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Yeast protocols, molecular biology protocols, media and general protocols used by the Cell Cycle Group
     Yeast Protocols | Molecular Biology Protocols | Media | General Protocols

Post-alkaline Extraction for Western Time-course Experiments

Solutions

Sample buffer
– 0.06 M Tris-HCl pH 7.0
– 5% glycerol
– 2% SDS
– 4% ß-mercaptoethanol
– Few grains of bromophenol blue
Can be kept at room temperature but after a month check that ß-mercaptoethanol is okay by smelling buffer.


Procedure

  1. Collect 2.5 OD and spin down.
  2. Resuspend in 200 µl dH2O.
  3. Incubate 3 min at room temperature.
  4. Spin 15 s at 12000 rpm and discard supernatant.
  5. Resuspend in 70 µl sample buffer.
  6. Boil 3 min.
  7. Spin 15 s at 12000 rpm and retain supernatent in new tube (can be frozen and reboiled) or loaded immediately (cool for a bit, never chill).
  8. Load about 5 µl per lane.
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