c*t
发帖数: 1063 | 1 被问到一个问题，不知道怎么回答，特来版上求助。
Immunostaining showed two proteins co-localize in the membrane, question is
to confirm that the two proteins directly bind each other.
First, I was thinking about co-ip, but this method does not exclude the
possibility other proteins are also involved, so it's hard to tell direct
protein binding with co-ip.
Second method I was thinking about is yeast two-hybrid, which is just to
detect the interaction of two proteins. But drawback is false positive.
Third method is a combination with co-ip, mass spectrometry. co-ip
will show both proteins are in one complex, and ideally if no other proteins are involved, mass
spectrometry will only show the two proteins that are studied.
The fourth method is BiFC, label both proteins with half of GFP, when they
bind together, there will be GFP signal. Problem for this method is that can
we say the two proteins directly bind with each other?
请教大家的建议，谢谢！ | m******r
发帖数: 854 | 2 PULL-DOWN ASSAY, No.1 method | c*t
发帖数: 1063 | 3 But the question asked is to confirm direct protein binding, I don't know if
I am "钻牛角尖"。 | X******n
发帖数: 914 | | w***e
发帖数: 269 | 5 Your lists are fine. I don't think there is a "perfect" method. You just
have to cross-validate direct binding with results from multiple essays. | c********r
发帖数: 189 | 6
Re
For direct binding, the only way is in vitro pull down assay with purified
proteins
【在 X******n 的大作中提到】
: In vitro binding
| k****o
发帖数: 589 | 7 surface plasmon resonance |
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