That's an extremely useful link - thanks to Will Stanley for posting
that one.
For a VP-ITC machine I'd guess that you need to load the injector with
about 500ul of protein at a concentration of 80x the Kd or more.
Notice that Alan Cooper was injecting 10 microliters of protein at 2mM
with a 12 microMolar dissociation constant, per injection. You would
probably want to maintain that approximate ratio - ~170 because it's
mostly a question of measuring deltaH with a decent signal-to-noise per
injection.
I recall that it takes up to 500 microLiters to load the injection
syringe on a VP-ITC without air gap between plunger tip and injection
point - unless someone's got a nice trick to reduce that.
The rule of thumb from the VP-ITC manual - and from practical experience
on our machine here - for A+B <=> AB is using at least 10x the Kd in the
sample chamber and about 80x the Kd in the injector. That's not exactly
the same situation, but 80x vs 170x suggests the the considerations are
much the same.
Phil Jeffrey
Princeton
On 2/14/14 12:52 PM, Keller, Jacob wrote:
What a nice idea this ITC dilution is--a great example of a wet lab technique
learned en passant on the ccp4bb.
I wonder what range of Kds could feasibly be measured with existing calorimeter
sensitivities?
JPK
-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Will
Stanley
Sent: Friday, February 14, 2014 12:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] KD of dimerization, off topic
Hi Careina,
Since alternative methods are being suggested...
ITC can be good for quantitating a monomer-dimer equilibrium by diluting dimers
out from a concentrated solution (which obviously favours the dimer) - and
presuming a reasonable Kon/Koff.
Alan Cooper has kindly figured out the data fitting for the rest of us:
http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf
I think Alan was using a VP-ITC when he was doing this stuff. Lower volumes -
and presumably concentrations if the KD is small enough - are feasible in an
ITC200. The protein is recoverable anyway.
All the best,
Will.
