14.リガンド結合実験（坂中千恵）
GTPγS binding
1.GTPγS solution
1mM GTPγS(cold) 0.1ml
[35S]GTPγS 15μl
Buffer* 1.885ml
use 2μl for 1 assay
2.reaction mixture
1M Tri-HCl(pH8.0) 2.0ml final 20mM
0.5M EDTA(pH8.0) 0.2ml 1mM
1M DTT 0.1ml 1mM
5M NaCl 2.0ml 100mM
10% LubrolPX 1.0ml 0.1%
1M MgCl2 2.5ml 25mM
20mM App(NH)p 0.5ml 0.1mM
2M NH4(SO4)2 12.5ml 250mM
water up to 48ml
use 48ml for 1 assay
3.binding assay
reaction mixture(2) 48μl
GTPγS solution(1) 2μl
sample(membrane etc.) 50μl
→ mixing and incubate at 37℃ for 60min
→ transfer onto nitrocellulose membrane(vacume trap)
→ wash membrane with 2ml of washing baffer(ice cold) three times
→ dry up membrane at 80℃
→ add liquid scintilator→ count
付）
Buffer*（1）＝ 10mM Tris-HCl(pH8.0),10mM DTT
washing buffer= 20mM Tris-HCl(pH8.0) ,25mM MgCl2,100mM NaCl