GST pull down vs immunoprecipitation - (Oct/27/2005 )

Dear people,

I am facing problems comparing results of my IPs vs. my GST pull downs. Maybe some of you experienced the same kind of problems:

1) My IP efficiency is way lower than my pull down efficiency

2) Are GST PD a good way to find out roughly what's binding and forming complexes in a cell?

3) My GST pull downs are much more cleaner in terms of non-specificity than my IPs.

I am tired of trying every interaction in both systems... and speculating on the reasons for the differences.

Has anyone an opinion on what is sufficient to consider an interaction "valid"?

Thanks!

Dapo

Hi Dapo,

I was just reading about both techniques and what books say it that the difference between pull down and coIP is that coIP is the only one that allows you to look for a physiological in vivo interaction.

However, if you do your coIP, overexpressing your protein, coIP comes to be the same as a pull down but with less efficiency and recovery because it depends on one more interaction...If you need to overexpress your protein, it's better to use pulldown. If you want to know what's really going on in vivo, maybe you have to grow many liters of culture and do an coIP..

Hope it helps,

Carlota