原创力文档- 1、本文档共1页，可阅读全部内容。
- 2、原创力文档（book118）网站文档一经付费（服务费），不意味着购买了该文档的版权，仅供个人/单位学习、研究之用，不得用于商业用途，未经授权，严禁复制、发行、汇编、翻译或者网络传播等，侵权必究。
- 3、本站所有内容均由合作方或网友上传，本站不对文档的完整性、权威性及其观点立场正确性做任何保证或承诺！文档内容仅供研究参考，付费前请自行鉴别。如您付费，意味着您自己接受本站规则且自行承担风险，本站不退款、不进行额外附加服务；查看《如何避免下载的几个坑》。如果您已付费下载过本站文档，您可以点击 这里二次下载。
- 4、如文档侵犯商业秘密、侵犯著作权、侵犯人身权等，请点击“版权申诉”（推荐），也可以打举报电话：400-050-0827(电话支持时间：9:00-18:30)。
查看更多
In vitro ubiquitination assay
A.
Total 15ul
Reaction setup Rxn-mix (ul) E1-mix (ul)
5x Buffer 2.0 1
ATP (100mM) 0.5 -
Ub (10mg/ml) 1.0 -
UbcH7 (~10mg/ml) 0.5 -
E1 - 0.5
E3 Sf1 (5ul) Sf2 (3ul) 5.0 -
Compounds (5-10uM) + (final DMSO can be up to 20% for Smurfs)
dH2O adjust to total 15ul
B.
Mix reaction-mix by vortex briefly, spin down and aliquot appropriate amount base on calculation into 1.5ml eppendorf tube containing Smurfs. Mix reaction by tapping. Add equivalent amount of compounds and mix.
C.
Pipette E1 into 5x Buffer in a separate labeled tube (E1-mix), mix properly and spin down. Aliquot E1 to the lid of the 1.5ml tube and close gently. Spin all tubes at the same time and incubate at room temperature (lab bench) for 1 hour.
Terminate reaction by adding sample buffer. Either store away immediately at –20C or boil for 4 min at 95C.
Resolve on 7.5% SDSand incubate in Anti-Ub antibody (1:2000).
1x Buffer
50mM Tris-Cl pH7.5
2mM ATP (supplement to 5mM before the reaction is initiated)
10mM MgCl2
50uM DTT
Stock 5 X buffer
250 mM Tris-Cl pH7.5
10mM ATP
50mM MgCl2
250?M DTT
文档评论（0）