a****l
发帖数: 125 | 1 Does anyone know a good way to do large scale high-throughput screen to see
how the stability of a protein is controlled? Doing western blot is too slow!
thinking some fluorescence based experiment. Anyone knows? |
p****s
发帖数: 3153 | 2 看看这个，
http://pubs.acs.org/doi/abs/10.1021/ja8049063
High-Throughput Thermal Scanning: A General, Rapid Dye-Binding Thermal Shift
Screen for Protein Engineering
see
slow!
【在 a****l 的大作中提到】
: Does anyone know a good way to do large scale high-throughput screen to see
: how the stability of a protein is controlled? Doing western blot is too slow!
: thinking some fluorescence based experiment. Anyone knows?
|
a****l
发帖数: 125 | 3 谢快速回贴，偶可能没说清楚：偶想看蛋白stability 是如何调控的。可以做siRNA or
chemical screen, 但readout 是什么？不能总做western, 太慢了。 |
p****s
发帖数: 3153 | 4 我没看懂你的帖子，蛋白稳定性为什么要用siRNA?你想做in vivo?那我就不懂了，不知
道连gfp行不行
or
【在 a****l 的大作中提到】
: 谢快速回贴，偶可能没说清楚：偶想看蛋白stability 是如何调控的。可以做siRNA or
: chemical screen, 但readout 是什么？不能总做western, 太慢了。
|
a****l
发帖数: 125 | 5 是in vivo, 细胞水平。已知一个蛋白不稳定， 想找到pathway or factors (
mechanisms) that can regulate its stability. 可以用siRNA 一个一个敲。 GFP
tag 行吗？ 有没有人试过？？ |
S****r
发帖数: 982 | 6 LZ可能说的是筛选，什么能调控该蛋白降解，不是结构的稳定性。 |
n********k
发帖数: 2818 | 7 GFP/RFP fusion proteins or luciferase-fusion protein(maybe hard)...Check
Stephen ellege and, Westbrook's study on NRSF/REST...
see
slow!
【在 a****l 的大作中提到】
: Does anyone know a good way to do large scale high-throughput screen to see
: how the stability of a protein is controlled? Doing western blot is too slow!
: thinking some fluorescence based experiment. Anyone knows?
|
s******s
发帖数: 13035 | 8 guess你想做fusion protein，但是fusion protein实际上保护了蛋白，
做stability分析有缺点。另外，你做transgene那么很多readout都是控制
transgene表达的基因，而不是你要的。
western也可以high throughput, 比如microwestern array
see
slow!
【在 a****l 的大作中提到】
: Does anyone know a good way to do large scale high-throughput screen to see
: how the stability of a protein is controlled? Doing western blot is too slow!
: thinking some fluorescence based experiment. Anyone knows?
|
g****1
发帖数: 261 | 9
see
slow!
If I understand your question correctly, you might be able to expand this
method to parallel/large scale:http://www.ncbi.nlm.nih.gov/pubmed/19995983
【在 a****l 的大作中提到】
: Does anyone know a good way to do large scale high-throughput screen to see
: how the stability of a protein is controlled? Doing western blot is too slow!
: thinking some fluorescence based experiment. Anyone knows?
|
a****l
发帖数: 125 | 10 seems to be the way to go. 但是否有很多人使用此方法?
【在 n********k 的大作中提到】
: GFP/RFP fusion proteins or luciferase-fusion protein(maybe hard)...Check
: Stephen ellege and, Westbrook's study on NRSF/REST...
:
: see
: slow!
|
