GST pull down 实验细节问题求教 有关GST-pull down和双向电泳问题
GST pull down\u5b9e\u9a8c\u7ed3\u679c\u600e\u4e48\u770b\u554a\u8fd9\u5f20\u56fe\u7684\u4e3b\u8981\u76ee\u7684\u662f\u4e3a\u4e86\u8bc1\u660eGST-Atrogin-1-wt\uff08\u91ce\u751f\u578b\uff09\u548c\u03b2-catenin\u5728\u4f53\u5916\u7684\u76f8\u4e92\u4f5c\u7528\uff0c
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\u4e0b\u56fe\u514d\u75ab\u5370\u8ff9\u7684\uff0c\u4e5f\u5c31\u662fwestern blot\u7684\u7ed3\u679c\uff0c\u5c06\u4e0a\u56fe\u540c\u6837\u7684\u80f6\u8f6c\u819c\u4e4b\u540e\uff0c\u7528\u662fFLAG\u6297\u4f53\u514d\u75ab\u5370\u8ff9\u7684\u7ed3\u679c\uff0c\u5176\u4e2d\u5b9e\u9a8c\u7ec4FLAG-\u03b2-catenin\u5b58\u5728\uff0c\u800c\u5bf9\u7167\u7ec4GST\u5e76\u6ca1\u6709\u62c9\u4e0b\u03b2-catenin\uff0c\u6240\u4ee5\u76f8\u4e92\u4f5c\u7528\u662f\u5b58\u5728\u7684\u3002
\u6700\u5de6\u680f\u7684input\u662f\u53d6\u81eaFLAG-\u03b2-catenin\u7684\u7ec6\u80de\u88c2\u89e3\u6db2\u7684\u6837\u54c1\uff0c\u53ef\u4f5c\u4e3a\u9633\u6027\u5bf9\u7167\uff0c\u4e0b\u9762\u7684WB\u7684\u7ed3\u679c\u8bf4\u660e\u4e86\u88c2\u89e3\u6db2\u4e2d\u5b58\u5728FLAG-\u03b2-catenin\u3002\u6b63\u5e38\u60c5\u51b5\u4e0b\uff0c\u5b9e\u9a8c\u4e2d\u63a7\u5236input\u7684\u603b\u86cb\u767d\u91cf\u4e0eGST\u6216\u8005GST-Atrogin-1-wt\u662f\u4e00\u6837\u591a\u7684\uff0c\u8fd9\u6837\u53ef\u4ee5\u8868\u660e\u88c2\u89e3\u6db2\u4e2d\u6709\u591a\u5c11FLAG-\u03b2-catenin\u88abGST-Atrogin-1-wt\u62c9\u4e0b\u6765\u3002
GST\u76f8\u5f53\u4e8e\u9634\u6027\u5bf9\u7167\uff0c\u62c9\u4e0b\u76ee\u7684\u86cb\u767d\u7684\u91cf\u662f0%
input\u76f8\u5f53\u4e8e\u9633\u6027\u5bf9\u7167\uff0c\u542b\u6709\u76ee\u7684\u86cb\u767d100%
\u5b9e\u9a8c\u7ec4\u5c31\u662fGST-Atrogin-1-wt\uff0c\u5728\u4e25\u683c\u63a7\u5236\u53cd\u5e94\u86cb\u767d\u7684\u91cf\u65f6\uff0c\u53ef\u4ee5\u770b\u51fa\u5176\u4f5c\u7528\u5f3a\u5f31
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细胞蛋白裂解液,洗脱液: PBS及PBS+1%Triton-100 PBS (1L) NaCl: 8g KCl: 0.2g Na2HPO4: 1.44g KH2PO4: 0.24g 加入800ml蒸馏水,用HCl调节溶液的pH值至7.4,最后加蒸馏水定容至1L即可,高温高压灭菌!4℃保存备用! PMSF(苯甲基磺酰氟) MW:174.19 工作浓度0.1-1mM,这里使用1mM,储存浓度100mM,将0.174g PMSF溶于10ml无水乙醇混匀即可!保持于-20℃或者4℃ (以下流程仅供研究已知原核蛋白A和真核过表达蛋白B相互作用) 1:原核融合蛋白A的获得 1.1:将编码蛋白A与GST的重组质粒化转BL21(DE3)菌株 1.2:挑取单个克隆到含有5mlLB(+100ug/mlAmp)的10ml试管里,37℃培养过夜 1.3:将培养菌液转移到含有500ml LB(+100ug/mlAmp)的1L锥形瓶中,37℃,225rpm培养至OD600≈1.0-1.5左右,加入适当浓度的IPTG,在适当温度下培养适当时间(诱导条件需要根据不同的蛋白做调整).6000g,10分钟,4℃离心收集细菌,去尽上清,将菌体至于-20℃放置O/N 1.4:室温冻融菌体,马上置于冰上,每500ml培养液加入10-20ml细菌裂解液(PBS+1%Triton-100+PMSF),吹打混匀 1.5:冰上超声破碎,开2秒,停9秒,总40-60分钟。至裂解液充分清凉 1.6:11000rpm,15分钟,4℃离心分离上清, -80℃保存备用 2:真核融合蛋白B的获得
2.1:将编码B蛋白的碱基序列克隆到编码标签蛋白(如HA,或者myc)的真核表达载体上,进行细胞转染 xq 3:48小时后,取适量融合蛋白GST-A冰上冻融 4:取50-70ulGST-Beads(Immobilized Glutathione)到EP管中,用800ul PBS+1%Triton-100润洗一次,将冻融融合蛋白GST-A与之混匀,4℃层析柜旋转结合1小时。 5:PBS+1%Triton-100洗3次,PBS洗3次。留取20ul( PBS+Beads)作Offer。 6:同时,裂解真核融合蛋白B,去尽培养基,用PBS(RT)洗一次,加入300ul裂解液(以6well为例,PBS+1%Triton-100+ Cocktaier),4℃放置30分钟。 7:吹打收集至1.5mlEP管,超声破碎。13000rpm,15分钟,4℃离心取上清。 8:BCA蛋白定量(optional)留取20ul做Offer,其余样品加入到已纯化蛋白的EP管中,用PBS补足液体到600ul左右,以便蛋白之间能充分结合!4℃旋转结合O/N9:PBS+1%Triton-100洗3次,PBS洗3次。 10:40ul 5×loading buffer溶解beads上的蛋白,煮沸3分钟,高速离心,Run –SDS PAGE做Western Blot检测。用HA或者myc Blot。 注意GST-pull down的对照问题。(以A-GST和B-6*His为例) 1. 跑SDS-PAGE时点一个 input的蛋白(即B-6*His) 用于做阳性对照 看一下western操作过程中,试剂,操作步骤有没有问题。 2. 谷胱甘肽琼脂珠上只固定GST蛋白,input B-6*His,用于做阴性对照 看一下是否GST会与input的蛋白相互作用 Protocol for Immunoprecipitation or GST-pull down 1. Wash the cell monolayer once with cold PBS. 2. Treat the cells with 2mM 3,3’-dithiolbissulfosuccinimidylpropionate/PBS at 4ºC for 25 min to crosslink the associated proteins. (optional) 3. Scratch off the cells, and spin down at 4ºC at 1000rpm for 5 min. 4. Decant the supernatant. 5. Resuspend the cell pellet in appropriate amount of IP-Buffer A, sonicate at 40%
output for 10 sec, and incubate the lysate at 4ºC for 45min. 6. Spin at highest speed at 4ºC for 5 min. 7. Recover the supernatant and detect protein concentration with Bradford Reagent (Bio-Rad). 8. Take 500ug proteins, mixed with 4 volume of IP-Buffer B. 9. Preclear the lysate by adding 1ug normal IgG and 5ul protein-G beads (Sigma), incubate on a rotator at 4ºC for 1hr. For GST-pull down assay, add 1ug GST protein and 5ul glutathione-sepharose 4B beads instead. 10. Spin down the beads at 4ºC at 10000rpm for 3min. 11. Recover the supernatant, add 1ug specific antibody or normal IgG, and incubate on a rotator at 4ºC for 2hr. For GST-pull down assay, add 1ug GST-fused specific protein or equal molar of GST protein instead. 12. Add 5ul of 5ul protein-G or glutathione-sepharose 4B beads, and incubate for another 2hr or overnight. 13. Spin down the beads at 4ºC at 2000rpm for 3min. Caution: There will be some white pellets just above the beads on the tube wall, which are protein precipitates due to denature. Remove away these precipitates, otherwise they will give false binding band in both control and test groups, affecting the explanation of the results. Check this phenomena at every washing step. 14. Wash the beads with 1ml of IP Washing buffer on a rotator at 4ºC for 5min. 15. Spin down the beads at 4ºC at 2000rpm for 3min. 16. Repeat step 14 and 15 for three more times. 17. Resuspend the pellet in 25ul 1xSDS loading buffer and incubate at RT for 30min. 18. Spin at 3000rpm at RT for 5min. 19. Recover the supernatant and put at –20ºC or subjected to SDS-PAGE running. 20. For the input control, 25ug protein will be included in gel running. Caution: to avoid the interference of IgG heavy chain with specific proteins with MW around 50KD, 1x SDS loading buffer without beta-mercaptoethanol or DTT can be used instead. At this condition, the interchain cysteine-cysteine bond will be maintained, giving IgG band at 100KD or so. IP-Buffer A: 50mM Tris-Cl PH 7.5 150mM NaCl 1mM EDTA PH8.0 0.5% Triton X-100 plus Protease Inhibitors (Roche) IP-Buffer B: IP-Buffer A minus Triton X-100. IP Washing Buffer: 50mM Tris-Cl PH 7.5 150mM NaCl
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