Question Detail
When extracting protein from animal tissues, how long does PMSF remain effective after being added to the lysis buffer?
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Tags：#Phenylmethylsulfonyl fluoride #Protease inhibitor #Animal tissue #Protein extraction #Methodology
All Answers4
T
Tube
2026-09-13
浓度方面，常规终浓度1mM足够。100mg小鼠腿肌建议用0.8-1ml裂解液，每ml加10μl 100mM母液。若目标蛋白易降解，可先做1mM与2mM对比，但2mM可能影响后续酶活，不建议盲目提高。裂解液pH控制在7.0-7.5，避免碱性加速PMSF水解。
刘
刘文佳8676
2026-09-23
母液配成100mM异丙醇溶液放-20℃很稳定，失效主要发生在稀释到水相之后。可把样品分3-4批，每批裂解液使用前单独加PMSF，不要把PMSF先加到大瓶裂解液里放1小时。腿肌纤维多，可视情况匀浆前加一次，匀浆后视降解情况少量补加。
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LeoScience6186
2026-07-22
担心失效可做时间点对照：同一批裂解液分别在0、30、60min加入PMSF后处理样品，比较目标蛋白条带。如果60min与0min差异不大，说明冰上条件可接受；若有降解则改为分批加。同时避免把PMSF配在水里长期存放，使用前再从异丙醇母液稀释。
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Lab7669
2026-06-28
建议分批加。PMSF在水相裂解液里半衰期较短，冰上放置1小时活性可能明显下降。20多个样品不要一次全加。按100mg组织加约0.5-1ml裂解液，临用前每管补加PMSF至终浓度1mM，100mM母液按1:100稀释。先研磨的样品先加，后面批次用时再加。
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