GST融合蛋白的纯化步骤整理：Disun 来源：互联网 一、制取细胞的裂解物：
1.每100ml培养物的细胞沉淀悬于4ml PBS缓冲液;
2.加入溶菌酶至最终浓度1mg/ml，冰上或冷藏放置30min;
3.用针筒将10ml0.2%TritonX-100强行注入细胞裂解物中，剧烈震动数次混匀；
座便器结构
4.加入DNase和RNase至终浓度5ug/ml,4℃震动并温育10min;
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微型显示器5.4℃3000g(5000r/min)离心30min;
6.上清转移到一只新试管，加入DTT至终浓度为1mmol/L;
二、纯化GST融合蛋白：
1.细胞裂解物与50%谷胱甘肽-琼脂糖树脂匀浆混合，每100ml细胞培养物加2ml树脂，于室温下轻摇30min; 2.混合物于4℃以500g(2100r/min)离心5min，小心去掉上清并留样少许进行SDS-PAGE;
3.沉淀中加入10倍标准体积的PBS，颠倒离心管数次混匀，洗去未与树脂结合的蛋白；
4.4℃以500g(2100r/min)离心5min，小心去掉上清并留样少许进行SDS-PAGE;
文件加密存储5.重复步骤3和4两次；
6.结合的GST融合蛋白可用谷胱甘肽洗脱缓冲液洗脱，也可用凝血酶，肠激酶或Xa因子切割，释放靶蛋白； 三、用谷胱甘肽洗脱洗脱融合蛋白：
1.沉淀中加入1倍柱床体积的谷胱甘肽洗脱缓冲液，室温轻轻搅动10min，洗脱树脂上结合的蛋白；
2.4℃以500g(2100r/min)离心5min，上清移至新管中;
3.重复步骤a和b两次，合并3次的上清；
四、蛋白酶解从结合的GST融合蛋白上回收靶细胞：
1.在结合了融合蛋白的树脂中加入凝血酶，肠激酶或Xa因子。每毫升树脂加入50单位1mlPBS的蛋白酶，颠倒离心管数次混匀，室温下震荡2～16h，用小规模实验确定精确时间；
2.4℃以500g(2100r/min)离心5min，上清小心移至新管中;
3.10%SDS-PAGE分析每一步（细胞抽提，洗涤和洗脱）样品的蛋白质组成。
五、谷胱甘肽琼脂糖树脂的处理：
1.轻轻颠倒盛有谷胱甘肽-琼脂糖树脂的容器，将树脂混成匀浆；
2.取部分匀浆放入15ml聚丙烯管（每100ml细菌培养物需要2ml匀浆）；
3.4℃以500g(2100r/min)离心5min，小心去掉上清;
4.在树脂中加入10倍柱床体积的冷的PBS，颠倒数次，混合匀浆，4℃以500g(2100r/min)离心5min，小心去掉上清；
5.每毫升树脂加入1ml冷的PBS，制成50%匀浆，颠倒数次，悬浮冰上放置待用。
GST融合蛋白纯化方法 Purification of GST Fused Proteins
Materials and Reagents
STE Buffer
10 mM Tris-HCl, pH 8.0
1 mM EDTA
150 mM NaCl
Lysozyme solution
10 mg/ml in water (make fresh)
PBS
Elution Buffer
50 mM Tris.Cl, pH 9.0
20 mM GSH
10% Sarkosyl in STE Buffer
10% Triton X-100 in STE Buffer
1 M DTT
100 mM IPTG
Procedure
Day 1
Set up an overnight culture in 50 ml 2XTY with 150 mg/ml of ampicillin.
Day 2
Seed 5 ml of overnight culture to 500 ml 2XTY with 150 mg/ml of ampicillin.
Grow at 37oC to an A600 of 0.6 to 0.8.
Induce with 0.1 mM to 2 mM of IPTG. Grow for 3 hr
仓储室内定位系统at 37oC or grow overnight at room temperature.
Lower IPTG concentrations and lower growing temperatures tend to produce greater solubility at the expense of yield.
Pellet cells by centrifuging at 3000 g, 4oC for 10 min. Decant media and resuspend cells in 30 ml ice-cold PBS to wash. Transfer to a 40-ml Oak Ridge tube and centrifuge at 3000 g, 4oC for 10 min. Decant PBS.
This is a convenient point to stop and to store pellets at -80oC. Else continue to lyse cells.
Thaw pellet on ice if cells are frozen else proceed to the next step.
Resuspend pellet in 10 ml of ice cold STE Buffer.
Add 100 ml of freshly prepared lyozyme solution, incubate on ice for 15 min. Just before sonication, add 100 ml of 1 M DTT and 1.4 ml of 10% Sarkosyl. Mix thoroughly by inversion and sonicate for a total time of 1 min.
Centrifuge 16,000 rpm for 20 min on the SS34 rotor to pellet debris. Transfer supernatant to a 50-ml conical tube and discard the pellet. Add 4 ml of 10% Triton X-100 and top up with STE Buffer to 20 ml. The effective concentration of Sarkosyl and Triton X-100 will be 0.7% and 2% respectively. Incubate at room temperature for 30 min.
Pour the lysate to 1 ml bed of prepared Glutathione Sepharose in PBS. Incubate at room temperatur
e for 30 min to 1 hr with agitation.海水防腐涂料
To prepare the 50% slurry, shake up the media and pipette 2 ml to a 50 ml tube. Fill to 50 ml with PBS, invert tube a few times. Centrifuge to 2000 rpm on a swing bucket centrifuge then switch off. Carefully suck off PBS and resuspend beads with 1 ml of PBS.
Wash the beads with 3 X 50 ml of PBS. Finally resuspend in 5 ml of PBS. Pour to a dispo-column. Wash the 50-ml conical tube with an additional 5 ml of PBS. Pool with the first 5 ml in the dispo-column.
To wash, use the same centrifugation technique for preparing the beads. When transferring beads to column, do not pipette but pour. The beads tend to stick to pipette tips.
If desired, elute with 10 x 1 ml fractions of Elution Buffer. Determine desired fractions with SDS PAGE
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