f*******e
发帖数: 354 | 1 求教：
手里研究的A蛋白影响了B蛋白的蛋白水平，但不影响mrna水平，本想做降解的，MG132
虽然可以恢复一部分，但是就是内源性降解曲线（CHX）没有明显差异。现在想要不要
转向翻译水平，但是没有full length 的mrna，只有coding区，因为也不知道是否在非
编码区的调控，所以单独试coding区意义不大。
所以如下假设，不知道有没有可能。
先将原核表达纯化的蛋白（如GST和GST-A）加入到IVT系统中，再加入相同ug的细胞
total mRNA,翻译一定时间后Western检测B蛋白，不知道这样是否可能，有没有达人做
过，一般都是特定的mrna，这样总rna会不会目的基因拷贝数太少而检测不到。
非常感谢！ |
s******9
发帖数: 283 | 2 total mRNA for cell-free translation is a bad idea, unless you have single-
molecule detection sensitivity. You have to fish out the cDNA, add a strong
promoter and synthesize a large amount of mRNAs for the translation if you
can only do western. |
g*********5
发帖数: 2533 | |
a*****x
发帖数: 901 | 4 CHX does not work for proteins with long half life. Try pulse-chase -- it's
a giant pain but the reviewers will ask for it. If it's super long half life
protein, you can also use siRNA, which worked well in my hand. |
r*******y
发帖数: 48 | 5 I ever used CHX for 36 hours and detected a decrease in the level of a very
stable protein, while the housekeeping protein was not affected.
s
life
【在 a*****x 的大作中提到】
: CHX does not work for proteins with long half life. Try pulse-chase -- it's
: a giant pain but the reviewers will ask for it. If it's super long half life
: protein, you can also use siRNA, which worked well in my hand.
|
