t**********8
发帖数: 223 | 1 我现在用的protocol，是crosslinking后用0.5%的SDS lysis cells。我见网上很多
protocol都是用1%的SDS，因为我的蛋白可能比较难抽，我就改用1%的SDS做，结果比0.
5%的SDS回收率还低。大家一般是用哪个浓度啊？谢谢！ | k*****n
发帖数: 323 | 2 我们用的是rick young 的protocol。
Lysis Buffer 1 Add protease inhibitors just before use, filter and keep cold
. Consists of 50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol,
0.5% NP-40, 0.25% Triton X-100, 1× protease inhibitors Lysis Buffer 2 Add
protease inhibitors just before use, filter and keep cold. Consists of 10 mM
Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1× protease
inhibitors
Lysis Buffer 3 Add protease inhibitors just before use, filter and keep cold
. Consists of 10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0
.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1× protease inhibitors | t**********8
发帖数: 223 | 3 谢谢你的热心回复。你冒似用的是两步裂解。你最后用的是RIPA buffer,不是SDS。我
的蛋白不用SDS可能抽不出来，我决定还是用1％SDS吧
cold
glycerol,
Add
mM
cold
0
【在 k*****n 的大作中提到】
: 我们用的是rick young 的protocol。
: Lysis Buffer 1 Add protease inhibitors just before use, filter and keep cold
: . Consists of 50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol,
: 0.5% NP-40, 0.25% Triton X-100, 1× protease inhibitors Lysis Buffer 2 Add
: protease inhibitors just before use, filter and keep cold. Consists of 10 mM
: Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1× protease
: inhibitors
: Lysis Buffer 3 Add protease inhibitors just before use, filter and keep cold
: . Consists of 10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0
: .1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1× protease inhibitors
|
|
