McClean:Western Blot

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Contents

Overview

This is a protocol to perform rapid yeast protein prep for SDS PAGE and Western.

Materials

  • Beta- mercaptoethanol
  • 4X LDS sample buffer(Invitrogen NP0008)
  • Protease inhibitor(Roche catlog number: 11836170001)
  • Phosphatase inhibitor(Fisher catlog number: 78420)
  • 20X NuPAGE running buffer(Invitrogen NP0001)
  • Prestained Protein Ladder(Invitrogen catlog number:10748-010)
  • Magic Marker(Invitrogen catlog number:LC5602)
  • NuPage pre-cast gel
  • PVDF membrane(Invitrogen catlog number:LC2005)
  • Methanol
  • 20X NuPAGE buffer(Invitrogen NP0006)
  • 1M Tris, pH 8.0
  • 2.5M NaCl
  • Tween 20
  • Nonfat dry milk
  • Primary and secondary antibody

Stock Solutions

Stock Solution 1

  • 4X LDS sample buffer(Invitrogen NP0008)

Stock Solution 2

  • 20X NuPAGE running buffer(Invitrogen NP0001)

Stock Solution 3

  • 1M Tris, pH 8.0

Stock Solution 4

  • 2.5M NaCl

Protocol

Cell growth and protein extraction

  1. 1.Grow cells to mid-log (~1x107 cells/ml; A600 = 0.7 or klett 80) and collect 1.5 ml cells in 1.5 ml microfuge tube (1 minute, 14000xg). It is important not to grow the cells to a high density as this method will not work well.
  2. 2.Resuspend cells in 100 microliters sample buffer(Bring 4X sample buffer to 1X with BME(final concertration 10%), protease inhibitor, phosphatase inhibitor and water).
  3. 3.Heat at 95 deg C for 5 minutes.

Notes

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References

Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.

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