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gst fusion protocol

A Dohlman Lab Protocol

GST Fusion Protein Purification from Yeast

Lysis Buffer (20 ml)
3.3 M* Triethanolamine (pH 7.2)
 194 µl *
 40 mM
 0.5 M EDTA (pH 8)
 80 µl
 2 mM
 5 M NaCl
 600 µl
 150 mM
  0.1 M DTT
 400 µl
 2 mM
 10 mM AEBSF
 0.4 ml
 0.2 mM
 1.5 mg/ml leupeptin
200 µl
 15 µg/ml
 0.5 mg/ml pepstatin
 20 µl
20 µg/ml
 1 M benzamidine
  20 µl
 1 mM
 0.5 mg/ml aprotinin
 400 µl
 10 µg/ml
 100 mM b-glycerolphosphate
 20 µl
 100 µM
 50 mM Na-o-vanadate
 200 µl
 0.5 mM
HOH to 20 mls


Thanks to Paul DiBello and Jiyoung Cha for their refinements of this protocol.

* Indicates a correction from an eariler version of the protocol. 

1. For lysis in the presense of GDP and GTP I use a final concentration of 10 uM GDP or 20 uM GTPgammaS and a final concentration of 3 mM MgCl2 in the lysis buffer.

2. You can substitute protease inhibitor cocktail (Sigma P8215) for individual protease inhibitors (AEBSF, leupeptin, pepstatin, benzamidine, aprotinin).

3. For lysis to determine phosphorylation, you may wish to add more phosphatase inhibitors (in addition to beta-glycerolphosphate and Na-o-vanadate) to the lysis buffer, or you may wish to omit phosphatase inhibitors altogether:
 50mM Na-M-Vanadate
 100mM Na-pyrophosphate
 2 ml
 2mg/ml Phosvitin

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