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上海乾思生物科技有限公司
中級會員 | 第17年

18916423352

細胞庫
優(yōu)勢品牌
Encapsula NanoSciences Quidel Bioporto Salimetrics Research Products International Corp Quanta BioDesign Peninsula Laboratories International Golden West Diagnostics Ocean NanoTech HaemoScan affinitybiologicals Cytodiagnostics Innovagen AB CLIAwaived Svar Abbexa ABMgood AffinityImmuno AGScientific AHF analysentechnik AG Alomone 英國Antibodies AstaTech Avidity Badrilla BioAcademia Biomatik Biosynth BioTeZ Biotrend Calbiotech CaroteNature Cell Sciences Click Chemistry Tools Cohesion Biosciences CordenPharma Cosmo Bio Creative Biomart Crystal Chem Crystalgen Diagnovum DiaMetra Dianova Diatheva Eagle Manufacturing Echelon Biosciences Edgebio Emfret Epoch Life Science Equitech-Bio Eramask Euro Diagnostica Exocell Fine Science Tools GenwayBio Giotto Biotech Glen Research Glycosynth Gold Biotechnology Golden West Biologicals Haemtech(HTI) Herolab IHC WORLD ImmuChrom GmbH Immunostep Immunotools Immunovision INCELL Corporation Innovative Research Innovrsrch(IRA) Insitus Iris Biotech GmbH Isosep IST Engineering Inc J.G. Finneran Kainos Kamiya Biomedical Labor Diagnostika Nord Larodan Laysanbio Lc laboratories Lee BioSolutions LGC Biosearch Technologies Lifecore List Labs lumiprobe Macrocyclics Medix Biochemica Meridian Life Science Microbix Milenia Biotec Moscerdam Mybiosource Nanoimmunotech Nanoprobes Neuro Probe Paratechs Physitemp Polypure Polysciences Probetex Progen ProScience Inc ProSpec QAbio Qorpak Quanta BioSciences QuickZyme RevMAb Biosciences SCHMIDT BioMedTech Seven Hills Foundation Smartox Biotechnology Spherotech Surgimax Swant SYSY Ted Pella Torpac Trilink USBio VIDIA Ltd ViroStat Virusys VP-scientific Xcessbio XENOMETRIX Zeptometrix ZYTOVISION
自產(chǎn)ELISA試劑盒,試劑盒
進口ELISA試劑盒
人細胞,人細胞系,人細胞株
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SIGMA現(xiàn)貨,SIGMA代理

免疫共沉淀|實驗技術(shù)服務(wù)

時間:2015/8/1閱讀:324
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關(guān)鍵詞:免疫共沉淀|實驗技術(shù)服務(wù)
簡介:世界*品牌免疫共沉淀|實驗技術(shù)服務(wù)原裝,,質(zhì)量保證,*,。
免疫共沉淀|實驗技術(shù)服務(wù)——pCzn1質(zhì)粒+Arctic Express宿主菌低溫表達體系。
我們具備豐富的蛋白表達設(shè)計經(jīng)驗及實驗操作技巧,,承諾客戶不成功不收費,。我們所有的實驗數(shù)據(jù)真實可信,我們提供原始的表達菌株和克隆質(zhì)粒,,客戶可以依據(jù)我們的實驗報告重復(fù)我們的實驗結(jié)果,。
產(chǎn)品品牌:   http://www.。,。com/goodsid/fenleiyi/2868603/1.html    
測序?qū)嶒灹鞒蹋?br>各種成分在工作液中的終濃度:
* Tris-HCl: 50 mM, pH 7.4
* NP-40: 1%
* 去氧膽酸鈉:0.25%
* NaCl: 150 mM
* EDTA: 1 mM
* PMSF: 1 mM
* 抑蛋白酶肽,,亮抑酶肽,胃蛋白酶抑制劑: 各1 μg/ml
* Na3VO4: 1 mM
* NaF: 1 mM
實驗流程為:
1.用磷酸鹽緩沖液洗30塊10 cm培養(yǎng)板上的適宜細胞,。刮去每塊板上的細胞到1 ml冰冷的EBC裂解緩沖液中。
2.將每毫升細胞懸液轉(zhuǎn)移到微量離心管中,,在微量離心機上4℃以zui大速度離心15 min,。
3.收集上清(約30 ml)并加入30μg的適當(dāng)抗體,4℃搖動免疫沉淀物1 h,。
4.加入0.9 ml的蛋白質(zhì)A-Sepharose 懸液,,4℃搖動免疫沉淀物30 min。
5.用含900 mmol/L NaCl的NETN洗蛋白A-Sepharose混合物,,再重復(fù)洗5次,。zui后,用NETN洗一次,。
6.吸出混合物的液體部分,。加入800μl的1×SDS膠加樣緩沖液到球珠中,煮沸4 min,。
7.將樣品加入到大孔的不連續(xù)SDS-PAGE梯度膠中,,在10 mA的恒定電流下電泳。
8.通過考馬斯藍染色觀察蛋白質(zhì)泳帶,。
9.從膠上切下目標帶,,將其放到微量離心管中,用1ml 50%乙腈洗兩次,,每次3 min,。
10. 用胰蛋白酶消化膠中的蛋白質(zhì),再將肽電洗脫,。
11. 通過窄孔液相色譜分離肽,。將收集的肽在ABI 477A或494A機器上進行自動Edman降解測序。
在免疫共沉淀實驗中要保證實驗結(jié)果的真實性,,應(yīng)注意以下幾點:
(1) 確保共沉淀的蛋白是由所加入的抗體沉淀得到的,,而并非外源非特異蛋白,單克隆抗體的使用有助于避免污染的發(fā)生,;
(2) 要確??贵w的特異性,即在不表達抗原的細胞溶解物中添加抗體后不會引起共沉淀,;
(3) 確定蛋白間的相互作用是發(fā)生在細胞中,,而不是由于細胞的溶解才發(fā)生的,這需要進行蛋白質(zhì)的定位來確定,。

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