リガンド結合実験（坂中千恵）
　　GTPγS binding

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第二生化マニュアル目次

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

第二生化マニュアル目次