Alpha Synuclein Quantification in Tissue Homogenates
Dear Researcher,

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).

Table 1: Alpha-synuclein concentrations in rat brain extracts obtained with the Biosensis Alpha-Synuclein RapidTM ELISA Kit (BEK-2216) using 2 extraction buffers.
*Total Protein was measured by Bradford

The results illustrate that RIPA buffer is more powerful for total protein extraction (9.1 vs. 2.2 mg/mL), but also for extracting α-synuclein (233.0 vs. 120.8 ng/mL). Both extraction buffers yield significantly different results in relation to α-synuclein concentration per extracted protein (25.7 vs. 54.4 ng/mg) and tissue weight (233 vs. 121 ng/g). This study clearly demonstrates why the choice of extraction buffer matters, and highlights the importance of comparing published literature with caution in relation to extraction buffer and reporting of results.
 
Biosensis takes pride in manufacturing high-quality ELISA kits. Our passion is to help the research community to “make sense” of experimental outcomes. Biosensis has an excellent Technical Support and a range of Technical Notes that will assist you with your research! Please visit our website or contact us to find out more about our products.
 
Good Luck with your research,
 
The Biosensis Team

Reference
  1. Chahine et al., Neurology. 2020 Sep 1;95(9):e1267-e1284. https://n.neurology.org/content/95/9/e1267
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