Dear colleagues,

I'm a bit puzzled by the result of a GST pulldown assay I have done a while ago using GST-Rac wild type (WT) or the constitutive active mutant Q61L, immobilised on bead and mixed with cell lysate containing my protein of interested (GFP-tagged). 

Turned out that my protein binds specifically to Q61L Rac1 but not to WT Rac1 - Pretty exciting for me.

When I repeated this experiment using Rac1 loaded with a non-hydrolysable GTP analogue (GppNHp), I failed to pull down my protein. 

The positive control in these 2 experiments is the commonly used CRIB domain of Pak and binds as efficiently both Q61L Rac1 and GppNHp-loaded Rac1.

Would it be possible that overall electrical charges might be different and for any reason, my protein is sensible to this?

Tried to look for paper mentioning a difference between these 2 forms of Rac1 but could not find any. 

Any help would be appreciate.

Cheers,