J*****n
发帖数: 1041 | 1 I have two proteins, and the binding is not very strong. IP and pull-down
assay showed positive results. But we want to have other evidence of the
binding?
Is ITC a good tool to study protein-protein interaction?
And is there any other approaches to study protein-protein interaction? Size
exclusion chromatography? SPR? ....
Many thanks in advance! |
p*****n
发帖数: 981 | 2 spr
itc is not good for weak interactions.
sec may work. get a hplc sec column.
Size
【在 J*****n 的大作中提到】
: I have two proteins, and the binding is not very strong. IP and pull-down
: assay showed positive results. But we want to have other evidence of the
: binding?
: Is ITC a good tool to study protein-protein interaction?
: And is there any other approaches to study protein-protein interaction? Size
: exclusion chromatography? SPR? ....
: Many thanks in advance!
|
e****s
发帖数: 1125 | 3 Sec won"t work for weak association.
Weak protein-protein interaction is always hard to confirm.
If one protein is small, you may try FP.
【在 p*****n 的大作中提到】
: spr
: itc is not good for weak interactions.
: sec may work. get a hplc sec column.
:
: Size
|
l**********1
发帖数: 5204 | |
k******0
发帖数: 1073 | 5 Your answer is quite right.
【在 e****s 的大作中提到】
: Sec won"t work for weak association.
: Weak protein-protein interaction is always hard to confirm.
: If one protein is small, you may try FP.
|
A****w
发帖数: 244 | 6 For ITC, you need milligrams of protein.
SPR, you need much less.
Another easy one is chemical cross-linking... |
H*g
发帖数: 2333 | 7 There are many ways to show protein-protein interaction; However, I would
suggest u to forget any additional in vitro assays. You already got IP and
pull-down, so do not waste time on other in vitro ones.
In your case, if I were the reviewer, I would directly ask for evidence from
other perspectives, in addition to all those in vitro assays. I would ask
for assays testing in vivo, real-time interactions, such as BiFC or FRET, as
well as any evidence demonstrating genetic interaction to support that the
physical interaction is biological meaningful.
Size
【在 J*****n 的大作中提到】
: I have two proteins, and the binding is not very strong. IP and pull-down
: assay showed positive results. But we want to have other evidence of the
: binding?
: Is ITC a good tool to study protein-protein interaction?
: And is there any other approaches to study protein-protein interaction? Size
: exclusion chromatography? SPR? ....
: Many thanks in advance!
|
J*****n
发帖数: 1041 | 8 Thank you all for the kindly replies.
Two of my proteins, one is 44 kd and another is 67, therefore I do not think
it is appropriate to use FP.
ITC did not give me positive results.
SEC showed binding but the peaks are ambiguous.
We might try MALS...not sure if it will be successful.
Actually, to be more specific, we want to check the binding stoichiometry of
these two proteins. Any suggestion?
Many thanks! |
