We use Isothermal Titration Calorimetry (ITC) for studying translational GTPases for quite some time now, and it turnes out one can do one more translation-related things with it: measure thermodynamics of subunit association with it (Osterman Biochimie 2011).
Quite amazing, actually. Sample is mixed at 200 rpm, ∼2 μM 50S are titrated with ∼11 μM 30S subunits, and yet seemingly there are no problems with precipitation and heat generated by it.
I would suspect that this means that one can follow initiation complex formation too...
References:
Ilya A Osterman, Petr V Sergiev, Philipp O Tsvetkov, Alexander A Makarov, Alexey A Bogdanov, Olga A Dontsova. Methylated 23S rRNA nucleotide m(2)G1835 of Escherichia coli ribosome facilitates subunit association. Biochimie 2011 PIMD 21237242
Hello,
ReplyDeleteI am a phD Student in Spain and i´m currently starting to work with ITC. What I want is to stablish Kd between my protein and some inhibitors. Actually I´m having quite a lot of problems because i´m not able to establish the optimal concentrations conditions to see the union. Could you explain me, please, how do you to to stablish which are your optimal conditions to work?
Thank you in advance.
Marta
Hi Marta,
ReplyDeletethe big issue with ITC is that your protein might aggregate during the titration curve and this hides the titration curve. Can you please send me some example curves of a) ligand titration b) solute without ligand titrated into the protein by email?