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蛋白提取及WB
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==蛋白提取== #弃去贴壁细胞的培养基,1 mLPBS清洗,弃去,加入400 ulPBS, #胰酶或者细胞刮板分离底部粘璧cell,移液枪转移至上述ep管中。流式不可采用刮板,会损伤cell;样品间刮板用PBS清洗; #配制细胞裂解液:配制100 mM的PMSF in IPA,临用前1:100加入用商品化的细胞裂解液。 #按照12孔板每孔cell加入25 ul细胞裂解液的比例向ep管加入cell裂解液,4 ℃裂解10-30min、14k rpm离心15min。(6孔板每孔50ul裂解液) #可取部分上清用于BCA测定 #用上清稀释loading buffer(按照商品化loading buffer 的要求),金属浴or沸水浴 5 - 10min,防止ep管加热过程开盖 #分装,-40--80℃保存,防止降解。
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