6 replies to this topic

[luigialfano]

Hi,
I have a problem with co-ip experiment. I identify the interaction between my protein and its target by pull down assay. Now i want to characterize this interaction by co-ip experiment but it isn't works. I use differents antibodies that recognize the proteins in different portions but i don't see the interaction. These antibodies works very good to precipitate my protein. I use different buffers but it isn't work. Can you help me? Do you think that the pull down works because the protein used as bait is to' mutch respect to' protein immunoprecipitate with the antibodies. There Are some solutions? Thank you

[bob1]

So your initial pull-down assay was not an IP?  Do you know anything about the relative frequency of this interaction?  If the interaction is only a small proportion of the protein in the cell, you may not be able to detect it, unless your antibodies are very sensitive.  You could try using more cells to IP from, change the detergents you use in the IP and wash buffers and don't agitate the beads too much, as this can physically break interactions.

[luigialfano]

Hi,
thank you to answer. No my initial pull down is not and IP because I used a recombinant protein GST-fused. Now I want to try more cells to IP and I try to don't agitate the beads
thank you

[bob1]

I see.  Did you check that the tag was not interfering with the GST pulldown by running an untagged construct through a GST pulldown?

[luigialfano]

Dear bob1,

my pull down assay works very good my endogenous Co-IP no

[bob1]

Yes, what I mean is that perhaps the GST (alone) is interacting with the other protein, and causing the pull down in your initial trials, which is why you don't see IP now.

How abundant are the endogenous proteins?  Can you IP (not co-IP) the endogenous proteins separately?

[luigialfano]

Sera bob1,
I tried to do the pull down with all controls: gst alone and glut. sepharosio beads but they are negative
I tried to ip only one proteins and it works very good