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上海乾思生物科技有限公司
中級會員 | 第17年

18916423352

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Western Blotting技術(shù)服務(wù)|實(shí)驗(yàn)技術(shù)服務(wù)

時間:2015/7/1閱讀:192
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關(guān)鍵詞:Western Blotting技術(shù)服務(wù)|實(shí)驗(yàn)技術(shù)服務(wù)
簡介:世界*品牌Western Blotting技術(shù)服務(wù)|實(shí)驗(yàn)技術(shù)服務(wù)原裝,,質(zhì)量保證,*,。
Western Blotting技術(shù)服務(wù)|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,我們也會提供相應(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:
免疫蛋白質(zhì)印跡(Western blotting)是根據(jù)抗原抗體的特異性結(jié)合檢測復(fù)雜樣品中的某種蛋白的方法,。寶賽生物為您提供全套的常規(guī)免疫印記技術(shù)服務(wù),同時本公司采用**的LICOR Odyssey雙色紅外激光成像系統(tǒng)和雙色熒光染料標(biāo)記的二抗推出高靈敏度的紅外激光免疫印跡服務(wù),。與常規(guī)化學(xué)發(fā)光方法比較,,靈敏度可達(dá)到皮克級(pg),還具有雙色成像,、定量,、成像清晰的優(yōu)點(diǎn)。同時具備功能強(qiáng)大的軟件系統(tǒng),,可快速判定樣品分子量大小并進(jìn)行定量分析,。
主要實(shí)驗(yàn)步驟如下: 
1. 蛋白質(zhì)抽提
    a.實(shí)驗(yàn)對象為組織樣品,取適量(250~500mg)新鮮組織樣品或正確保存的組織樣品,,加1ml含蛋白酶抑制劑的總蛋白抽提試劑(或核蛋白抽提試劑),,勻漿后抽提總蛋白(或核蛋白)。
    b.實(shí)驗(yàn)對象為細(xì)胞樣品,,每份樣品取1×106~1×107細(xì)胞,,PBS清洗細(xì)胞,,去PBS加0.1ml~1ml含蛋白酶抑制劑的總蛋白抽提試劑(或核蛋白抽提試劑)抽提總蛋白(或核蛋白)。 
2. 蛋白質(zhì)定量
    按KCTMBCA蛋白質(zhì)定量試劑盒操作說明操作,,測定樣品濃度,。
3. 變性聚丙烯酰胺不連續(xù)凝膠電泳(SDS-PAGE)  
    將準(zhǔn)備好的樣品液和生物素標(biāo)記的蛋白質(zhì)分子量標(biāo)準(zhǔn)分別上樣,標(biāo)準(zhǔn)加進(jìn)*個孔中,,電泳分離蛋白,。
4. 蛋白質(zhì)轉(zhuǎn)移到硝酸纖維膜或PVDF膜
    按Bio-Rad蛋白轉(zhuǎn)移裝置說明組裝濾紙凝膠纖維素夾層,30mA恒流條件下,,4°C轉(zhuǎn)移,。
5. 膜的封閉和抗體孵育
    a.膜在5%BSA溶液中室溫孵育1小時以封閉膜上的非特異結(jié)合。
    b.封閉過的膜加入一級抗體室溫孵育1.5小時,,抗原抗體結(jié)合,。 
    c.加入HRP標(biāo)記的二級抗體以結(jié)合一級抗體及HRP標(biāo)記的抗生物素抗體以結(jié)合分子量標(biāo)準(zhǔn),室溫孵育膜1小時,。加入HRP標(biāo)記的GAPDH抗體可同時檢測GAPDH含量。,。
6. 結(jié)果檢測
    化學(xué)發(fā)光法檢測,,膜與化學(xué)發(fā)光底物孵育,經(jīng)X膠片曝光顯影,。圖片掃描保存為電腦文件,,并用ImageJ分析軟件將圖片上每個特異條帶灰度值的數(shù)字化。
7. 數(shù)據(jù)分析
    目的蛋白的灰度值除以內(nèi)參GAPDF的灰度值以校正誤差,,所得結(jié)果代表某樣品的目的蛋白相對含量,。
8. 提供實(shí)驗(yàn)報告
包括詳細(xì)的實(shí)驗(yàn)方法及免疫印跡實(shí)驗(yàn)結(jié)果的相關(guān)圖表.

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