GST Fusion Protein Production
GST Fusion Protein Production
GST Fusion Protein Production
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NaCl 80g 137mM<br />
KCl 2g 2.7mM<br />
Na 2 HPO 4 11.5g 4.3mM<br />
KH 2 PO 4 2g 1.4mM<br />
PH to 7.5<br />
F, a/c<br />
5. STE (Sodium chloride, Tris, EDTA)<br />
Stock Sol'n Final [ ]<br />
(a) 5M NaCl<br />
150mM<br />
(b) 1M Tris (7.9)<br />
10mM<br />
(c) 0.5M EDTA<br />
1.0mM<br />
(d) Glycerol 10%<br />
6. PMSF (Phenylmethylsulfonyl fluoride) and AEBSF (4-(2-amino-ethyl)- benzenesulfonyl fluoride<br />
hydrochloride), which is a non-toxic alternative to PMSF)<br />
Stock For 1 ml Final []<br />
PMSF (100mM) 17.4mg 1mM<br />
2-propanol (iso) 1.0 ml<br />
Store at –20 o C<br />
or<br />
AEBSF (100mM) 23.9mg 1mM<br />
dH 2 O<br />
1.0 ml<br />
Store at –20 o C<br />
7. Pepstatin A For 1 ml Final []<br />
Pepsatin A 1mg 1µg/ml<br />
Ethanol<br />
1ml<br />
8. DTT (Dithiolthreitol) 1M<br />
Stock For 10 ml Final []<br />
DTT 1.545g 1mM<br />
0.01M NaOAc 10ml<br />
(pH 5.2)<br />
Filter sterile. Store in 1.0 ml aliquots at –80 o C.<br />
(0.01M NaOAc is 33µl of 3M NaOAc in 9.67ml dH2O)<br />
9. Complete Mini, EDTA-free Protease inhibitor cocktail tablets (Roche Diagnostics Corp. cat # 1836170).<br />
B. Solutions for <strong>GST</strong> affinity chromatography:<br />
1. <strong>GST</strong>-CBF1 Lysis and Binding Buffer (Minus <strong>Protein</strong>ase inhibitor cocktail); PGE-<br />
Stock Sol'n Final [ ]<br />
Stockinger Lab; Version 02/16/01
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