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SRB|實(shí)驗(yàn)技術(shù)服務(wù)

時(shí)間:2015/7/1閱讀:307
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關(guān)鍵詞:SRB|實(shí)驗(yàn)技術(shù)服務(wù)
簡介:世界*品牌SRB|實(shí)驗(yàn)技術(shù)服務(wù)原裝,,質(zhì)量保證,*,。
SRB|實(shí)驗(yàn)技術(shù)服務(wù)——pCzn1質(zhì)粒+Arctic Express宿主菌低溫表達(dá)體系,。
我們具備豐富的蛋白表達(dá)設(shè)計(jì)經(jīng)驗(yàn)及實(shí)驗(yàn)操作技巧,,承諾客戶不成功不收費(fèi),。在獲得重組蛋白產(chǎn)品的同時(shí),,我們也會(huì)提供相應(yīng)的原始數(shù)據(jù)和原始圖片,不需要再額外花費(fèi)精力重復(fù)實(shí)驗(yàn)。另一方面,,我們所有的實(shí)驗(yàn)數(shù)據(jù)真實(shí)可信,,我們提供原始的表達(dá)菌株和克隆質(zhì)粒,,客戶可以依據(jù)我們的實(shí)驗(yàn)報(bào)告重復(fù)我們的實(shí)驗(yàn)結(jié)果。
產(chǎn)品品牌:   http://www.,。,。com/goodsid/fenleiyi/2868603/1.html    
測序?qū)嶒?yàn)流程:
1.SRB檢測方法的原理
磺酰羅丹明B(Sulforhodamine B,SRB)比色法,,主要用來檢測細(xì)胞增殖情況,。SRB是一種粉紅色陰離子染料,易溶于水,,在酸性條件下可特異性地與細(xì)胞內(nèi)組成蛋白質(zhì)的堿性氨基酸結(jié)合;在540 nm波長下產(chǎn)生吸收峰,,吸光值與細(xì)胞量成線性正相關(guān),故可用作細(xì)胞數(shù)的定量檢測,。SRB染色后不會(huì)像MTT法那樣很容易變色,,細(xì)胞固定染色后在96孔板中可以放置較長時(shí)間,因而受測定時(shí)間的影響較小,。用Tris-base溶液溶解的SRB也可穩(wěn)定較長時(shí)間,。因此不同時(shí)間點(diǎn)固定的96孔細(xì)胞培養(yǎng)板可在同一時(shí)間測定,測定的吸光值結(jié)果不會(huì)受到明顯影響,。吸光值與SRB濃度作圖時(shí),,在OD單位1.5-2.0以下為線性,當(dāng)超出線形范圍時(shí),,稀釋后重新讀數(shù),。雖然SRB法比其他檢測方法操作步驟繁瑣,但是由于時(shí)間可以自己掌握,,不受限制,,因此適用于高通量篩選。
2.SRB檢測的操作步驟
1)細(xì)胞固定:藥物作用時(shí)間終點(diǎn)時(shí),,每孔加入50μL4℃預(yù)冷的TCA溶液(30%,,w/v)固定細(xì)胞,TCA溶液的終濃度為10%,。靜置5 min移入4℃冰箱中固定1 h,,取出用去離子水沖洗5遍,室溫晾干,。
2)染色:待96孔板室溫下晾干后,,每孔加入0.4%(w/v)的SRB染液(1%的乙酸配制)70μL,染色30 min后倒掉染液,,用1%(v/v)乙酸沖洗4次,,去除未結(jié)合的染料,室溫晾干,。
3)檢測:用100μL非緩沖Tris-base堿液(10mM,,pH=10.5)溶解與細(xì)胞蛋白結(jié)合的染料,,水平搖床上振蕩20min,采用酶標(biāo)儀540nm處測定光吸收值,。操作中,,應(yīng)注意洗除染料時(shí)操作需迅速,避免用移液器吸取液體,,防止動(dòng)作過慢造成與細(xì)胞蛋白結(jié)合的染料解吸附,。
3.SRB檢測的計(jì)算公式
根據(jù)美國國立癌癥研究所(National Cancer Institute,NCI)關(guān)于SRB檢測的介紹,,細(xì)胞增殖百分率的計(jì)算存在以下兩種情況:1)Tx-T0>0,PG=100×(Tx-T0)/(C-T0)
2)Tx-T0<0,,PG=100×(Tx-T0)/T0
其中,,
T0:藥物作用前的細(xì)胞經(jīng)過固定、染色后測得平均吸光值;
C:培養(yǎng)基中加有等體積的DMSO,,沒有藥物作用的陰性對照的平均吸光值(陰性對照);
Tx:藥物作用終點(diǎn)時(shí)細(xì)胞經(jīng)過固定,、染色后測得平均吸光值。
PG即Percentage Growth,,是指藥物作用的樣品孔與陰性對照孔中細(xì)胞增殖量的百分率,。

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