Several people have already pointed out that a "pull-down" is not
a rigorous, quantitative method. Mackay et al (TIBS 32, 530-531, 2007)
have some interesting comments to the effect that many reported
interactions found by pull-down or immunoprecipitation are simply
artefacts. Pull-downs are also
Hi Brett,
As Filip pointed out, pulldown assay is not an equilibrium
experiment. In addition, one of protein should be attached to the
resin in the pulldown assay, which might not be sometimes favorable.
You might not want to make very quantitative analysis on the pulldown
results.
If I
Dear Artem,
Sorry I didn't want to scare people with too may details.
The pulldowns and ITC were done with the same protein samples
expressed in bacteria and purified by the usual ways. For both mutant
and wildtype the stoichiometry from ITC was approximately 1:1 (from
about 0.85 to about
Dear Brett,
since your deltaH contribution gets weaker, and the deltaS contribution
changes from negative to neutral, you could have a case of enthalpy-entropy
compensation (see e.g.Dunitz JD (1995) Win some, lose some: enthalpy-entropy
compensation in weak intermolecular interactions. *Chem Biol*
Dear CCP4 Community,
My apologies for the non-crystallography biochemical
question but it occurred to me that there are many people
on this list who are also very good biochemists.
We have just performed an ITC experiment with two proteins
and measured a Kd of 150 nM, deltaH of -15 kCal/mol,
