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elisa試驗中關于培養(yǎng)細胞樣品裂解的方法

時間:2011/12/23閱讀:480
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 elisa試驗中關于培養(yǎng)細胞樣品裂解的方法

對于培養(yǎng)細胞樣品:
1.融解RIPA裂解液,混勻。取適當量的裂解液,在使用前數(shù)分鐘內(nèi)加入PMSF,使PMSF的zui終濃度為1mM。
2.對于貼壁細胞:去除培養(yǎng)液,用PBS、生理鹽水或無血清培養(yǎng)液洗一遍(如果血清中的蛋白沒有干擾,可以不洗)。按照6孔板每孔加入150-250微升裂解液的比例加入裂解液。用槍吹打數(shù)下,使裂解液和細胞充分接觸。通常裂解液接觸細胞1-2秒后,細胞就會被裂解。
對于懸浮細胞:離心收集細胞,用手指把細胞用力彈散。按照6孔板每孔細胞加入150-250微升裂解液的比例加入裂解液。再用手指輕彈以充分裂解細胞。充分裂解后應沒有明顯的細胞沉淀。如果細胞量較多,必需分裝成50-100萬細胞/管,然后再裂解。
3.充分裂解后,10000-14000g離心3-5分鐘,取上清,即可進行后續(xù)的PAGE、Western和免疫沉淀等操作。
裂解液用量說明:通常6孔板每孔細胞加入150微升裂解液已經(jīng)足夠,但如果細胞密度非常高可以適當加大裂解液的用量到200微升或250微升。

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