Abstract
The current protocol details the preparation of soluble and insoluble protein lysates from mouse brain or spinal cord samples. In detail, tissue homogenization and sequential protein extraction are described. This procedure yields soluble and insoluble protein extracts that can be further processed in down-stream applications like ELISA or Western blotting.
Keywords: Protein extraction, Homogenization, Soluble fraction, SDS, Brain
Background
This simple and reproducible protocol of brain tissue protein fractionation details the initial separation of a total protein homogenate into a soluble and an insoluble fraction. It can also be applied also to other tissue samples and yields a soluble fraction containing hydrophilic proteins and an insoluble fraction consisting of more hydrophobic proteins. Following an initial homogenization in a lysis buffer containing no detergent, the supernatant including the soluble protein fraction is removed and the pellet containing the insoluble fraction can be further extracted using Sodium Dodecyl Sulfate (SDS) as detergent to ensure entire cell lysis (see Figure 1). This approach can facilitate the analysis of low-abundance proteins by reducing the complexity of the sample. Figure 1. Flow-chart describing the sequential extraction procedure
Materials and Reagents
Equipment
Procedure
Notes:
Data analysis
Protein fractions obtained by this protocol can be further used in downstream applications like Western blot or enzyme-linked immunosorbent assays (ELISA) (for an example of potential further use in Western blots see Härtig et al., 2014; Saul and Wirths, 2017).
Recipes
Acknowledgments
Financial support from the Alzheimer Forschung Initiative e.V. is gratefully acknowledged.
References
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Dear Niti Sharma,I apologize but we have no data on whether the indicated protocol works for the estimation of antioxidant enzymes as this is something we have never tested.Kind regards,Oliver Wirths