酶的活性
酶热失活
什么是热失活?
热失活,英文是Heat inactivation,也叫热灭活,是一种化学现象,特别是在蛋白质和酶的领域中较为常见。它指的是蛋白质或酶在高温条件下失去其活性或功能的过程。这
种失活通常发生在蛋白质或酶的最适反应温度以上。
热失活是停止限制性核酸内切酶反应的便捷方法。例如,在65°C下孵育20分钟,可以使大多数**孵育温度为37°C的限制性核酸内切酶失活。在65°C下不能失活的酶通常可以通过在80°C下孵育20分钟而失活。下表显示了酶是否可以被热灭活以及所需的温度。
对于在65°C或80°C下无法热失活的酶,我们建议使用色谱柱进行清除(例如Monarch®PCR&DNA清除试剂盒),或在琼脂糖凝胶上进行反应,然后提取DNA(建议使用Monarch凝胶提取试剂盒),或进行苯酚/氯仿提取。
下面是一个类似热灭活的典型实验案例。
将含有适当NEBuffer,0.5µg小牛胸腺DNA和5或10µl限制性核酸内切酶的50µl反应混合物在37°C孵育60分钟,然后在65°C或80°C孵育20分钟。将0.5µg底物DNA(通常为λ)添加到反应混合物中,并在酶的**反应温度下孵育60分钟。
利用琼脂糖凝胶电泳观察,如果第二次孵育后底物DNA出现任何的消化(包括完全或部分),均可以认为是不完全热灭活。
酶一般在什么温度下失活?
酶的失活温度和时间大全
酶的名称 | 热失活温度 | 热失活所需时间 |
AatII | 80°C | 20 minutes |
Acc65I | 65°C | 20 minutes |
AccI | 80°C | 20 minutes |
AciI | 65°C | 20 minutes |
AclI | No | -- |
AcuI | 65°C | 20 minutes |
AfeI | 65°C | 20 minutes |
AflII | 65°C | 20 minutes |
AflIII | 80°C | 20 minutes |
AgeI | 65°C | 20 minutes |
AhdI | 65°C | 20 minutes |
AleI | 80°C | 20 minutes |
AleI-v2 | 65°C | 20 minutes |
AluI | 80°C | 20 minutes |
AlwI | No | -- |
AlwNI | 80°C | 20 minutes |
ApaI | 65°C | 20 minutes |
ApaLI | No | -- |
ApeKI | No | -- |
ApoI | 80°C | 20 minutes |
AscI | 80°C | 20 minutes |
AseI | 65°C | 20 minutes |
AsiSI | 80°C | 20 minutes |
AvaI | 80°C | 20 minutes |
AvaII | 80°C | 20 minutes |
AvrII | No | -- |
BaeGI | 80°C | 20 minutes |
BaeI | 65°C | 20 minutes |
BamHI | No | -- |
BamHI | No | -- |
BanI | 65°C | 20 minutes |
BanII | 80°C | 20 minutes |
BbsI | 65°C | 20 minutes |
BbsI | 65°C | 20 minutes |
BbvCI | No | -- |
BbvI | 65°C | 20 minutes |
BccI | 65°C | 20 minutes |
BceAI | 65°C | 20 minutes |
BcgI | 65°C | 20 minutes |
BciVI | 80°C | 20 minutes |
BclI | No | -- |
BclI | 65°C | 20 minutes |
BcoDI | No | -- |
BfaI | 80°C | 20 minutes |
BfuAI | 65°C | 20 minutes |
BfuCI | 80°C | 20 minutes |
BglI | 65°C | 20 minutes |
BglII | No | -- |
BlpI | No | -- |
BmgBI | 65°C | 20 minutes |
BmrI | 65°C | 20 minutes |
BmtI | 65°C | 20 minutes |
BpmI | 65°C | 20 minutes |
Bpu10I | 80°C | 20 minutes |
BpuEI | 65°C | 20 minutes |
BsaAI | No | -- |
BsaBI | 80°C | 20 minutes |
BsaHI | 80°C | 20 minutes |
BsaI | 65°C | 20 minutes |
BsaIv2 | 80°C | 20 minutes |
BsaJI | 80°C | 20 minutes |
BsaWI | 80°C | 20 minutes |
BsaXI | No | -- |
BseRI | 80°C | 20 minutes |
BseYI | 80°C | 20 minutes |
BsgI | 65°C | 20 minutes |
BsiEI | No | -- |
BsiHKAI | No | -- |
BsiWI | 80°C | 20 minutes |
BsiWI | 80°C | 20 minutes |
BslI | No | -- |
BsmAI | No | -- |
BsmBI-v2 | 80°C | 20 minutes |
BsmFI | 80°C | 20 minutes |
BsmI | 80°C | 20 minutes |
BsoBI | 80°C | 20 minutes |
Bsp1286I | 65°C | 20 minutes |
BspCNI | 80°C | 20 minutes |
BspDI | 80°C | 20 minutes |
BspEI | 80°C | 20 minutes |
BspHI | 80°C | 20 minutes |
BspMI | 65°C | 20 minutes |
BspQI | 80°C | 20 minutes |
BsrBI | 80°C | 20 minutes |
BsrDI | 80°C | 20 minutes |
BsrFI | No | -- |
BsrFI-v2 | No | -- |
BsrGI | 80°C | 20 minutes |
BsrI | 80°C | 20 minutes |
BssHII | 65°C | 20 minutes |
BssKI | 80°C | 20 minutes |
BssSI | 80°C | 20 minutes |
BssSI-v2 | No | -- |
BstAPI | 80°C | 20 minutes |
BstBI | No | -- |
BstEII | No | -- |
BstNI | No | -- |
BstUI | No | -- |
BstXI | 80°C | 20 minutes |
BstYI | No | -- |
BstZ17I | No | -- |
BstZ17I | No | -- |
Bsu36I | 80°C | 20 minutes |
BtgI | 80°C | 20 minutes |
BtgZI | 80°C | 20 minutes |
BtsCI | 80°C | 20 minutes |
BtsI | 80°C | 20 minutes |
BtsI-v2 | No | -- |
BtsIMutI | 80°C | 20 minutes |
Cac8I | 65°C | 20 minutes |
ClaI | 65°C | 20 minutes |
CspCI | 65°C | 20 minutes |
CviKI-1 | No | -- |
CviQI | No | -- |
DdeI | 65°C | 20 minutes |
DpnI | 80°C | 20 minutes |
DpnII | 65°C | 20 minutes |
DraI | 65°C | 20 minutes |
DraIII | 65°C | 20 minutes |
DraIII | No | -- |
DrdI | 65°C | 20 minutes |
EaeI | 65°C | 20 minutes |
EagI | 65°C | 20 minutes |
EarI | 65°C | 20 minutes |
EciI | 65°C | 20 minutes |
Eco53kI | 65°C | 20 minutes |
EcoNI | 65°C | 20 minutes |
EcoO109I | 65°C | 20 minutes |
EcoP15I | 65°C | 20 minutes |
EcoRI | 65°C | 20 minutes |
EcoRI | 65°C | 20 minutes |
EcoRV | 80°C | 20 minutes |
EcoRV | 65°C | 20 minutes |
Esp3I | 65°C | 20 minutes |
FatI | 80°C | 20 minutes |
FauI | 65°C | 20 minutes |
Fnu4HI | No | -- |
FokI | 65°C | 20 minutes |
FseI | 65°C | 20 minutes |
FspI | No | -- |
HaeII | 80°C | 20 minutes |
HaeIII | 80°C | 20 minutes |
HgaI | 65°C | 20 minutes |
HhaI | 65°C | 20 minutes |
HinP1I | 65°C | 20 minutes |
HincII | 65°C | 20 minutes |
HindIII | 80°C | 20 minutes |
HindIII | 80°C | 20 minutes |
HinfI | 80°C | 20 minutes |
HpaI | No | -- |
HpaII | 80°C | 20 minutes |
HphI | 65°C | 20 minutes |
Hpy166II | 65°C | 20 minutes |
Hpy188I | 65°C | 20 minutes |
Hpy188III | 65°C | 20 minutes |
Hpy99I | 65°C | 20 minutes |
HpyAV | 65°C | 20 minutes |
HpyCH4III | 65°C | 20 minutes |
HpyCH4IV | 65°C | 20 minutes |
HpyCH4V | 65°C | 20 minutes |
I-CeuI | 65°C | 20 minutes |
I-SceI | 65°C | 20 minutes |
KasI | 65°C | 20 minutes |
KpnI | No | -- |
MboI | 65°C | 20 minutes |
MboII | 65°C | 20 minutes |
MfeI | No | -- |
MluCI | No | -- |
MluI | No | -- |
MlyI | 65°C | 20 minutes |
MmeI | 65°C | 20 minutes |
MnlI | 65°C | 20 minutes |
MscI | 80°C | 20 minutes |
MseI | 65°C | 20 minutes |
MslI | 80°C | 20 minutes |
MspA1I | 65°C | 20 minutes |
MspI | No | -- |
MspJI | 65°C | 20 minutes |
MwoI | No | -- |
NaeI | No | -- |
NarI | 65°C | 20 minutes |
Nb.BbvCI | 80°C | 20 minutes |
Nb.BsmI | 80°C | 20 minutes |
Nb.BsrDI | 80°C | 20 minutes |
Nb.BssSI | No | -- |
Nb.BtsI | 80°C | 20 minutes |
NciI | No | -- |
NcoI | 80°C | 20 minutes |
NcoI | 80°C | 20 minutes |
NdeI | 65°C | 20 minutes |
NgoMIV | No | -- |
NheI | 80°C | 20 minutes |
NlaIII | 65°C | 20 minutes |
NlaIV | 65°C | 20 minutes |
NmeAIII | 65°C | 20 minutes |
NotI | 65°C | 20 minutes |
NotI | 65°C | 20 minutes |
NruI | No | -- |
NsiI | 65°C | 20 minutes |
NsiI | 80°C | 20 minutes |
NspI | 65°C | 20 minutes |
Nt.AlwI | 80°C | 20 minutes |
Nt.BbvCI | 80°C | 20 minutes |
Nt.BsmAI | 65°C | 20 minutes |
Nt.BspQI | 80°C | 20 minutes |
Nt.BstNBI | 80°C | 20 minutes |
Nt.CviPII | 65°C | 20 minutes |
PI-PspI | No | -- |
PI-SceI | 65°C | 20 minutes |
PacI | 65°C | 20 minutes |
PaeR7I | No | -- |
PaqCI | 65°C | 20 minutes |
PciI | 80°C | 20 minutes |
PflFI | 65°C | 20 minutes |
PflMI | 65°C | 20 minutes |
PhoI | No | -- |
PleI | 65°C | 20 minutes |
PluTI | 65°C | 20 minutes |
PmeI | 65°C | 20 minutes |
PmlI | 65°C | 20 minutes |
PpuMI | No | -- |
PshAI | 65°C | 20 minutes |
PsiI | 65°C | 20 minutes |
PsiI-v2 | 65°C | 20 minutes |
PspGI | No | -- |
PspOMI | 65°C | 20 minutes |
PspXI | No | -- |
PstI | 80°C | 20 minutes |
PstI | No | -- |
PvuI | No | -- |
PvuII | No | -- |
PvuII | No | -- |
RsaI | No | -- |
RsrII | 65°C | 20 minutes |
SacI | 65°C | 20 minutes |
SacII | 65°C | 20 minutes |
SalI | 65°C | 20 minutes |
SalI | 65°C | 20 minutes |
SapI | 65°C | 20 minutes |
Sau3AI | 65°C | 20 minutes |
Sau96I | 65°C | 20 minutes |
SbfI | 80°C | 20 minutes |
ScaI | 80°C | 20 minutes |
ScaI | 80°C | 20 minutes |
ScrFI | 65°C | 20 minutes |
SexAI | 65°C | 20 minutes |
SfaNI | 65°C | 20 minutes |
SfcI | 65°C | 20 minutes |
SfiI | No | -- |
SfoI | No | -- |
SgrAI | 65°C | 20 minutes |
SmaI | 65°C | 20 minutes |
SmlI | No | -- |
SnaBI | 80°C | 20 minutes |
SpeI | 80°C | 20 minutes |
SphI | 65°C | 20 minutes |
SphI | 65°C | 20 minutes |
SrfI | 65°C | 20 minutes |
SspI | 65°C | 20 minutes |
StuI | No | -- |
StyD4I | 65°C | 20 minutes |
StyI | 65°C | 20 minutes |
SwaI | 65°C | 20 minutes |
TaqI-v2 | No | -- |
TfiI | No | -- |
TliI | No | -- |
TseI | No | -- |
Tsp45I | No | -- |
Tsp509I | No | -- |
TspMI | No | -- |
TspRI | No | -- |
Tth111I | No | -- |
WarmStart | 80°C | 20 minutes |
XbaI | 65°C | 20 minutes |
XcmI | 65°C | 20 minutes |
XhoI | 65°C | 20 minutes |
XmaI | 65°C | 20 minutes |
XmnI | 65°C | 20 minutes |
ZraI | 80°C | 20 minutes |
表1. 酶的失活温度和时间 (美格生物)
扩展阅读
如何找到**的PCR反应体系?PCR技术所需要的酶和原料的调整