McClean: Fluorescent Folding Time Assay

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Contents

Overview

This is a protocol to measure the in vivo folding time of fluorescent proteins in yeast. The gene for the fluorescent protein must be behind a reliably inducible promoter. In this specific protocol yMM736 is used, which has GFP behind pSTL1 and tdTomato behind pFUS1. Before doing this experiment you need to know how soon after induction the earliest detectable amount of fluorescent protein is translated. To do this use the fluorescent protein start protocol.

Materials

  1. yMM736

Protocol

  1. Fill falcon tubes with 800 microL PBS + .1% tween, put on ice
  2. Grow up 25ml of yMM736 to mid-log phase in a klett flask.
  3. At t=0 minutes induce with 2.5 microL of 1 mg/ml alpha-factor for a concentration of 100 ng/ml alpha-factor (for sorbitol induce with 12.5 ml of 2M sorbitol in LFM for a concentration of 0.66M sorbitol).
  4. At t=10 minutes add cyclohexamide to a final concentration of 100 microg/ml cyclo (so 50 microL of 50 mg/ml cyclo to the 25 ml sample (or 75 microL to the 37.5 ml sorb induced sample)).
  5. Take 250 microL samples at t=15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 minutes, add these to the ice cold PBS + tween.
  6. Sonicate samples briefly
  7. FACS sort samples

Notes

  • Base the time points around how fast you expect the protein to fold. The above time points are for measuring GFP folding, but for tdTomato folding you would want to do a longer timecourse with less frequent time points.
  • It may be a good idea to do this experiment twice for each protein, doing a long but low resolution time course the first try and then using that information to intelligently design the time points for the second time.
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