Ann Short Rep | Volume 2, Issue 3 | Research Article | Open Access
Denis Rousseau*
Applied and Fundamental Bioenergetic Laboratory, Grenoble Alpes University, France
*Correspondance to: Denis Rousseau
Fulltext PDFWestern-blot analysis is a very powerful and widely used technic for protein analysis and quantification. However, most of the sample preparation procedures involved a centrifugation step after lysis, in order to remove insoluble materials. These pellets are insoluble DNA/chromatin complexes and proteins, like integral membrane ones, or tightly chromatin-bound transcription and replication factors. Therefore, discarded as pellets, these proteins are rarely considered and analyzed, generating indeed several artefacts. We present here a simple technic to extract and analyze total proteins by western-blot, from cell sample or tissues. This technic simply involves the lysis of the sample in SDS 1%/NaCl 0.5M/Tris 10 mM pH 7.5, at room temperature with protease inhibitors, and the direct protein dosage and western-blot analysis. As an example, we analyzed the quality of the technic by detecting ATAD3, a mitochondrial integral membrane protein which is mainly insoluble in 1% triton/0.5M NaCl. This technic avoids therefore all the possibilities to lose part or all of the target protein because of the high level of lysis/solubilization and the suppression of clarifying centrifugation step. Also, limitations of the procedure are discussed.
Rousseau D. A Simplified Technique for Western-Blot Analysis of Whole-Cell or -Tissue Protein Extracts. Ann Short Reports. 2019;2:1041 .