Neuromethods DOI 10.1007/7657_2018_13 © Springer Science+Business Media New York 2018 Detecting BDNF Protein Forms by ELISA, Western Blot, and Immunofluorescence Stefano Donega` and Enrico Tongiorgi Abstract The neurotrophin brain-derived neurotrophic factor (BDNF) is extensively expressed in neural cells and other cell types. BDNF displays a support function in both central CNS and peripheral PNS where it is involved in most neuronal mechanisms, from development to neurodegeneration, cellular growth, neuro- protection, survival, and network signaling. In the last decades, decreased levels of BDNF were directly linked to Parkinson’s, Alzheimer’s, and Huntington’s disease, while BDNF upregulation was found in patients affected by epilepsy, autism, and multiple sclerosis. Accordingly, determination of BDNF expression can contribute to gain insight in several pathological models both in vivo and in vitro. In this manuscript, we provide detailed description of the main techniques for BDNF detection in cultured neurons and in brain slices, using different methods such as ELISA, Western blot, and immunofluorescence. Keywords BDNF pro-peptide, Brain-derived neurotrophic factor, Mature BDNF, Neurotrophins, proBDNF, Proteolytic processing, Truncated BDNF 1 Introduction 1.1 The Different Protein Forms of BDNF and Their Proteolytic Cleavage The neurotrophin brain-derived neurotrophic factor (BDNF) was discovered in 1982, focusing on brain extracts with neurite- promoting effects on the nodose ganglion [1]. It is present in various body districts under four different protein forms: proBDNF, mature BDNF, truncated BDNF, and the BDNF pro-peptide [2]. Similar to other members of the neurotrophin protein family, BDNF is initially synthesized as a precursor protein (preproBDNF) that is structurally composed of a signal sequence, a pro-domain, and the mature domain [3]. Mammalian preproBDNF is a protein of 248 amino acids having a molecular weight of 32 kDa that is N-glycosylated at one residue in the proBDNF domain. After cleavage of the signal peptide (pre-region) in the endoplasmic retic- ulum, proBDNF can be further processed to generate either a truncated BDNF form of 28 kDa (truncBDNF) or the C-terminal mature BDNF protein of 14 kDa BDNF (mBDNF) together with the N-terminal BDNF pro-peptide (pBDNF) of about the same molecular weight [2]. Production of these BDNF forms depends upon two different proteolytic cleavages. Cleavage at the