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免疫分子生物技术制作步骤?

信息分类:生命科学资讯    作者:yiyi发布 

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MIANYIFENZISHENGWUJISHU

JIANJIE:

RENTINEIDEXIBAOYUKANGYUANSHI, HUISHOUCIJIFANZHI, ERCHANSHENGDALIANGYOUZHUANYIXINGDEKANGTI(monoclonal antibody). ERKANGYUANCHANGYINQIBUTONGDEBXIBAOCHANSHENGKANGTI, GUZAIXUEYEZHONGDEKANGTISHIJISHANGSHIDUOYUANXINGKANGTI(polyclonal antibodies), QIYUANYINSHICIKANGYUANCHANGHANYOUBUZHIYICHUZHIKANGTIDEBUWEI(epitopes), MEIGEBUWEIDOUKEYINQIYIGEBUTONGBXIBAOKANGTI.

KANGTIYOUWUZHONG, IgG, IgA, IgD, IgEJIIgM, DUITONGYIGERENERYAN, CI5ZHONGKANGTIJUNDUITONGYIKANGYUANYOUZUOYONG. KANGTIDEXINGZHUANGRUYZIXING, YOUERTIAOQINGLIAN, ERTIAOZHONGLIAN. QINGLIANYOUYOUKHEλERZHONG, (DANTONGYIKANGTIZHIHANQIYI), DANZHONGLIANZEZHIYOUYIZHONG. JIIgGSHIγ, IgASHIα, IgDSHIδ, IgMSHIμ, IgESHIε.

BULUNQING, ZHONGLIANJUNHANYOUBIANYIDUAN(variable region)YUHENGCHANGDUAN(constant region), YUKANGYUANZUOYONGDEBUFENSHIZAIQING, ZHONGLIANBIANYIDUANDEGAODUBIANYI(hypervariable region)BUFEN, DAYUEZHAN15Å×20Å×15ÅDEKONGJIANDAXIAO.

KANGTIZHISUOYINENGYOURUCIDUOBIANHUA, BINGFEIRENDEJIYINYOURUCIDALIANG, ERSHIZAIBXIBAOCHENGSHUGUOCHENGZHONG, QIJIYINZHONGZU(YOUHENDUOHENGCHANGDUAN, BIANYIDUAN, LIANJIEDUAN, BIANHUAdiversityDUANDEJIYIN, KEYIZHONGZUPAILIE), KEYIGOUCHENGBUTONGDEKANGTI.

JIANGMEIXIANJIEZHIKANGTIZHIJISHU

JIANGMEIXIANJIEZHIKANGTISHANG, SHISHIMEI(LIRUhorweradish peroxidase HRPO, HUOalkaline phosphatase)YUKANGTIXINGCHENGWENDINGDE. QIEBUYINGXIANGMEIYUKANGTIZHIGONGNENG.

YIHRPOXIANJIEWEILI, QIHUAXUEBIANHUARUXIA:

CIYIJISHU, KEYONGYUEnzyme-linked immounoworbent assay (ELISA)JIwestern boltting, QIFANGFARUXIA:

CAI    LIAO:

1mg/ml antibody solution

0.1M phosphate buffer, pH6.8

Horseradish peroxidase (HRPO)

0.1M Carbonate buffer, pH9.2

Sodium periodate (NaIO4) solution, freshly prepared

Sodium borohydride (NaBH4) solution, freshly prepared

Saturated ammonium sulfate (NH4)2SO4 solution

Tris/EDTA/NaCl (TEN) buffer, pH7.2

BSA

Glycerol

Dialysis membrane

Pasteur pipet fitted with glass wool

Sephadex G-25, medium

FANG    FA:

1.     JIANG1mg/mlKANGTIYI2GONGSHENG0.1M phosphate buffer pH6.8TOUXI, GUOYE,     4℃ HUANHUANJIAOBAN. (KANGTILIANGKEYIA280/1.44=mg Ig/mlLAIJISUAN) (TOUXIMOYING    50﹪ethanolJINYIXIAOSHI, ZAIYI10mM NaHCO3JINYIXIAOSHI, ZAIYI1mM EDTAJINYIXIAOSHI, ZAIYIdH2OCHONGXI, ZAIBAOCUNZAIphosphaed bufferZHONG, 4℃, WEIFANGXIJUNSHENGZHANG, bufferZHONGKEJIARU0.01﹪DEsodium azide).

2.     RONG10mgDEGRPOYU1mlDE0.1M carbonate buffer, pH9.2ZHONG.

3.     JIANG0.25mlXINPEIHAODENaIO4YEYU0.25mlDE10mg/ml HRPO/carbonateYEHUNHE, GAIJINGAIZI, ZHISHIWEN2hr(BIMIANGUANGZHAO).

4.     ZAIYIZHIPasteur pipetteZHONGFANGRUSHILIANGglass wool, JIANGCHUKOUCHUYIparafilmBAOHAO, ZAIJIANG1mlYITOUXIGUODEantibodyYE, JIARU0.5mlDE10mg/ml HRPOYEZHONG, ZAIJIANG0.25gDESephadex G-25JIARUCIantebody/HRPOHUNHEYEZHONG (CIYIBUZHOUKEZENGJINMEIYUKANGTIZHIJIEHE). JIANGCIHUNHEWUJIARUCIPasteur pipetteZHONG.

5.     ZHIYUANSHI, SHIWEN3hr.

6.     JIANGCIColumnYI0.75mlDECarbonate bufferJIANGconjugateXICHU, BAOCUN.

7.     ZAICISHICHUYEZHONG, JIARU38μlDEXINPEIHAOZHINaBH4YE, ZAISHIWEN, ANSHI, ZHI30min.

8.     ZAIJIARU112μlDENaBH4YE, ANSHI, ZHI60min.

9.     ZAIJIARUBAOHE0.9ml (NH4)2SO4YE, HUANHUANJIAOBAN30min, 4℃, LIXIN15min, 10,000xg.

10.DAOQUSHANGQING, JIANGCHENDIANWURONGYU0.75mlDETENZHONG.

11.TOUXICIYE, 4℃, 2GONGSHENGDETEN, GUOYE, DIERTIANHUANGUOYICITENYE, ZAITOUXI4hr.

12.JIANGTOUXIMONEIWUQUCHU, JIARUSHILIANGBSA, SHICIYEZHONGZUIZHONGBSALIANGWEI20mg BSA/ml.

13.JIARUDENGLIANGZHIglycerolBINGBAOCUNZAI-20℃.

PEIFANG:

0.1M Carbonate buffer, pH9.2:

1.36g Sodium carbonate

7.35g Sodium bicarbonate

950ml H2O

YI1M HClHUO1MDENaOHDIAOZHENGpHZHI9.2ZAIJIAdH2OSHITIJICHENGWEI1GONGSHENG.

0.1M phosphate buffer, pH6.8:

BAOCUNARONGYE:0.2M:31.2g NaH2PO4YU1GONGSHENGDEdH2OZHONG

BAOCUNBRONGYE:0.2M:28.39g NaH2PO4YU1GONGSHENGDEdH2OZHONG

HUNHE51mlDEAYEYU49mlDEBYE, JI100mlDEdH2OCHENGWEISHIYONGRONGYE.

BAOHEammonium sulfate (NH4)2SO4YE:

JIANG1.21g Tris baseJIARU990ml dH2OZHONG, SHICHENG0.01M TrisYE, DIAOZHENGpHZHI7.0, ZAIJIAdH2O, SHIZONGTIJIWEI1GONGSHENG, LIANG767gDE(NH4)2SO4JIARUCITrisYEZHONG,JIAOBAN, BINGJIAWEIRE, SHIRONG, DIAOZHENGpHZHI7.0, BINGBAOCUNZAI4℃, (NH4)2SO4DEJIEJINGHUICHENDIANCHULAI.

Sodium borohydride (NaBH4)YE:

5mg NaBH4/ml dH2O, BIXUYONGQIANCAIPEI

Sodium periodate (NaIO4)YE:

1.71mg NaIO4/ml dH2O (YONGQIANCAIPEI)

Tris/EDTA/NaCl(TEN)YE, pH7.2:

ZAI930ml dH2OZHONGJIARU:

0.06g Tris base

0.37g Na2 EDTA

8.77g NaCl

DIAOZHENGpHZHI7.2(YIHClDIDING)

JIAdH2OSHITIJICHENGWEI1GONGSHENG

FUZHU:

1.    SHIYONGCIFA, YUE1ng/mlZHI10ng/mlDEKANGYUANKEYIZHENCEDAO.

2.    ZAISHIYONGSHI, KEJIANGJIEGUOXISHI100ZHI10,000BEI. SHIXIAOGUORUHEERDING.

ZHUNBEIXIJUNKANGYUAN

CAI    LIAO:

E.Coli(ZHANGYUPEIYANGYEZHONG)

Cell resuspending buffer

Lysozyme solution

Tris/EDTA/NaCl(TEN) buffer

10﹪SDS

8M urea 

FANG    FA:

1.     JIANG5mlDEE.ColiYEZAIZHUOMIANLIXINJILIXIN2,500rpm, 10min, DAOQUSHANGQING, JIANGCHENDIANXIJUNRONGYU5ml resuspending bufferZHONG, CHONGFENHUNHE(Votex).

2.     XICHU1mlXIJUNYERU1.5mlDEWEILIANGLIXINGUANZHONG, ZHIBINGSHANGZAIJIARU0.2mlDElysozymeYE, DAI5min.

3.     LIXIN3,000rpm, 5min, BAOLIUSHANGCENG, JIANGCHENDIANWUYI1.2ml TENRONGJIEZHI. (YINWEIYOUXIEDANBAIZHISHIBURONGYUSHUI, GUERZHEJUNYINGBAOLIU)

4.     ZAIMEIYIZHONGYANGBENZHONGGEJIARU65μlDE10﹪SDS, ZHI37℃, 10min, CISHIYANGBENJIKESHIYONG, RUOBUSHIYONGYINGLENGDONGBAOCUN, RUOYOUBIYAOKEYI8M ureaYE(4.8g ureaJIARU10mlYEZHONG), CHULIDANBAIZHI.

PEIFANG:

cell resuspending buffer (10mM HEPES)

2.38g HEPES

JIAdH2OHUO1 liter

FUZHU:

1.     ZAIMIANYIXUESHIYANZHONG, Enzyme-Linked Immunolorbent Assays(ELISA)SHICHANGYONGYUZHENCEXIJUNHUOKANGYUANDEFANGFA. YIBANFENWEIZHIJIEFA, JIJIANGKANGYUANFANGRUmicro titer plate, SHIFUZHEZAINEIBI, ZAIYIdiluting bufferSHIWEIBEIFUZHECHUNENGBUJIESHOUKANGTIFUZHE, JIEZHEJIARUYIXIANJIEMEIDEKANGTI, ZAIXIQUDUOYUDEKANGTI, ZAIJIARUMEIDEZUOYONGWU, SHICHANSHENGFANYING.

LINGYIZHONGWEIJIANJIEFA, JIXIANJIANGKANGTIFANGRUmicro titer plateZHONG, SHIYUNEIBIFUZHE, ZAIYIdiluitng bufferCHULI, SHIWEIBEIFUZHEDEBIBUHUIJIESHOUKANGYUAN, ZAIJIARUKANGYUANSHIYUKANGTIZUOYONG, ZAIXIQUDUOYUDEKANGTI, ZAIJIARUYIXIANJIEMEIDEKANGYUAN, ZAIXIQUDUOYUDE, ZAIJIARUMEIDEZUOYONGWUSHICHANSHENGFANYING.

ZHIJIEFADEQUEDIANSHIXIANGCEDEYOUZHUANYIXINGDEKANGYUANXUYUQITAZAZHIHUZHENGFUYUNEIBI, YIZHIYOUSHILIANGSHAO, ERJIANJIEFAZEKEXUANZEXINGDELIUXIASHOUCEDEKANGYUAN.

2.     ZHIYUKANGTI, KEYIYONGKANGYUANPEIHEadjuvatYITONGZHUSHERUTIZHONG, 4ZHOUHOUZAIZENGQIANGZHUSHEYICI, 2ZHOUHOUZAIZENGQIANGYICI, YIHOUMEICICHOUXUEQIAN7TIANZHUSHEYICIJIKEQUDEHANCIKANGTIDEXUEYE. CIXUEYEJINGLIXIN, BAOCUNXUEQING, ZAIYIammonium sulfateCHENDIANKANGTI(YUE33﹪DENONGDUJIKECHENDIANCHUIgG), ZAILIXIN, BAOCUNCHENDIANWU, ZAITOUXIQUYAN, JIKEDEKANGTI

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