4.2 Article

Cost-effective and rapid lysis of Saccharomyces cerevisiae cells for quantitative western blot analysis of proteins, including phosphorylated eIF2

Journal

YEAST
Volume 34, Issue 9, Pages 371-382

Publisher

WILEY
DOI: 10.1002/yea.3239

Keywords

Saccharomyces cerevisiae; cell lysis; immunoblotting; time course experiment; protein kinase; Gcn2

Funding

  1. Health Research Council of New Zealand Emerging Researcher grant
  2. Auckland Medical Research Foundation
  3. Maurice and Phyllis Paykel Trust
  4. Massey University Research Fund
  5. Marsden Fund Council [MAU0607]
  6. CNPq [153660/2010-4]
  7. CAPES-PSDE [1915-13-4]
  8. Massey University doctoral scholarship
  9. Institute of Natural and Mathematical Sciences

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The common method for liberating proteins from Saccharomyces cerevisiae cells involves mechanical cell disruption using glass beads and buffer containing inhibitors (protease, phosphatase and/or kinase inhibitors), followed by centrifugation to remove cell debris. This procedure requires the use of costly inhibitors and is laborious, in particular when many samples need to be processed. Also, enzymatic reactions can still occur during harvesting and cell breakage. As a result low-abundance and labile proteins may be degraded, and enzymes such as kinases and phosphatases may still modify proteins during and after cell lysis. We believe that our rapid sample preparation method helps overcome the above issues and offers the following advantages: (a) it is cost-effective, as no inhibitors and breaking buffer are needed; (b) cell breakage is fast (about 15min) since it only involves a few steps; (c) the use of formaldehyde inactivates endogenous proteases prior to cell lysis, dramatically reducing the risk of protein degradation; (d) centrifugation steps only occur prior to cell lysis, circumventing the problem of losing protein complexes, in particular if cells were treated with formaldehyde intended to stabilize and capture large protein complexes; and (e) since formaldehyde has the potential to instantly terminate protein activity, this method also allows the study of enzymes in live cells, i.e. in their true physiological environment, such as the short-term effect of a drug on enzyme activity. Taken together, the rapid sample preparation procedure provides a more accurate snapshot of the cell's protein content at the time of harvesting. Copyright (c) 2017 John Wiley & Sons, Ltd.

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