Apparent kDs - why are they only apparent?? (Jan/19/2011 )

Hi,

I have recently been told I cannot refer to K_D values calculated through binding assays withour calling them apparent K_Ds (or K_Dapp). I think this is because a standard binding assay only measures association rates and not disassociation rates. Can anyone confirm this? I've been googling for half an hour now and I can't find any good explanations for the use of the term apparent!

Thanks
P

The terms are super confusing and interchangeable. Biacore has a lot of really good application/technical notes that really go into binding kinetics - that may be a good resource for information. Dissociation rates can be measured directly when using SPR-like technologies.

antibodymania on Wed Jan 19 18:03:40 2011 said:

Hi,
I've spoken to some SPR experts and they say that, although SPR gives good estimates, it can still only provide apparent binding affinities and the only technique which gives actual K_Ds is Isothermal Titration Calorimetry (ITC). But I still don't know what is really meant by apparent K_D and how it's different from K_D - any ideas??

Thanks,
P

I guess SPR would be measuring interactions on a solid phase platform which may effect such sensitive calculations.

Hmmm...I am having the same issues as you are in searching for more information. But perhaps you may have answered your own question? Maybe apparent Kd is an indirect measure (such as data obtained from SPR) vs Kd is an actual measure (data obtained from interaction measurements in solution such at ITC). This is a guess...and I do apologize for not having an absolute answer for you =(

As a side note, GraphPad Prism also offers some stuff on kinetics http://www.curvefit.com/association_.htm

It appears that none of these methods accurately measure K_D. Thus one would refer to such measurements as "the K_D as measured by this method" or "apparent K_D". This seems directly analogous to measuring the MW of a protein by any number of not-completely-accurate methods -- they're referred to as "apparent MW", or "relative MW (aka MW_R)".

Thanks guys,

I think my main source of confusion stems from the fact that most publications use the term K_D when in reality the term apparent K_D should be used. I guess its standard to do this and most journals don't worry about it.

The reason I'm asking is because I'm writing my thesis and I always use the term apparent K_D. I wanted to be sure this was correct before I have to explain to an examiner why my calculations are only apparent.

Thanks again!
P