromegaly (or NFPA) cells were seeded in a 96-well dish, collected for different time-lengths and kept at −80 °C. Secreted GDNF levels were measured with a Human GDNF DuoSet ELISA (DY212, R&D Systems, USA) according to the manufacturer's protocol. Briefly, a 98-well microplate was coated with the capture antibody and incubated overnight at room temperature. Wells were blocked for 1 h at room temperature and standards and culture medium samples added and incubated for 2 h at room temperature. Detection antibody was added during for 2 h at room temperature. Avoiding exposure to light, streptavidin-HRP was added for 2 min, followed by the substrate solution for a further 20 min. After termination of the enzyme reaction with the stop solution, the plate was read at A450/A550nm in a microplate reader (Mithras LB 940, Berthold, Germany).

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Manufacturer protocol

Download the product protocol from R&D Systems for Human GDNF DuoSet ELISA below.

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