Alpha Synuclein (α-synuclein) is highly expressed in different regions of the brain and found concentrated in neuronal synapses and nuclei (hence its name “synuclein”!). Quantification of α-synuclein in tissue extracts can be used to investigate its patho-physiological role1 but published literature does not always agree on the actual α-synuclein levels (in fact, this is true for almost any other protein!). While the choice of a reliable ELISA kit manufacturer is crucial, a thorough understanding of sample preparation is equally important to draw accurate experimental conclusions (and “make sense” of published data”).
The Choice of an Extraction Buffer Makes a Difference!
Tissue extraction buffers can differ in their ability to solubilize the target protein, disrupt protein-protein interactions and “concentrate” certain cellular sub-structures. For example, detergents such as Triton-X100 may help to release the target protein from binding partners, thus making it available for detection. At the same time, detergents solubilize hydrophobic cell membranes, resulting in higher total extracted protein, potentially causing a “dilution” of the target protein with irrelevant proteins. As a result, detection levels and target protein concentration may vary.
Empirical testing is often needed to address the choice of extraction buffer(s). The following case study exemplifies this. Two standard extraction buffers (neutral TRIS buffer without detergents vs. RIPA buffer containing Triton-X100) were compared for quantification of α-synuclein in whole rat brain tissue after extraction with a bead homogenizer (Table 1).