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DNA-Protein交互作用的研究、细胞核物质抽提

信息分类:生命科学资讯    作者:yiyi发布 

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DNA-ProteinJIAOHUZUOYONGDEYANJIU

许多基因功能控制受到与此基因中某一片断与某特殊蛋白质的结合之影响,
为研究此种关係而研发出一些技术. 要研究此一主题, 首先必须先进行细胞核与细胞质中物质的抽提.

XIBAOHEWUZHICHOUTI

CAI    LIAO:

BURULEIDONGWUXIBAO(KEYONGZUZHIPEIYANGXIBAORUHela)

PBS

Hypotonic buffer

Low-salt buffer

High-salt buffer with 1.2M KCl

Dialysis buffer

Liquid nitrogen

BOLIZHIDounce homogenizer

Dialysis membrane tubing (KONGDONGFENZILIANG≦14,000)

导电测量

FANG    FA: (CIFANGFAJUNZAI4℃JINXING)

1.     XUYONG5~10×108cells/literDEZUZHIPEIYANGXIBAO, YI250mlLIXINGUAN, LIXIN20min, 4,000rpm. (RUOWEImonolayerXIBAO, YINGYISUJIAOGUADAOJIANGXIBAOGUAXIAFANGRULIXINGUANZHONG, XIBAOYAOXIANYIPBSXIGUOZAIGUAXIA.) DAOQUSHANGQING.

2.     GUJICHENDIANWUZHIDALVETIJI, JIARUTIJILIANG5BEIDEPBS, HUNHEHOU, LIXIN10min, 4,000rpm, DAOQUSHANGQING.

3.     JIANGCHENDIANXIBAORONGYUhypotonic buffer(YUECHENDIANTIJI5BEILIANG), LIXIN5min, 4,000rpm, DAOQUSHANGQING. CIYIBUZHOUDONGZUOYAOKUAI, YIMIANXIBAOPOLIESUNSHICHOUTIWUZHI.

4.     JIARUYUANCHENDIANXIBAOTIJI3BEIDEhypotonic buffer, ZHIBINGSHANG10min, (CIWAIYUANCHENDIANTIJILIUZAIDIERBUZHOUSHISUODETIJI, YINZAIDISANBUZHOUSHITIJIYINXIBAOPENGZHANGERZENGDALE).

5.     JIANGXIBAODAORUDounce homogenizer, SHANGXIAYAZHAXIBAO(CHULISHI, DONGZUOQING, YIMIANSHANGJIXIBAOHE).

6.     JIANGJUNZHIHOUDEXIBAODAORULIXINGUAN, LIXIN15min, 4,500rpm, FENKAIBAOLIUSHANGQINGYUCHENDIANWUERZHE. SHANGQINGYONGYUCHOUTIXIBAOZHIWU, CHENDIANWUYONGYUCHOUTIXIBAOHENEIWUZHI.

7.     GUJICHENDIANWUTIJI, JIARUYUETIJI1/2BEIDElow-salt buffer, RANHOUYIBIANJIAOBAN, YIBIANYIDIYIDIJIARUhigh-salt buffer, ZHIDAOYUE1/2DECHENDIANWUTIJIWEIZHI.

8.     HUANHUANZHENDONG, 30min, ZAILIXIN15,000rpm, 30min, SHANGQINGYEJIUSHIHENEICUIQUYE.

9.     JIANGCUIQUYEFANGRUTOUXIMONEI, YI50BEIYETOUXI, ZHIDAOKEYIDAODIANQILIANGCHUDAINEIWAIZHIDAODIANDUXIANGTONGWEIZHI. (2mlYIXIALIANGYUE1hr, 100mlYUE5hr)

10.QUCHUCUIQUWU, LIXIN15,000rpm, 20min, BAOLIUSHANGQING. YONGXIAOLIANGCEDANBAIZHINONGDU, QIYUBAOCUNZAI-70℃.

JIEZHEJIANGQIANSHUBUZHOU6ZHONGDESHANGQINGWU, YONGLAITIQUXIBAOZHICUIQUWU.

CAI    LIAO:

10x cytoplasmic extract buffer

FANG    FA:

1.     QUXIBAOZHI(SHANGQINGYE)LIANG0.11BEITIJIDE10x cytoplasmic extract bufferJIARUHUNHE.

2.     LIXIN16,000rpm, 1hr, JIANGSHANGQINGYEDAORUTOUXIMONEI, ZHIMONEIWAIDAODIANDUXIANGTONGWEIZHI.

3.     QUCHUCUIQUWU, LIXIN15,000rpm, 20min, BAOLIUSHANGQING, BAOCUNYU-70℃.

PEIFANG:

10x cytoplasmic extract buffer:

0.3M HEPES, pH7.9, 4℃

1.4M KCl

0.03M MgCl2

Dialysis buffer:

20mM HEPES, pH7.9, 4℃

20﹪glycerol

0.2mM EDTA

0.2mM PMSF (phenyl methylsulfonyl fluoride)

※ CIHUAHEWURONGYUisopropanol, YINGXIANDIAO0.2MDECHUCUNYEBEIYONG, ZAISHIYONGQIANCAIDIRU.

0.5mM DTT (Dithiothreitol)

※ BIXUSHIYONGQIANCAIJIARU.

High salt buffer:

20mM HEPES, pH7.9, 4℃

25﹪glycerol

1.5mM MgCl2

0.8, 0, 1.2, 1.4HUO1.6M KCl (YONGYUCESHIHEZHONGNONGDUXIAKECUIQUZUIGAOLIANGDEWUZHI, RUOWUZUGOUSHIJIAN, QU1.0HUO1.2MSHIZHI)

Hypotonic buffer:

10mM HEPES, pH7.9, 4℃

1.5mM MgCl2

10mM KCl

0.2mM PMSF (SHIYONGQIANJIARU)

0.2mM DTT (SHIYONGQIANJIARU)

Low salt buffer:

PEIFANGTONGhigh salt buffer, ERKClYONG0.02MJIKE.

FUZHU:

1.     YICIFANGFACEDEJIEGUOYUE8mg/mlXIBAODANBAIZHI.

2.     ZUODNA-ProteinJIAOHUZUOYONGYANJIU, FANGFAWEIJIANGHEHUOXIBAOZHITIQUZHIDANBAIZHIYUXIANGYANJIUDEDNAPIANDUANJIEHE, BINGYIDIANYONGCHULI, SHOUJIEHEDEDNAHUIPAODEMAN. LINGWAI, YIKEYIDnase ICHULI, FANYOUJIEHEDEDNABUSHOUQIEGE, FANGFAHENDUO, RONGHOUZAIJIESHAO.

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