D******9
发帖数: 2665 | 1 I would like to study the cell signaling pathway initiated by a cell surface
ligand. However, I do not know its receptor. What is the best way to find
its receptor? Any good ideas? Thanks a lot. |
p*****m
发帖数: 7030 | 2 expression cloning, the classic method
or RNAi screen, or educated guess, or to test known receptor agonist/
antigonist library
surface
【在 D******9 的大作中提到】
: I would like to study the cell signaling pathway initiated by a cell surface
: ligand. However, I do not know its receptor. What is the best way to find
: its receptor? Any good ideas? Thanks a lot.
|
D******9
发帖数: 2665 | 3 many thanks for your answer. Can you give a little bit details about
expression cloning? Is the receptor agonist/antigonist library very specific
? Thx |
D******9
发帖数: 2665 | 4 Is it possible to add the ligand to the cell culture, of couse I know the
cells are responsive to this ligand, then crosslink, IP and send for MASS
SPEC analysis? |
p*****m
发帖数: 7030 | 5 1 你google一下从EPO receptor到serotonin receptor到TRP channel怎么搞出来的
就行了 简单来说就是搞一个没有receptor的cell line，把有receptor表达的cDNA
library转进去 然后加ligand，看下游的phenotype就行了 可以是binding activ
ity，也可以是downstream signaling,等等
2 没有 但是如果你有个范围可以大概用agonist/antigonist猜猜看
specific
【在 D******9 的大作中提到】
: many thanks for your answer. Can you give a little bit details about
: expression cloning? Is the receptor agonist/antigonist library very specific
: ? Thx
|
p*****m
发帖数: 7030 | 6 你是什么性质的ligand? 也不是不行 但是实际上会很困难
【在 D******9 的大作中提到】
: Is it possible to add the ligand to the cell culture, of couse I know the
: cells are responsive to this ligand, then crosslink, IP and send for MASS
: SPEC analysis?
|
D******9
发帖数: 2665 | |
p****l
发帖数: 291 | 8 yeah, it is really difficult. One of my projects is quite similar.
I finished the step crosslink and IP, and sent the sample for MASS.
But to confirm the receptor is the protein reported by MASS, still need a
lot of time.
I prefer the method you recommended, transfer cDNA library to a cell line
which does not express the receptor. Screen the positive clone.
【在 p*****m 的大作中提到】
: 你是什么性质的ligand? 也不是不行 但是实际上会很困难
|
k****o
发帖数: 589 | 9 我用的是educated guess法，撞大运找对了
没空玩screening的话，多查查文献也许有头绪 |
s******y
发帖数: 28562 | 10 There is back-ward and forward screening methods
For forward screening, you can do crosslinking and pulldown of candidate
proteins, and then send the products for mass spec
For back-ward screenings, you can use random KO library (usually with yeast)
to select the clones that are resistant to your ligand (with proper
screening assay), and then figure out what gene is required for the signal
pathway.
Either way it is not a small work
surface
【在 D******9 的大作中提到】
: I would like to study the cell signaling pathway initiated by a cell surface
: ligand. However, I do not know its receptor. What is the best way to find
: its receptor? Any good ideas? Thanks a lot.
|
