I*y
2 楼
都不太靠谱。
split gfp是说BiFC? 这个如果work了就work了，算你走运，如果不work，你连
troubleshooting都不知道咋下手。fret 相对还靠谱一点。
vs
【在 e***o 的大作中提到】
: 做蛋白互做检测时这2种方法，那种得灵敏度更高些？还是都不太靠铺？ Split GFP vs
: FRET
: 谢谢。
split gfp是说BiFC? 这个如果work了就work了，算你走运，如果不work，你连
troubleshooting都不知道咋下手。fret 相对还靠谱一点。
vs
【在 e***o 的大作中提到】
: 做蛋白互做检测时这2种方法，那种得灵敏度更高些？还是都不太靠铺？ Split GFP vs
: FRET
: 谢谢。
b*y
3 楼
FRET is within 5nm. If your proteins are large, the chance of success is low
, too.
, too.
e*o
4 楼
谢谢！我也担心这些东西有点不靠铺。除了IP以外，不知道有人用过Mammalian
Two-Hybrid System没有，希望这个能管用一些。
Two-Hybrid System没有，希望这个能管用一些。
X*n
5 楼
Mammalian Two-Hybrid System的sensitivity 大概是25％，false positive偏高
MAPPIT的sensitivity 大概是35％，false positive据说很低，
如果蛋白是在细胞核的，可以试试Y2H，但sensitivity大概也是30％，而且会有假阳性
问题，
SPICA 据说sensitivity 非常高 75％以上，但是我估计假阳性也会很高，
PCA（或者BiFC),sensitivity <25%,有假阳性问题，
invitro binding assay
MAPPIT的sensitivity 大概是35％，false positive据说很低，
如果蛋白是在细胞核的，可以试试Y2H，但sensitivity大概也是30％，而且会有假阳性
问题，
SPICA 据说sensitivity 非常高 75％以上，但是我估计假阳性也会很高，
PCA（或者BiFC),sensitivity <25%,有假阳性问题，
invitro binding assay
i*0
6 楼
Have you considered about beta-Galactosidase complement assay? you can test
luminescence by adding substrate. You basically fuse your proteins of
interest with omega- and alpha-subunit of beta-Gal, if there are
interactions between the two protein, beta-gal activity will be restored,
and you will see high luminescence compared to control. Any read out from
enzyme enlargement will give much higher signal/background ratio compared to
fluorescence from GFPs.
luminescence by adding substrate. You basically fuse your proteins of
interest with omega- and alpha-subunit of beta-Gal, if there are
interactions between the two protein, beta-gal activity will be restored,
and you will see high luminescence compared to control. Any read out from
enzyme enlargement will give much higher signal/background ratio compared to
fluorescence from GFPs.
e*o
7 楼
wow!! 好专业的回答！
你说的spica是不是2011年底nature method的那篇文章。看起来好象不错，不知道做起
来是否靠铺，因为还没有人重复。另外Luciferase reconstruction based 方法，做
live cell imaging和FACS比不上XFP。上大规模可能活比较多。 看起来MAPPIT比较可
靠，但是是transcription based,可能不够dynamic.应该不能做live cell imaging。
另外，你认为Y2H做核外的，大概会有多少！
btw, 你知道有什么好的方法可以检测RNA Protein 互做的吗？ 除了RNA-IP, CHIP?
谢谢！
【在 X******n 的大作中提到】
: Mammalian Two-Hybrid System的sensitivity 大概是25％，false positive偏高
: MAPPIT的sensitivity 大概是35％，false positive据说很低，
: 如果蛋白是在细胞核的，可以试试Y2H，但sensitivity大概也是30％，而且会有假阳性
: 问题，
: SPICA 据说sensitivity 非常高 75％以上，但是我估计假阳性也会很高，
: PCA（或者BiFC),sensitivity <25%,有假阳性问题，
: invitro binding assay
你说的spica是不是2011年底nature method的那篇文章。看起来好象不错，不知道做起
来是否靠铺，因为还没有人重复。另外Luciferase reconstruction based 方法，做
live cell imaging和FACS比不上XFP。上大规模可能活比较多。 看起来MAPPIT比较可
靠，但是是transcription based,可能不够dynamic.应该不能做live cell imaging。
另外，你认为Y2H做核外的，大概会有多少！
btw, 你知道有什么好的方法可以检测RNA Protein 互做的吗？ 除了RNA-IP, CHIP?
谢谢！
【在 X******n 的大作中提到】
: Mammalian Two-Hybrid System的sensitivity 大概是25％，false positive偏高
: MAPPIT的sensitivity 大概是35％，false positive据说很低，
: 如果蛋白是在细胞核的，可以试试Y2H，但sensitivity大概也是30％，而且会有假阳性
: 问题，
: SPICA 据说sensitivity 非常高 75％以上，但是我估计假阳性也会很高，
: PCA（或者BiFC),sensitivity <25%,有假阳性问题，
: invitro binding assay
e*o
8 楼
Thanks! Does this work well in vivo?
test
to
【在 i***0 的大作中提到】
: Have you considered about beta-Galactosidase complement assay? you can test
: luminescence by adding substrate. You basically fuse your proteins of
: interest with omega- and alpha-subunit of beta-Gal, if there are
: interactions between the two protein, beta-gal activity will be restored,
: and you will see high luminescence compared to control. Any read out from
: enzyme enlargement will give much higher signal/background ratio compared to
: fluorescence from GFPs.
test
to
【在 i***0 的大作中提到】
: Have you considered about beta-Galactosidase complement assay? you can test
: luminescence by adding substrate. You basically fuse your proteins of
: interest with omega- and alpha-subunit of beta-Gal, if there are
: interactions between the two protein, beta-gal activity will be restored,
: and you will see high luminescence compared to control. Any read out from
: enzyme enlargement will give much higher signal/background ratio compared to
: fluorescence from GFPs.
e*o
9 楼
I thought for FRET, it is 10 nm. How about split gfp/BiFC? Thanks！
People are using split GFP (called GRASP) to reconstruct pre/post synaptic
split GFP and get good signal. The distance of synapse is around 20-40nm.
The extracellular part of XFG isnt big. I am wondering 10nm is good for split gfp/BiFC?
low
【在 b******y 的大作中提到】
: FRET is within 5nm. If your proteins are large, the chance of success is low
: , too.
People are using split GFP (called GRASP) to reconstruct pre/post synaptic
split GFP and get good signal. The distance of synapse is around 20-40nm.
The extracellular part of XFG isnt big. I am wondering 10nm is good for split gfp/BiFC?
low
【在 b******y 的大作中提到】
: FRET is within 5nm. If your proteins are large, the chance of success is low
: , too.
X*n
10 楼
是这篇文章，但是我估计假阳性会非常高。
不知道你的目的是discovery还是confirmation?
如果要做dynamic interaction,肯定还是ＸＦＰｂａｓｅｄ比较现实。
Y2H:如果是膜蛋白，可以用split-ubiquitin membrane yeast two-hybrid system。
如果不是膜蛋白，不需要postranslational modicication,应该还是可以试一下的，因
为这方法最便宜。
看过一些ＲＮＡ ｐｒｏｔｅｉｎ 的方法，但是自己没做过，不敢乱说。不过如果你
想做large scale interaction mapping的话，这确实是个很好的ｎｉｃｈｅ，因为
protein-protein , protein DNA interaction 做的人太多了，如果你没有很好的资源
的话 （比如说ＯＲＦｏｍｅ），没有多少优势。但Protein RNA和Protein LIPID才刚
起步。如果能在方法上有突破的话，还是能做大文章的。
【在 e***o 的大作中提到】
: wow!! 好专业的回答！
: 你说的spica是不是2011年底nature method的那篇文章。看起来好象不错，不知道做起
: 来是否靠铺，因为还没有人重复。另外Luciferase reconstruction based 方法，做
: live cell imaging和FACS比不上XFP。上大规模可能活比较多。 看起来MAPPIT比较可
: 靠，但是是transcription based,可能不够dynamic.应该不能做live cell imaging。
: 另外，你认为Y2H做核外的，大概会有多少！
: btw, 你知道有什么好的方法可以检测RNA Protein 互做的吗？ 除了RNA-IP, CHIP?
: 谢谢！
不知道你的目的是discovery还是confirmation?
如果要做dynamic interaction,肯定还是ＸＦＰｂａｓｅｄ比较现实。
Y2H:如果是膜蛋白，可以用split-ubiquitin membrane yeast two-hybrid system。
如果不是膜蛋白，不需要postranslational modicication,应该还是可以试一下的，因
为这方法最便宜。
看过一些ＲＮＡ ｐｒｏｔｅｉｎ 的方法，但是自己没做过，不敢乱说。不过如果你
想做large scale interaction mapping的话，这确实是个很好的ｎｉｃｈｅ，因为
protein-protein , protein DNA interaction 做的人太多了，如果你没有很好的资源
的话 （比如说ＯＲＦｏｍｅ），没有多少优势。但Protein RNA和Protein LIPID才刚
起步。如果能在方法上有突破的话，还是能做大文章的。
【在 e***o 的大作中提到】
: wow!! 好专业的回答！
: 你说的spica是不是2011年底nature method的那篇文章。看起来好象不错，不知道做起
: 来是否靠铺，因为还没有人重复。另外Luciferase reconstruction based 方法，做
: live cell imaging和FACS比不上XFP。上大规模可能活比较多。 看起来MAPPIT比较可
: 靠，但是是transcription based,可能不够dynamic.应该不能做live cell imaging。
: 另外，你认为Y2H做核外的，大概会有多少！
: btw, 你知道有什么好的方法可以检测RNA Protein 互做的吗？ 除了RNA-IP, CHIP?
: 谢谢！
e*o
11 楼
我主要还是想做筛选。但是目前方法要么假阳性太高，要么不敏感。或者太贵了。
X*n
12 楼
做筛选的话，可能就ｃｏＡＰ－ＭＳ或者Ｙ２Ｈ比较现实，因为其它的方法没有很好的
Ｌｉｂｒａｒｙ。ＭＡＰＰＩＴ现在估计有不到３０００个蛋白，ＳＰＩＣＡ就更少了
。
Ｌｉｂｒａｒｙ。ＭＡＰＰＩＴ现在估计有不到３０００个蛋白，ＳＰＩＣＡ就更少了
。
l*1
13 楼
Y-2-H 用多重标记 做对照和比较
比如以下的 split-luciferase+GST+HA-epitope tagged
三重标记
they found new proteins interaction paper:
//www.ncbi.nlm.nih.gov/pubmed/21530591
Protocol details:
>the wheat germ in vitro translation system for use in the splitlu
ciferase
assay and co-purifications with glutathione S transferase
(GST)- and HA-epitope tagged proteins (Fig. 2A).
>
In the split-luciferase assay, proteins of interest are fused to
N- and C-terminal fragments of luciferase. If the proteins interact,
the N- and C-terminal luciferase fragments associate to create
a functional luciferase enzyme [10]. Though a number of variants
of this system have been described, for these experiments
we used fragments of firefly luciferase that were optimized to
yield a high signal to background ratio and sufficient enzymatic
activity to be easily measured [11].
ignored
New found part:
>
Surprisingly, there was no detectable homology between
PfMyb2 and Cef1 in the PFC0365w/PRP19 binding domains, even
though the yeast orthologs of PfMyb2 and PFC0365w also interact.
In fact, the only significant homology between Cef1 and PfMyb2
was in the first 233 amino acids that encode the Myb DNA-binding
domain motifs (49% identical, 70% similar). This result was unexpected
because protein interaction interfaces are generally thought
to evolve more slowly, since changing residues in one protein
would require compensating changes in the binding partner. The
interaction between PfMyb2 and PFC0365w demonstrates that this
is not always the case. A similar observation has been made for
three pairs of interacting proteins from Caenorhabditis elegans [15].
Despite the lack of homology between PfMyb2 and Cef1 C-terminal
to the MYB DNA-binding domain, the fact that PfMyb2 binds to
the P. falciparum PRP19 homolog suggests that PfMyb2 is the functional
homolog of Cef1. Since Cef1 is primarily involved in RNA
splicing, we propose that PfMyb2 functions in a similar manner
and is unlikely to act as a transcription factor.
比如以下的 split-luciferase+GST+HA-epitope tagged
三重标记
they found new proteins interaction paper:
//www.ncbi.nlm.nih.gov/pubmed/21530591
Protocol details:
>the wheat germ in vitro translation system for use in the splitlu
ciferase
assay and co-purifications with glutathione S transferase
(GST)- and HA-epitope tagged proteins (Fig. 2A).
>
In the split-luciferase assay, proteins of interest are fused to
N- and C-terminal fragments of luciferase. If the proteins interact,
the N- and C-terminal luciferase fragments associate to create
a functional luciferase enzyme [10]. Though a number of variants
of this system have been described, for these experiments
we used fragments of firefly luciferase that were optimized to
yield a high signal to background ratio and sufficient enzymatic
activity to be easily measured [11].
ignored
New found part:
>
Surprisingly, there was no detectable homology between
PfMyb2 and Cef1 in the PFC0365w/PRP19 binding domains, even
though the yeast orthologs of PfMyb2 and PFC0365w also interact.
In fact, the only significant homology between Cef1 and PfMyb2
was in the first 233 amino acids that encode the Myb DNA-binding
domain motifs (49% identical, 70% similar). This result was unexpected
because protein interaction interfaces are generally thought
to evolve more slowly, since changing residues in one protein
would require compensating changes in the binding partner. The
interaction between PfMyb2 and PFC0365w demonstrates that this
is not always the case. A similar observation has been made for
three pairs of interacting proteins from Caenorhabditis elegans [15].
Despite the lack of homology between PfMyb2 and Cef1 C-terminal
to the MYB DNA-binding domain, the fact that PfMyb2 binds to
the P. falciparum PRP19 homolog suggests that PfMyb2 is the functional
homolog of Cef1. Since Cef1 is primarily involved in RNA
splicing, we propose that PfMyb2 functions in a similar manner
and is unlikely to act as a transcription factor.
e*o
14 楼
谢谢！这个假阳性应该比较低，但不知道假阴性会不会比较高。漏掉一些蛋白。
【在 l**********1 的大作中提到】
: Y-2-H 用多重标记 做对照和比较
: 比如以下的 split-luciferase+GST+HA-epitope tagged
: 三重标记
: they found new proteins interaction paper:
: //www.ncbi.nlm.nih.gov/pubmed/21530591
: Protocol details:
: >the wheat germ in vitro translation system for use in the splitlu
: ciferase
: assay and co-purifications with glutathione S transferase
: (GST)- and HA-epitope tagged proteins (Fig. 2A).
【在 l**********1 的大作中提到】
: Y-2-H 用多重标记 做对照和比较
: 比如以下的 split-luciferase+GST+HA-epitope tagged
: 三重标记
: they found new proteins interaction paper:
: //www.ncbi.nlm.nih.gov/pubmed/21530591
: Protocol details:
: >the wheat germ in vitro translation system for use in the splitlu
: ciferase
: assay and co-purifications with glutathione S transferase
: (GST)- and HA-epitope tagged proteins (Fig. 2A).
l*1
15 楼
Above paper its pp58
While we cannot dismiss the possibility that the split-luciferase
and the GST pulldown results are false-negatives, the interaction is
not supported by other evidence and is of lower confidence.
楼主 不会用排列组合吗?
萤火虫萤光标记N and C terminal a class
glutathione S transferase GST b class
HA-epitope tagged c class
then a must be used
positive control group:
(a,b,c)
(a,b)
(a,c)
negative control group:
(null, b,c)
(null, b)
(null, c)
Ps: if you are satisfied to this replied. BAOZI one please transfer to my
account.
【在 e***o 的大作中提到】
: 谢谢！这个假阳性应该比较低，但不知道假阴性会不会比较高。漏掉一些蛋白。
While we cannot dismiss the possibility that the split-luciferase
and the GST pulldown results are false-negatives, the interaction is
not supported by other evidence and is of lower confidence.
楼主 不会用排列组合吗?
萤火虫萤光标记N and C terminal a class
glutathione S transferase GST b class
HA-epitope tagged c class
then a must be used
positive control group:
(a,b,c)
(a,b)
(a,c)
negative control group:
(null, b,c)
(null, b)
(null, c)
Ps: if you are satisfied to this replied. BAOZI one please transfer to my
account.
【在 e***o 的大作中提到】
: 谢谢！这个假阳性应该比较低，但不知道假阴性会不会比较高。漏掉一些蛋白。
e*o
16 楼
这几天比较忙，现在一并转baozi。不过我没有多少包子。
l*1
17 楼
BAOZI got
Merci beaucoup.
【在 e***o 的大作中提到】
: 这几天比较忙，现在一并转baozi。不过我没有多少包子。
Merci beaucoup.
【在 e***o 的大作中提到】
: 这几天比较忙，现在一并转baozi。不过我没有多少包子。
l*0
18 楼
可以试试，Duolink，我才做过，还不错
e*o
19 楼
谢谢，
这个技术看起来很ｃｏｏｌ，那个ＰＬＡ ＰＬＵＳ ＭＩＮＵＳ的连接主要还是看两
者之间的距离吗？如果近的话就能连？那个ｒｏｌｌｉｎｇ ｃｙｃｌｅ做起来贵不贵？
【在 l********0 的大作中提到】
: 可以试试，Duolink，我才做过，还不错
这个技术看起来很ｃｏｏｌ，那个ＰＬＡ ＰＬＵＳ ＭＩＮＵＳ的连接主要还是看两
者之间的距离吗？如果近的话就能连？那个ｒｏｌｌｉｎｇ ｃｙｃｌｅ做起来贵不贵？
【在 l********0 的大作中提到】
: 可以试试，Duolink，我才做过，还不错
g*5
20 楼
we try positive control with Duolink
the same protein,
mAb, And Pab.
no red spot visible...
don't know the reason...
【在 l********0 的大作中提到】
: 可以试试，Duolink，我才做过，还不错
the same protein,
mAb, And Pab.
no red spot visible...
don't know the reason...
【在 l********0 的大作中提到】
: 可以试试，Duolink，我才做过，还不错
g*5
21 楼
erpao (paopao) 的大作中提到: 】
Gel shift.
Gel shift.
b*s
22 楼
不知道split GFP怎么做，FRET相当不好做，需要很仔细的control。 我们这里一个不
走运的博士生花了4年make it work。
vs
【在 e***o 的大作中提到】
: 做蛋白互做检测时这2种方法，那种得灵敏度更高些？还是都不太靠铺？ Split GFP vs
: FRET
: 谢谢。
走运的博士生花了4年make it work。
vs
【在 e***o 的大作中提到】
: 做蛋白互做检测时这2种方法，那种得灵敏度更高些？还是都不太靠铺？ Split GFP vs
: FRET
: 谢谢。
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