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GFP 融合蛋白

GFP 融合蛋白# Biology - 生物学

b*92010-09-07 07:09

1 楼

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guangshui (shuiche) 于 (Wed Apr 6 20:17:12 2011, 美东) 提到:
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bless
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big bless!
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Bless!
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Bless
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p*o2010-09-07 07:09

2 楼

没有装多看，
主要是想弄几个文件夹，把自己的PDF分分类，
比如专业的，小说的等等
在网上SEARCH了一下，没找到合适的信息
感觉那个新建COLLECTION不是我想要的
大家用什么办法？
谢谢

A*e2010-09-07 07:09

3 楼

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*a2010-09-07 07:09

4 楼

原系统无解
amen

【在 p*****o 的大作中提到】

: 没有装多看，
: 主要是想弄几个文件夹，把自己的PDF分分类，
: 比如专业的，小说的等等
: 在网上SEARCH了一下，没找到合适的信息
: 感觉那个新建COLLECTION不是我想要的
: 大家用什么办法？
: 谢谢

k*c2010-09-07 07:09

5 楼

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

e*y2010-09-07 07:09

6 楼

有个pc端管理collection的工具, 可以根据文件夹目录生成collection label
不过还是多看方便

【在 p*****o 的大作中提到】

s*y2010-09-07 07:09

7 楼

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*e2010-09-07 07:09

8 楼

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*e2010-09-07 07:09

9 楼

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*y2010-09-07 07:09

10 楼

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*e2010-09-07 07:09

11 楼

Thanks to sunnyday again. Your explain makes sense.