Active BDNF (Human, Rat) ELISA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Bring all reagents to room temperature before use.	- If crystals have formed in the Wash Buffer concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. -Streptavidin-HRP Conjugate and Substrate solution protect from light.

Upstream tips
-Bring all reagents to room temperature before use.
Protocol tips
- If crystals have formed in the Wash Buffer concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. -Streptavidin-HRP Conjugate and Substrate solution protect from light.

Publication protocol

BDNF levels were also quantified using a sandwich ELISA kit (Aviscera Bioscience, SK00752-
01) that has been validated to show good selectivity for mature BDNF over proBDNF (Polacchini et al.,
2015). Within each experiment, the plates used were from the same lot and were run simultaneously.
Samples were assayed in duplicate (protein loaded per well: 500 µg for hippocampus, 800 µg for
neocortex for non-acid treated samples; 200 µg for hippocampus, 400 µg for neocortex for acid treated
samples) following the provided instructions. Briefly, samples were incubated for 2 h in a 96-well plate
precoated with a monoclonal antibody against BDNF. The plate was then incubated for 2 h with the
biotinylated detection antibody, followed by a 1 h incubation with streptavidin-HRP conjugate.
Tetramethylbenzidine (TMB) substrate solution was added, and colour was developed for 10-18 min
(depending on the experiment) before addition of the stop solution (0.5 M HCl). All incubations were
conducted at room temperature on an orbital shaker. Absorbance was measured at 450 nm using a
microplate reader (Wallac 1420 Victor2
, Perkin Elmer Life Sciences; or FilterMax F5 Multi-Mode
Microplate Reader, Molecular Devices, depending on the experiment). BDNF concentrations were
determined based on a standard curve and converted to pg of BDNF per mg of total protein.

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Manufacturer protocol

Download the product protocol from Aviscera Bioscience for Active BDNF (Human, Rat) ELISA Kit below.

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