m**********d 发帖数: 137 | 1 IHC及IF显示蛋白A在tumor cell的细胞核里高表达,蛋白A的canonical function是与
蛋白B bind并激活B。一些preliminary data提示蛋白A有新的功能,就是与蛋白C
bind而调控另一个重要的生物过程。
现在需要得到A与C直接作用的证据,下面这个用Dynal magnetic beads做CO-IP的
protocol:
10μl Dynal beads coated with protein A+10μl Dynal beads coated with
protein G each reaction, wash with ice cold PBS plus 5% BSA, three times.
Incubate with antibody against 蛋白A (20μl, approximately 4μg) in 500μl
PBS plus 5%BSA, overnight.
2^106 cells/10cm plate, treatment (which induces A and C interaction while
does not affect A/B interaction, according to preliminary data)
Add 1% formaldehyde, 37°C incubate 10min
Wash with ice cold PBS plus 5%BSA, another time with ice cold PBS
Scrape cells into 350μl PBS, 2000rpm×1min, add 350μl lysis buffer (1% SDS
, 10mM EDTA, 50mM Tris-HCl pH8.1 plus protease inhibitors) into cell pellet.
Incubate on ice for 20min, then spin down at 14,000rpm×15min.
Dilute SN 1:10 with dilution buffer (1% triton, 2mM EDTA, 150mM NaCl, 20mM
Tris-HCl pH 8.1)
Add 1ml diluted sample to dynal beads pre-incubated with antibody against 蛋
白A, incubate 24hrs.
Wash three times with RIPA buffer (50mM HEPES pH 7.6, 1mM EDTA, 0.7% Na
Deoxycholate, 1% NP-40, 0.5M LiCl). Wash twice with TE buffer.
Add 50μl elution buffer (8.34μl 6X loading bubber+ 41.66μl ddH2O) to each
sample, boil at 95°C for 10min, load 25μl on SDS-PAGE gel.
Western blotting for 蛋白B (作为positive control),及蛋白C
我对CO-IP实在没有经验,烦请版上的前辈们指点指点,这么做CO-IP有啥不妥之处,谢谢 | g*********5 发帖数: 2533 | 2 看的头昏眼花,有这么复杂吗?我的做法
直接用dynal protein G.
裂解细胞(裂解常用的detegent有tritionx100,NP40,chaps)
裂解液的配方要自己摸。看看文献蛋白A 别人用什么条件作ip。
用PBS洗珠子,最后一遍用裂解液洗。
lysate preclear control igG 1 HOUR, 加珠子半小时。
沉淀珠子,抗体和对照igG分别加到lysate中。2小时 ----overnight
加珠子沉淀。
洗珠子3-4次。
loading buffer 煮,100度3min
离心,上样。
包子。 | s****l 发帖数: 395 | 3 我觉得要证明是直接相互作用,怎么也要用纯化的蛋白吧,在体内的co-IP好像不能排
除是某种adapter介导的interaction
l
【在 m**********d 的大作中提到】![](/moin_static193/solenoid/img/up.png) : IHC及IF显示蛋白A在tumor cell的细胞核里高表达,蛋白A的canonical function是与 : 蛋白B bind并激活B。一些preliminary data提示蛋白A有新的功能,就是与蛋白C : bind而调控另一个重要的生物过程。 : 现在需要得到A与C直接作用的证据,下面这个用Dynal magnetic beads做CO-IP的 : protocol: : 10μl Dynal beads coated with protein A+10μl Dynal beads coated with : protein G each reaction, wash with ice cold PBS plus 5% BSA, three times. : Incubate with antibody against 蛋白A (20μl, approximately 4μg) in 500μl : PBS plus 5%BSA, overnight. : 2^106 cells/10cm plate, treatment (which induces A and C interaction while
| s******s 发帖数: 13035 | 4 //nod. 体内的只能证明是complex,除非去打质谱
【在 s****l 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我觉得要证明是直接相互作用,怎么也要用纯化的蛋白吧,在体内的co-IP好像不能排 : 除是某种adapter介导的interaction : : l
| g*********5 发帖数: 2533 | 5 you are right. need in vitro experiment.
【在 s****l 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我觉得要证明是直接相互作用,怎么也要用纯化的蛋白吧,在体内的co-IP好像不能排 : 除是某种adapter介导的interaction : : l
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