24小时热门版块排行榜

返回列表

dandan_hui

木虫 (正式写手)

应助: 0 (幼儿园)
金币: 5084.8
散金: 61
帖子: 346
在线: 16.1小时
虫号: 647551
注册: 2008-11-06
性别: GG
专业: 生物大分子结构与功能

[交流] 【求助/交流】重金求关于PULL-DOWN 的protocol一篇

求各位同仁给一篇pull-down 的protocol，在网上找了好久也没找到，谢谢大家啦

回复此楼

» 猜你喜欢

» 本主题相关价值贴推荐，对您同样有帮助:

熵变的求解关于粒子位置分布的随意性带来的熵已经有6人回复
求助关于土壤细菌分离鉴定的知识，希望朋友帮忙解答已经有8人回复
请教关于质谱的几个问题已经有10人回复
化学生物学的理论基础（求助！！！急！）已经有7人回复
求助sybyl关于添加形式电荷的问题已经有8人回复
求助首都师大的前辈已经有3人回复
重金求救：这个氧化镁条中加了什么添加剂，能烧成型，并能在磨床上任意加工已经有3人回复
求助：BJH计算孔容和单点法计算孔容？已经有7人回复
噬菌体展示相关文献和protocol 已经有75人回复
一种化合物的溶解问题费解重金求助已经有6人回复
【求助/交流】求nature protocol一片文章已经有3人回复
【求助/交流】pull down 已经有3人回复
己解决--DS v2.5 for Windows 的Protocol不能用! 已经有13人回复

1楼 2010-09-02 09:36:21

已阅回复此楼关注TA 给TA发消息送TA红花 TA的回帖

snowice

银虫 (小有名气)

应助: 0 (幼儿园)
金币: 300.6
帖子: 143
在线: 70.3小时
虫号: 996623
注册: 2010-04-14
性别: GG
专业: 医学分子遗传学

★ ★ ★
scelab(金币+3):谢谢交流！:tiger06: 2010-09-02 23:00:29
dandan_hui(金币+1): 2010-09-03 08:14:24

GST-Fusion Protein Purification and Pull Down Assay

1.  Grow BL21 bacteria containing plasmid(from a single colony or -70°C stock,) in 5ml LB plus 50ug/ml ampicillin at 37°C shaker for overnight;
2.  Inoculate 400ml of LB plus 50ug/ml ampicillin with 4mlo/n culture;
3.  Grow for 2-3 hrs until OD600 is 0.5;
4.  Add IPTG to a final concentration of 0.1 and grow at 37°C for 2-3hrs(update, except inducing GST-Rhoteckin at 30°C for 3hrs because of its unstability);
5.  Spin 6k rpm for 15 min 4°C and resuspend the pellet in 10ml of PBS plus 1mM PMSF and 10mM benzamidine;
6.  Sonicate 5x30sec, at maximum power; Incubate the sonicates in ice between sonications for 30sec;
7.  Add Triton X-100 to 1% and DTT to 1mM (both final concentration) and incubate for 30min in ice;
8.  Spin 7k rpm for 10min at 4°C. Take the supernatant for the next step and save the pellet for inclusion body purification.
9.  Transfer the supernatant into a 50 ml conical tube, add 25ml in PBS plus 1mM PMSF and 10mM Benzamidine and 2ml 1:1 slurry of glutathione-agarose beads. (If make GST fusion protein from 5ml culture, reduce the PBS volume to 1ml and add 50ul glutathione-agarose beads) The beads need to be preswollen overnight in PBS and washed 4 times with PBS before use.
10. Incubate at 4°C on a rotator for overnight;
11. Wash the beads with PBS at 4°C for 5 times.
12. Batch elute with 1 column volume of 20mM glutathione in 20mM Tris, pH8.0 for 6 times.
13. Pool and dialyze against 4 liters of PBS o/n at 4°C.
14. The GST fusion protein can be estimated by measuring the absorbance at 280nm. For the GST
affinity tag, 1A280=0.5mg/ml. Also, it can be determined by SDS-PAGE with standard BSA protein.
15.  Incubate about 500mg cell lysate with 2-5ug GST fusion protein and 40ul Gluthatione-agarose beads(if the GST fusion protein-binding beads are less than 40ul, use empty beads to 40ul) in a total 1ml volume(adjust to 1ml volume with RIPA buffer ) at 4°C on a rotator for at least 1hr.  Briefly wash the beads with Modified RIPA buffer for 3 times.
16. Elute the protein into the SDS loading buffer, then run SDS-PAGE gel.

Reagent:

1.  Glutathione-Agrose beads: G-4510 from Sigma.
2.  Glutathione, reduced form: G-6529 from Sigma.
3.  Benzamidine: B-6506 from Sigma, 1M stock solution.
4.  PMSF(phenylmethyl-sulfonyl fluoride): P7626 from Sigma, 0.1M stock solution in isopropanol.
5.  RIPA buffer:150mM NaCl, 50mM Tris.HCl(PH7.5), 1%NP-40, 0.25% Sodium Deoxyclolate, 2mM EGTA(stock solution 0.4M add before use), 1mM NaVO4(200mM stock solution, add before use)
6.  6XSDS loading buffer: 0.35M Tris-HCl(pH6.8), 10.28(w/v)%SDS, 36%(v/v) glycerol, 0.6M dithiothreitol(or 5%β-mercaptoethanol), 0.012%(w/v)bromophenol blue. Store in 0.5ml aliquots at -80°C.