?supernatant

supernatant發(fā)音

英：[?sju:p?'ne?t?nt]　　美：[?su:p?'ne?t?nt]

英：　　美：

supernatant中文意思翻譯

上層清液

supernatant詞形變化

副詞: supernally |

supernatant同義詞

ethereal | celestial | aerial

supernatant反義詞

infernal

supernatant常見(jiàn)例句

1 、Methods Expression vector pCTLA 4/Ig was transfected into COS 7 with lipid reagent. The expression of fusion protein in the supernatant of the cell culture media was detected with double antibody sandwich ELISA.───方法 將pCTLA 4/Ig表達質(zhì)粒轉染COS 7細胞 ,雙抗體夾心ELISA檢測細胞培養上清中融合蛋白表達 ;

2 、The results show that when PAC was used as the sole coagulant, supernatant with low turbidity can be achieved, however, only at very high dosage.───中文摘要臺灣地區因具高山陡峻、河川短促之地理特性，河川流速湍急，河岸沖刷嚴重，且洪、枯水量差異大；

3 、In this study,the gene of DCD-1L,a derivant of Dermcidin,was first cloned into plasmid pPIC9 and expressed in Pichia pastiros GS115.The supernatant extracted from the expressing system had the activities against E.───為了能快速并低成本地獲得該肽 ,DCD 1L基因 ,第一次被克隆到畢赤酵母載體pPIC9中 ,并在畢赤酵母GS1 1 5中進(jìn)行表達。

4 、(2) The supernatant of C6 glioma cells have the signal substance which can induce migration of NSCs in vitro.───(2)C6膠質(zhì)瘤細胞無(wú)血清培養的上清中存在著(zhù)能夠誘導神經(jīng)干細胞遷移的物質(zhì)。

5 、Methods Mouse IL-3 cDNA was cloned to retrovirus vector pLXSN and then the recombinant IL-3 cDNA was transfected on PA317 cells with lipofectin. The cells was selected with C418 after BMS was transfected by the supernatant containing virus.───方法 將小鼠IL－3cDNA克隆到逆轉錄病毒載體（pLX－SN）上，然后將重組的IL－3 cDNA用脂質(zhì)體轉染到包裝細胞PA317上，G418篩選后得到抗性克隆，將含病毒的上清液轉染骨髓基質(zhì)細胞。

6 、Two inactivated vaccine with different antigen concentration were prepared by using the supernatant materials obtained from centrifuging the ground emulsion of SPF whole embryos infected with AEV Van Rockel strain.───將感染了AEV－VanRockel株的SPF全胚磨制的乳劑離心分離，取上清液制成抗原含量不同的兩種滅活疫苗。

7 、Keywords EM bacteria material;garbage supernatant;active soil;bacteria membrane;───EM菌劑;垃圾滲瀝液;活性污泥;生物膜;

8 、Methods The LDH content in supernatant of endothelial cells after preservation-rewarming to determine the injury after preservation-rewarming.───方法 1. 測定內皮細胞經(jīng)保存-復溫后上清中乳酸脫氫酶（LDH）濃度，比較保存-復溫后損傷；

9 、Quantibody arrays can be used in cell culture supernatant, serum, plasma, CSF, tissue lysate, and other body fluid.───Quantibody芯片可用于培養細胞上清、血清、血漿、集落刺激因子、組織勻漿以及其他生物學(xué)液體。

10 、Experimental grouping is as follows: group A: DC+T+SMMC-7721 supernatant;───實(shí)驗分組如下：A組: DC+T+ SMMC7721上清；B組: DC+T。

11 、Study on the Influence of Immunosuppression of Supernatant of S180 by AMB and PUPS───中藥黃芪注射液、豬苓多糖注射液影響S180腫瘤細胞培養上清免疫抑制作用的研究

12 、In addition, some culture media supernatant of directly irradiated cells were transferred to the non-irradiated cells culture medium, forming the group P2.───另將直接受照細胞培養液轉移至未受照細胞培養瓶中形成旁效應2組。

13 、TES could maitain 85 cell"s subsistence.Maybe supernatant affect growth cycle of T cell line.───上清液能維持85細胞生存，可能與影響T細胞生長(cháng)周期有關(guān)。

14 、The carcinogenetic and tumor-promoting effects of concentrated Helicobacter pylori culture supernatant in rats.───幽門(mén)螺桿菌培養濾液誘癌和促癌作用的實(shí)驗研究。

15 、Study on Antitumor Activities in Supernatant Preparations from T-AK Cells in Culture───T-AK細胞上清液抑瘤活性的研究

16 、breast carcinoma supernatant───乳癌上清液

17 、Submandibular tissues of rats were incubated in vitro and different concentration of FSH was added.Enzyme link immunoassay was used to measure EGF and NGF in supernatant.───體外孵育大鼠下頜下腺組織并給予不同濃度FSH,應用酶聯(lián)免疫分析方法檢測上清液中NGF和EGF的含量。

18 、Keywords Lactobacillus acidophilus;spent culture supernatant （SCS）;diarrhea;───嗜酸性乳桿菌;耗盡培養上清液;腹瀉;

19 、Keywords olfactory ensheathing cells（OECs）;supernatant;glial cell line-derived neurotrophic factor （GDNF）;content;───嗅鞘細胞;上清;膠質(zhì)源性神經(jīng)生長(cháng)因子（GDNF）;含量;

20 、Once precipitated, the sample was centrifuged and the supernatant was removed.───如有沉淀，離心樣品，出去上層漂浮物。

21 、After enzymatic hydrolysis,the yield of XOS could be up to 84.93% of total carbohydrates of the supernatant.───上清液經(jīng)木聚糖酶酶解后,低聚木糖的含量可達到上清液總糖的84.93%。

22 、The supernatant was examined by SDS-PAGE protein electrophoresis .───取上清進(jìn)行SDS-PAGE蛋白質(zhì)電泳。

23 、Keywords Cordyceps militaris;Fermentation supernatant;Cordycepin;Adenosine;Analysis by HPLC;───北冬蟲(chóng)夏草;發(fā)酵液;蟲(chóng)草素;腺苷;HPLC分析;

24 、Keywords marrow stromal cells;supernatant;umbilical cord blood;hemapoietic cells;expand in vitro;suface markers;───關(guān)鍵詞骨髓基質(zhì)細胞;上清;臍血;造血細胞;體外擴增表面標志;

25 、Effects of spent culture supernatant of Lactobacillus acidophilus on intestinal flora in mice with antibiotic-associated diarrhoea───嗜酸性乳桿菌培養上清液對抗生素相關(guān)性腹瀉小鼠腸道菌群的影響

26 、Methods Peripheral blood mononuclear cells(PBMC) were cultured with different concentration of tumor cell culture supernatant.───方法將不同濃度腫瘤細胞培養上清液與體外分離的人淋巴細胞一起培養;

27 、The anisotropic of the supernatant was smaller than that of the bottom layer.───存放還引起粘度下降，估計這與液晶生成會(huì )使粘度下降有關(guān)。

28 、The culture supernatant(SN)of T6.6could augment NK activity significantly and the augmen-tation was more efficient on combination with IL-2.Theaugmentation could be blocked by anti-IL- 6 MoAb.───T6.6培養上清能顯著(zhù)增強自然殺傷（NK）活性，當與IL－2合用時(shí)增強效果更佳，抗IL－6單抗能阻斷這種增強作用。

29 、Retrovirus mediated gene transfection was carried out. First, the package cell line PT67 was transfected by lipofectAMINE TM2000, so as to obtain recombinant retrovirus supernatant, which was directly used in the transfection of MSCs.───反轉錄病毒介導的基因轉染,首先利用LipofectAMINETM 2000轉染包裝細胞系PT67,獲得重組反轉錄病毒上清,然后用病毒上清直接感染骨髓間充質(zhì)干細胞。

30 、Keywords Lactobacillus acidophilus;Spent culture supernatant;Antibiotic-associated diarrhoea;Intestinal flora;───嗜酸性乳桿菌;耗盡培養上清液;抗生素相關(guān)性腹瀉;腸道菌群;

31 、Effects of MSB 1 Culture Supernatant to MDV───MSB-1培養物上清液對MDV的作用

32 、The cytopathic effect(CPE),beating frequency of myocardial cells and the lactate dehydrogenase(LDH)levels in supernatant was tested at day 2,3,5 respectively after the treatment.───在處理的d 2,3,5觀(guān)察各組細胞病變效應(CPE)、細胞搏動(dòng)頻率并測定細胞上清液中乳酸脫氫酶濃度。

33 、In addition, BPF and GM-CSF could also be found in the supernatant collected on 24th day of culture.───培養第24天的上清液中還可檢出BPF和GM-CSF活性。

34 、The IL-6 secretion and IL-6 mRNA expression were significantly increased in FLS cultured with the supernatant from U937 cell treated with LPS.───LPS刺激的U937細胞培養液上清液可明顯增強FLS中IL 6蛋白分泌及mRNA表達 ;

35 、cardiac muscle lysate supernatant───心肌上清液

36 、It was found that the fixed cell of NIH3T3/TM TNFm, the supematant of NIH3T3/S TNFm, both fixed cell and supernatant of NIH3T3/Wt TNF could effectively kill tumor cell H22 in vitro.───在體外 ,NIH3T3/TM TNFm的固定細胞、NIH3T3/S TNFm的上清、NIH3T3/Wt TNF的固定細胞和上清均可有效殺傷腫瘤細胞H2 2。

37 、cultural supernatant───培養上清

38 、The recombinant protein, in the culture supernatant, designated C2-45 (IgG1), was purified by affinity chromatography, and characterized for the CEA binding specificity and the biological activity of CDC and ADCC.───應用免疫親和層析法,純化培養上清中表達的重組蛋白C245(IgG1),并鑒定其與CEA結合的能力和CH的生物學(xué)功能CDC及ADCC。

39 、Results COS?7 cells transfected with constructed PCIneo?hTPO expressed and secreted hTPO up to 48.28 ng/ml in the supernatant.───應用hTPOcDNA轉染的COS－7細胞表達產(chǎn)物對實(shí)驗性小鼠血小板減少癥進(jìn)行腹腔注射。 結果hTPOcDNA不僅可在COS－7細胞中獲得表達，且分泌到細胞培養上清中，最高表達量為48.28ng/ml。

40 、Methods The inhibition activity on P.gingivalis,P.intermedia and F.nucleatum of S.sanguis suspension and supernatant were measured by Pucci test and Agarose gel diffusion method.───方法分別采用交叉雙層培養基法和微量瓊脂糖彌散法測定血鏈球菌全菌細胞和培養上清液對牙齦卟啉單胞菌、中間普雷沃菌、具核梭桿菌的拮抗作用。

41 、Take 150 ml of supernatant for pH check and SCOD and sulfate analysis.───取出150ml上清液測定pH、SCOD和硫酸鹽。

42 、Methods: The culture supernatant of cord blood LAK cells and diluted E.coli suspension were cocultured in agar medium and the colonies of E.coli were counted.───方法將臍血LAK細胞培養上清液和稀釋的埃希氏大腸桿菌懸液混合,在培養皿內培養不同時(shí)間,計數各個(gè)培養皿內細菌菌落數。

43 、Spent culture supernatant(SCS)───發(fā)酵上清液

44 、ERP-SBR BIO-CHEMICAL PROCESS FOR NITROGEN AND PHOSPHORUS REMOVAL BY DRAINING OUT ANAEROBIC RICH PHOSPHATE SUPERNATANT───排出厭氧富**污水生物化學(xué)除**脫氮ERP-SBR系統研究

45 、when growing in liquid culture,its supernatant fluid was clear with granular sediment in it and the sediment couldn't disperse completely after shaking.───在液體培養基中,上清液清亮并有顆粒狀沉淀,搖動(dòng)沉淀不能完全散開(kāi)。除此之外,該菌的其它特性均符合典型大腸桿菌的特征。

46 、In addition to residual turbidity in the supernatant, the zeta potential of the floc formed and the volume of sludge were also monitored.───故后續之研究可考量添加助凝劑于濃縮槽，或將濾床反沖洗水與沉淀污泥分開(kāi)處理。

47 、The elevation of MDA content of hepatocytes and LDH level in supernatant of hepatocytes induced by CP were restored remarkably by HSS.───HSS明顯降低由CP升高的肝細胞培養上清液中LDH水平和肝細胞MDA含量。

48 、The fluorescence intensity of the supernatant was measured by spectrofluorometer. The data were described by two types of binding sites model,the binding parameters of BSM-R8 were obtained by Scatchard plots.───數據符合兩種不同結合位點(diǎn)的模型,經(jīng)Scatchardplots處理后得到R8與BSM的結合參數。

49 、SDS-PAGE results indicated that there was a clear target band in Muts and Mut+ recombinant Culture supernatant after 48 hours culture respectively.───實(shí)驗結果表明,誘導表達48h后,Mut+和MutS重組子表達的產(chǎn)物在SDS-PAGE膠上都出現了清晰的目的帶。

50 、The supernatant was collected for HCG assay. [Results] Ectopic HCG increased in EAC and JEC with prolonged culture period, especially in JEC.───[結果]隨著(zhù)培養時(shí)間的延長(cháng),兩株細胞自分泌的HCG量有所增加,特別JEC株表現更加明顯。

51 、Low Radioactivity Used in Night Soil Supernatant Treatment───利用低放射性處理糞便上清液技術(shù)

52 、The endothelial cells incubated with the supernatant of MBP-stimulated PBMC can promote T cell proliferation───MBP刺激的PBMC培養上清對內皮細胞促進(jìn)T細胞增殖的作用

53 、FL in the supernatant was detected by ELISA. MTT was adopted to determine the sensitivity of MCF 7/TK FL cells to GCV (ganciclovir).───方法 構建以IRES(內部核糖體進(jìn)入位點(diǎn) )相連的TK與FL的真核表達質(zhì)粒 ,脂質(zhì)體法轉入人乳腺癌細胞株MCF 7中。

54 、Isolation of rat hepatocytes and effect of BRL cell culture supernatant on rat hepatocytes proliferation───大鼠肝細胞的分離及BRL細胞培養上清對原代肝細胞增殖的影響

55 、Neutrophil chemotactic activity in sputum supernatant was higher than the pool of normal subjects both in chronic asthma and COPD patients.───慢性哮喘患者和COPD患者痰液上清液中的中性粒細胞趨化活性均較正常人高。

56 、AN AGGLUTINATE SUBSTANCE IN THE SUPERNATANT OF HUMAN MYELOID LEUKEMIC CELLS(HL-60)───人早幼粒白血病細胞hl_(60)能夠產(chǎn)生具有凝集活性的物質(zhì)

57 、The test status of calcium removal of carbide slag supernatant and sulfur and phosphorus removal of waste water was introduced.───介紹了電石渣上清液除鈣及工藝廢水除硫、**的試驗工作。

58 、anaerobic phosphate-enriched supernatant───厭氧富**上清液

59 、AP has caused widespread concern in the Derby, Guangdong, Shenzhen and drink supernatant team's 0-1 win against Guangzhou Shayuergui medical team.───搜狐體育訊引起廣泛關(guān)注的廣東德比中，深圳上清飲隊以0-1的比分負于廣州醫藥隊鎩羽而歸。

60 、Effect of sixpetal clematis root parenteral solution on interlukin-1 level in osteoarthritis joint fluid and chondrocyte culture supernatant───威靈仙注射液對骨關(guān)節炎關(guān)節液與軟骨白介素-1水平的影響

61 、Compared with normal MsC, the number of G2-M cells treated with DCN-containing supernatant decreased to 35%(P───DCN上清明顯抑制正常MsC的增殖,G2-M期細胞數明顯減少,僅為對照組的35%(P

62 、GT1-7 cells were incubated with galanin at a cohort of concentrations for different time points and GnRH released in the supernatant was determined by RIA.───(2 )將不同濃度的甘丙肽以不同時(shí)間與GT1- 7細胞孵育 ,用RIA法測定細胞上清液中GnRH含量。

63 、Bifidobacterium-cultivated supernatant───雙歧桿菌培養上清

64 、The mIL 12 in the supernatant was detected by ELISA after the pCmIL 12 was transfected into COS 7 cells.───p Cm IL - 12在體外轉染 COS- 7細胞后 ,經(jīng) EL ISA證實(shí)有 m IL- 12表達 ,其表達上清能在體外明顯增強小鼠 NK細胞活性。

65 、GTCC application on treatment for supernatant layer of vinasse of corn alcohol───殼聚糖季銨鹽處理玉米酒精清糟液的研究

66 、Effect of Helicobacter pylori broth culture supernatant on cell proliferation in rat gastric mucosa───幽門(mén)螺桿菌培養上清液對大鼠胃黏膜上皮細胞增殖的影響

67 、Keywords BNR;anaerobic supernatant;model;phosphorus recovery;strippant;struvite;───厭氧池上清液;模型;**回收;分離液;鳥(niǎo)糞石;

68 、MaAb culture supernatant───McAb培養上清

69 、You centrifuge the solution, decant the supernatant, and discard the pellet.───你然后對溶液離心，慢慢倒出上清，棄沉淀。對上清液你加更多的硫酸銨。

70 、Carefully transfer the supernatant of the flow-through to a new microcentrifuge tube (not supplied) without disturbing the cell-debris pellet in the collection tube.───小心將液體的上清移至一新的微量離心管中，不要攪動(dòng)收集管中的細胞殘渣沉淀。

71 、supernatant town is a millennium-old ancient town, located in the scenic Shangqing Gong, 25 km away from the urban Yingtan.───上清古鎮是一個(gè)具有千年歷史的古鎮，位于上清宮景區內，距鷹潭市區25公里。

72 、Keywords Lactic acid bacteria;supernatant;human pathogens;inhibitory action;───關(guān)鍵詞乳酸菌;上清液;人源腸道致病菌;抑菌特性;

73 、You proceed with the purification using the supernatant fraction that contains mostly intact mitochondria.───你繼續用含大多為完整的線(xiàn)粒體的上清液純化。

74 、However, Shenzhen supernatant drink in the team after getting over by Li Jianhua through a plug out of the ball, Johnson missed long-range De Qiuhou.───但是深圳上清飲隊在緩過(guò)勁后，由李建華塞出了一個(gè)穿越球，約翰森得球后遠射偏出。

75 、The splenocytes stimulated with supernatant from syngeneic or allogeneic MLTC of H22 tumor cells generated lower proportion of CD4+CD25+ T cells than that of D5 tumor cells（p───D5刺激的同系脾細胞上清培養的脾細胞中的CD4+CD25+T細胞與同種脾細胞上清相比明顯增多（p

76 、Using the saliva supernatant, they isolated RNA, performed two-round linear amplification with T7 RNA polymerase, and applied human genome U133A microarrays for profiling human salivary transcriptome.───使用唾液離心后的上清液，他們分離出RNA，以T7 RNA聚合脢進(jìn)行兩個(gè)回合的線(xiàn)性放大作用，以及應用人類(lèi)基因體U133A微陣列來(lái)分析人類(lèi)唾液轉錄體。

77 、The clear supernatant of rat macrophage cultivated in DMEM culture fluid containing 12.5 mol/L glucose could markedly promote the hyperplasia of mesangial cell(P───大鼠巨噬細胞在含12.5mol/L葡萄糖的DMEM培養液中培養24h,其上清明顯促進(jìn)系膜細胞增殖的(P

78 、RT PCR result showed that after 4h,12h,24h incubation respectively, 30% supernatant can not increased insulin messenger RNA levels significantly at the present of 16.7mmol/L glucose compared with INS 1 cells cultured with glucose alone.───RT PCR結果顯示 ,在 1 6 7mmol L葡萄糖刺激 4、1 2和 2 4h后 ,30%上清液對INS 1細胞胰島素mRNA水平均無(wú)明顯影響。

79 、Keywords homogenate supernatant;dentritic cells;endothelialization;───勻漿上清;樹(shù)突狀細胞;內皮化;

80 、The study shows the rhein can appreciably increase the survival rate of acinar cell and lower the levels of LDH,AMY and NAG in the supernatant.───大黃酸可提高相應腫瘤壞死因子濃度下的胰腺細胞的細胞活率,降低培養上清中 LDH、AMY 和 NAG 的水平。

GST pull down 實(shí)驗細節問(wèn)題求教

GST pull-down實(shí)驗是一個(gè)行之有效的驗證酵母雙雜交系統的體外試驗技術(shù),近年來(lái)越來(lái)越受到廣大學(xué)者的青睞。其基本原理是將靶蛋白-GST融合蛋白親和固化在谷胱甘肽親和樹(shù)脂上,作為與目的蛋白親和的支撐物,充當一種“誘餌蛋白”,目的蛋白溶液過(guò)柱,可從中捕獲與之相互作用的“捕獲蛋白”(目的蛋白),洗脫結合物后通過(guò)SDS-PAGE電泳分析,從而證實(shí)兩種蛋白間的相互作用或篩選相應的目的蛋白,“誘餌蛋白”和“捕獲蛋白”均可通過(guò)細胞裂解物、純化的蛋白、表達系統以及體外轉錄翻譯系統等方法獲得。此方法簡(jiǎn)單易行,操作方便。 GST：谷胱甘肽巰基轉移酶(glutathione S-transferase) 分子克隆第三版有詳細介紹，結合其中的示意圖很容易理解 GST融合蛋白沉降技術(shù)利用了GST對谷朧甘膚偶聯(lián)球珠的親和性，從非相互作用蛋白的溶液中純化相互作用蛋白。 GST融合的探針蛋白從細菌中表達和純化，并平行制備細胞裂解液(可被35S標記或非標記)，再將GST融合蛋白探針和細胞裂解液在谷朧甘膚瓊脂糖球珠存在下混合并孵育，以使蛋白結合。GST融合探針蛋白和任何結合分子被離心收集，獲得的混合物經(jīng)洗滌后，用過(guò)量游離的谷朧甘膚洗脫或直接在SDS-PAGE上樣緩沖液中煮沸。蛋白質(zhì)經(jīng)SDS-PAGE分離后進(jìn)行下一步的western印跡、放射自顯影及蛋白質(zhì)染色分析。GST沉降技術(shù)對探測蛋白在溶液中的相互作用特別有用，而這種相互作用在膜的分析中可能是檢測不到的。 GST沉降實(shí)驗通常有兩種應用:確定融合(或探針)蛋白與未知(或靶)蛋白間新的相互作用(Kaelin et al. 1991, Grlinick and Chao 1996),以及證實(shí)探針蛋白與已知蛋白質(zhì)間可疑的相互作用(例子請見(jiàn)Posern et al. 199$, Grgureaich et al. 1999, Hunteret al. 1999, Sun et al. 1999)。這兩種實(shí)驗的設計和實(shí)施都有所不同。  實(shí)驗目的：體外檢測蛋白質(zhì)與蛋白質(zhì)之間相互作用。用于驗證兩個(gè)已知蛋白的相互作用，或者篩選與已知蛋白相互作用的未知蛋白。 實(shí)驗原理：利用重組技術(shù)將探針蛋白與GST（Glutathione S transferase）融合，融合蛋白通過(guò)GST與固相化在載體上的GTH（Glutathione）親和結合。因此，當與融合蛋白有相互作用的蛋白通過(guò)層析柱時(shí)或與此固相復合物混合時(shí)就可被吸附而分離. 試劑：NaCl， KCl，Na2HPO4，KH2PO4，Triton-100，IPTG(Merck分裝)，PMSF（Amersco），Cocktaier（Merck 539134），Immobilized Glutathione（PIERCE 15160） 實(shí)驗操作程序： 材料及試劑 探針蛋白與GST融合的原核蛋白，裂解的細胞蛋白，或者組織蛋白提取物

細胞蛋白裂解液，洗脫液： PBS及PBS+1%Triton-100 PBS (1L) NaCl： 8g KCl： 0.2g Na2HPO4： 1.44g KH2PO4： 0.24g 加入800ml蒸餾水，用HCl調節溶液的pH值至7.4，最后加蒸餾水定容至1L即可，高溫高壓滅菌！4℃保存備用！ PMSF(苯甲基磺酰氟) MW:174.19 工作濃度0.1-1mM，這里使用1mM，儲存濃度100mM，將0.174g PMSF溶于10ml無(wú)水乙醇混勻即可！保持于-20℃或者4℃ （以下流程僅供研究已知原核蛋白A和真核過(guò)表達蛋白B相互作用） 1：原核融合蛋白A的獲得 1.1：將編碼蛋白A與GST的重組質(zhì)?；DBL21(DE3)菌株 1.2：挑取單個(gè)克隆到含有5mlLB(+100ug/mlAmp)的10ml試管里，37℃培養過(guò)夜 1.3：將培養菌液轉移到含有500ml LB(+100ug/mlAmp)的1L錐形瓶中，37℃，225rpm培養至OD600≈1.0-1.5左右，加入適當濃度的IPTG，在適當溫度下培養適當時(shí)間（誘導條件需要根據不同的蛋白做調整）.6000g，10分鐘，4℃離心收集細菌，去盡上清，將菌體至于-20℃放置O/N 1.4：室溫凍融菌體，馬上置于冰上，每500ml培養液加入10-20ml細菌裂解液（PBS+1%Triton-100+PMSF），吹打混勻 1.5：冰上超聲破碎，開(kāi)2秒，停9秒，總40-60分鐘。至裂解液充分清涼 1.6：11000rpm，15分鐘，4℃離心分離上清， -80℃保存備用 2：真核融合蛋白B的獲得

2.1：將編碼B蛋白的堿基序列克隆到編碼標簽蛋白（如HA,或者myc）的真核表達載體上，進(jìn)行細胞轉染 xq 3：48小時(shí)后，取適量融合蛋白GST-A冰上凍融 4：取50-70ulGST-Beads（Immobilized Glutathione）到EP管中，用800ul PBS+1%Triton-100潤洗一次，將凍融融合蛋白GST-A與之混勻，4℃層析柜旋轉結合1小時(shí)。 5：PBS+1%Triton-100洗3次，PBS洗3次。留取20ul（ PBS+Beads）作Offer。 6：同時(shí)，裂解真核融合蛋白B，去盡培養基，用PBS(RT)洗一次，加入300ul裂解液（以6well為例，PBS+1%Triton-100+ Cocktaier），4℃放置30分鐘。 7：吹打收集至1.5mlEP管，超聲破碎。13000rpm，15分鐘，4℃離心取上清。 8：BCA蛋白定量（optional）留取20ul做Offer，其余樣品加入到已純化蛋白的EP管中，用PBS補足液體到600ul左右，以便蛋白之間能充分結合！4℃旋轉結合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分鐘，高速離心，Run –SDS PAGE做Western Blot檢測。用HA或者myc Blot。 注意GST-pull down的對照問(wèn)題。（以A-GST和B-6*His為例） 1. 跑SDS-PAGE時(shí)點(diǎn)一個(gè) input的蛋白（即B-6*His） 用于做**對照 看一下western操作過(guò)程中，試劑，操作步驟有沒(méi)有問(wèn)題。 2. 谷胱甘肽瓊脂珠上只固定GST蛋白，input B-6*His，用于做陰性對照 看一下是否GST會(huì )與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

post-translation modification是什么意思

post-translation modification_翻譯

post-translation modification

[詞典] [醫]翻譯后修飾（分）;