REVIEW
2021年7月15日，由Promega公司举办的“激酶活性检测及药物筛选技术培训班”已圆满落幕，本次培训班我们特别邀请到Promega Corporation两位资深研发科学家从方案设计，实验问题剖析，技术工具介绍，以及新技术新产品等方面为大家带来了精彩演讲，受到了很多观众的好评！不过，错过直播的你也不用担心，回放及留言解答都为你备好啦！
内容回顾
SPEAKER
Kevin Hsiao, PhD
Promega 资深研发科学家
Dr.Hsiao 在Promega一直致力于激酶和磷酸酶相关检测系统的研究工作，拥有近28年的研发经验。
演讲主题
用于激酶药物研发的生化和细胞学检测工具
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Juliano Alves, PhD
Promega 资深研发科学家
Dr. Alves在Promega主要负责激酶系统，Succinate-Glo以及病毒研究相关检测系统的研发，拥有近10年的研究经验。
演讲主题
Promega激酶反应系统纵览
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留言解答
Q&A
由于培训班直播现场时间有限，嘉宾未来得及一一解答大家的问题，因此我们将直播间的留言进行了整理，并请我们的技术专家做了详细解答。希望这部分内容能为大家的研究工作带来参考。
为什么加药后读值比只加底物不加激酶的还要低呢，是药物抑制了底物的自身磷酸化了吗？
答：RLU went down, meaning there is no consuming ATP --- so you should do:
Set one: only compound;
Set two: only compound + substrate;
Set three: only substrate;
Set four: no compound but with solvent for compound + substrate.
– solvent might also cause problem --- I do not know how high (in %) concentration of solvent in the reaction. You have to keep solvent concentration constant in every reaction.
It might be too much substrate from beginning – so, you might need to do substrate titration (with and/or without kinase) to choose what concentration of substrate to use in kinase reaction.
温度要控制在多少？检测值偏低可能原因是什么？
答：You can set the kinase reaction up to 37ºC, and/or the reaction can be up to 90min if you would like to. However, you need to take plate (96 well) out about 5 ~ 10min before you are going to add ADP-Glo Detection Solution.
The RLU value low --- meaning:
1) Kinase was not too active at the temperature you set-up (you may increase reaction temperature – up to 37·C;
2) you can increase the reaction time up to 90min;
3) you may need to add more kinase in the reaction – due to low activity; and/or
4) your kinase might be no any activity.
检测ATP裂解细胞的同时会不会因为细胞受刺激导致ATP的瞬时增加或减少，从而影响实验结果？
答：ADP-Glo is biochemical kit --- can’t use for cell lysate – due to Adenosine nucleotides interference. Unless you can use immunoprecipitation to pull down your interested protein(s) then to do kinase reaction with PPT beats. However, two factors you need to keep in mind:
1) Antibodies choice --- not blocking out active site of enzyme;
2) once reaction down (through entirely ADP-Glo) you need to pipet out the reaction solution to a new solid white plate to read – due to the ppt beads may interfere the reading.
激酶反应中，ATP Km的滴定是否需要做时间曲线呢？
答：From literature you can find the Km value of your interested Kinase(s). otherwise, you need to do ATP titration curve to find out what is Km value for your interested kinase(s). it would be some changes in Km – due to:
1) reaction buffer;
2) reaction temperature;
3) reaction time; and/or
4) other protein/peptide substrate concentrations. The enzyme itself --- maybe different from original enzyme – such as mutant one.
NanoBRET和FRET证明功能与结构关系，比方说可以证明激酶被牵拉导致位移吗？
答：It could be --- however, depends on same enzyme or mutant --- may cause difference. Same sequence of enzyme length you uses comparing with others.
ATP在-20 保存多久可以正常？也会降解么？
答：Normally, we suggest and ask our customers to aliquot to small volume then re-store them at -80ºC. if you puts in -20ºC the ultra pure ATP will gradually degradation to ADP --- that is way you may have higher background and/or low enzyme activity. To prevent from degradation of ATP --- keep it in -80ºC. We have seen with a couple weeks in -20ºC it start to degrade.
不知道是不是操作不熟练的问题，如果重复不出说明书的application note的IC 50，那结果在什么范围可以接受？
答：In general speaking, IC50 within 2-3 folds would be ok --- due to reaction condition may change as enzyme itself, buffer, substrate(s). and/or temperature.
Promega有聚合酶类相关的检测方法吗？有黄嘌呤氧化酶的活性检测试剂盒吗？
答：Please give some more details about what kind of polymerase or xanthine oxidase, if you have exact product, you can directly ask Promega for more information.
ADP-Glo试剂盒对于高浓度ATP筛选，想得到好的信号窗口，有什么好的优化建议，高浓度ATP会有较高的背景吗？
答：How high of ATP would like to use? We have ADP-Glo Max (V7001 or V7002) can be use for some membrane ATPases. I think it can handle ATP high concentration as high as up to 5mM. Please check with our Tech Service as well Special Sale Rep.
NanoBRET技术是否适合应用于PROTAC药物的研发筛选？
答：Yes, but let us know what the target enzyme so we can help as much as we can before you would try it.
测完了，我还能洗掉抗体，样本用来做一下WesternBlot吗？
答：No, I don’t think so. You may try it if you really would like to: Trying to use higher NaCl concentration up to 500mM. But sometimes antibodies with target protein may have higher affinity --- hard to get all antibodies out. Besides, you may face higher and non-specific background or bends.
请问下贵公司有ATR靶点激酶测试试剂盒吗？
答：More specific ATR --- we might have as Protein Target Engagement of NanoBRET…. But we need more specific so we can check our list, or you can directly connect with China technical servise.
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有奖调研
SURVERY
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