R*s
发帖数: 2041 | | n***h
发帖数: 11 | 2 照我的记忆说来：
yeast 2 hybrid system是两个蛋白之间的binding
yeast 1 hybrid是蛋白质和DNA之间的binding ，就象ras说的，测出的是bait和
fragment之间的结合，可以象ras说的那样fragment是library, 来找和已知蛋白
结合的特定element，也可以特定的fragment，转cDNA library进去，找和这个
fragment 结合的蛋白。 （infact that's what I did)
回来说2hybrid。transcriptional factor一般至少有两个domain: binding domain
和activition domain. binding domain binds to DNA elements, while activation
domain activates the transcription of downstream genes. 我们要测试蛋白A
和蛋白B是否bind, 就做以下两个construct：
—————————— ————————— | R*s
发帖数: 2041 | 3 example:
--------------------- ----------------------------
| bait | activator| + |fragment A | reporter gene |
--------------------- ----------------------------
Bait ait is the gene u r intestered in, and is attached to a
transcription activator. fragment A is one of those from a DNA library
which is attached to a reporter gene.
Basically you transfect both the bait construct and library construct
into yeast (a and alpha respectively) and let the mate (each yest should have
only one of those library DNA fragment). If the bait can bind with one of
the fragments, then the transcription activator can be brought to the reporter
gene and transcribe the reporter gene. The expression of the reporter gene
will lead to blue color of the specific yeast colony. Then u pick the blue
colony, grow up culture, and purify DNA from it. You got the DNA that can
interact with your bait.
| n***h
发帖数: 11 | 4 照我的记忆说来：
yeast 2 hybrid system是两个蛋白之间的binding
yeast 1 hybrid是蛋白质和DNA之间的binding ，就象ras说的，测出的是bait和
fragment之间的结合，可以象ras说的那样fragment是library, 来找和已知蛋白
结合的特定element，也可以特定的fragment，转cDNA library进去，找和这个
fragment 结合的蛋白。 （infact that's what I did)
回来说2hybrid。transcriptional factor一般至少有两个domain: binding domain
和activition domain. binding domain binds to DNA elements, while activation
domain activates the transcription of downstream genes. 我们要测试蛋白A
和蛋白B是否bind, 就做以下两个construct：
—————————— ————————————
|A | binding domain | |B | activition domain |
--------------------- ------------------------
两个都是in-frame fusion，完全transcribed to protein. 这样如果AB bind each
other, binding domain and activition domain就被拉到一起，结合在DNA上，
activate这个transcriptional factor 所本应activate 的gene transcription.
一般我们就在这个transcriptional factor结合的地方下面加个reporter gene
这样如果reporter gene 表达，如lacZ就是菌落蓝色，就说明AB bind.如果不表达
AB就不bind.
【在 n***h 的大作中提到】
: 照我的记忆说来：
: yeast 2 hybrid system是两个蛋白之间的binding
: yeast 1 hybrid是蛋白质和DNA之间的binding ，就象ras说的，测出的是bait和
: fragment之间的结合，可以象ras说的那样fragment是library, 来找和已知蛋白
: 结合的特定element，也可以特定的fragment，转cDNA library进去，找和这个
: fragment 结合的蛋白。 （infact that's what I did)
: 回来说2hybrid。transcriptional factor一般至少有两个domain: binding domain
: 和activition domain. binding domain binds to DNA elements, while activation
: domain activates the transcription of downstream genes. 我们要测试蛋白A
: 和蛋白B是否bind, 就做以下两个construct：
| N****1
发帖数: 1117 | 5 Jon不服
have
【在 R*s 的大作中提到】
: example:
: --------------------- ----------------------------
: | bait | activator| + |fragment A | reporter gene |
: --------------------- ----------------------------
: Bait ait is the gene u r intestered in, and is attached to a
: transcription activator. fragment A is one of those from a DNA library
: which is attached to a reporter gene.
: Basically you transfect both the bait construct and library construct
: into yeast (a and alpha respectively) and let the mate (each yest should have
: only one of those library DNA fragment). If the bait can bind with one of
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