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Protein extraction for BDNF detection with ELISA

RJ Robin Ji
MS Morgan Smith
YN Yusuke Niimi
MK Maria E. Karakatsani
MM Maria F. Murillo
VJ Vernice Jackson-Lewis
SP Serge Przedborski
EK Elisa E. Konofagou
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Frozen brains sections were weighed and 10 mL per 1 g of tissue lysis buffer was added to each. Lysis buffer consisted of T-PER solution (Thermo Fisher Scientific, Waltham, MA, USA) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and 10uL of inhibitors per 1 mL of lysis buffer. Samples were homogenized in an ice bath in intervals of 30 seconds, with 10 seconds cooling periods to prevent tissue heating. Homogenates were then centrifuged at 14,000 g for 30 min in 4 °C, supernatant was then recovered and stored at −80 °C until used for ELISA. Before the ELISA was carried out, the total protein concentration was determined by Bradford assay. The BDNF measurement was carried out using the Abcam Human BDNF ELISA kit following the manufacturer’s protocol (ab99978).

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