A*******e
发帖数: 284 | 1 诸位达人们，困扰我的问题请教。构建GFP融合蛋白，GFP放在C段，GFP的ATG需要除掉
吗？不除掉的话细胞会不会把GFP前面我的基因当作5'utr 而只翻译free GFP出来吗？
一个lab mate构建GFP融合蛋白，GFP放在C段，WESTERN检测发现既有融合蛋白也有free
GFP,这如何解释？ |
k*******c
发帖数: 142 | 2 you will find little GFP-free peptides come out even there is not a RBS-like
sequence upstream of GFP: something may function as it is.
However, you should get the fused protein dominantly. Remove the ATG will
help a little bit.
Good luck |
s******y
发帖数: 28562 | 3 Yes, we do that all the time.
However, if you only use western blot for your dection technique, then you
don't have to worry, because the fusion protein has different size.
？
free
【在 A*******e 的大作中提到】
: 诸位达人们，困扰我的问题请教。构建GFP融合蛋白，GFP放在C段，GFP的ATG需要除掉
: 吗？不除掉的话细胞会不会把GFP前面我的基因当作5'utr 而只翻译free GFP出来吗？
: 一个lab mate构建GFP融合蛋白，GFP放在C段，WESTERN检测发现既有融合蛋白也有free
: GFP,这如何解释？
|
A*******e
发帖数: 284 | 4 Thanks to kelvincsc. I do not understand how this GFP-free peptides come out
. You mean there will be free gfp for any GFP-fused proteins? In this case,
subcellular localization using GFP fused protein will not work. You know,
free GFP, even very less, will disturb the protein subcellular localization
data. |
A*******e
发帖数: 284 | 5 Thanks to sunnyday. I know it's all right for western. However for protein
subcellular localization, it will not work if all gfp-fused proteins have
free gfp signal. There are lots of papers with protein subcellular
localization results from GFP-fused protein. If there is free gfp signal for
all gfp-fused protein, what's mean of these results? |
s******y
发帖数: 28562 | 6 1. not everybody is as careful as you or me.
2. they usually still put in a negative control of free GFP
When there is a ATG in front of the GFP, there is always a chance that you
will see free GFP getting expressed, no matter with or without IRS.
However, even removing the ATG and the IRS all together, sometimes you still
see the free GFP showing up on western blot. We believe that is because the
linker sequence between your protein and the GFP suffered a proteolytic
cleavage
for
【在 A*******e 的大作中提到】
: Thanks to sunnyday. I know it's all right for western. However for protein
: subcellular localization, it will not work if all gfp-fused proteins have
: free gfp signal. There are lots of papers with protein subcellular
: localization results from GFP-fused protein. If there is free gfp signal for
: all gfp-fused protein, what's mean of these results?
|
A*******e
发帖数: 284 | 7 Thanks to sunnyday again. Your explain makes sense. |
s**e
发帖数: 1523 | 8 我构建的没有出现过这问题，western和immunostaining都做过control。 |
