Hello,
OK, this all makes sense to me. So if the amount of volume added is only
a few percent of the original volume then I'd say we can ignore the
effect. But if it is 15-20% then that is definitely significant. We will
need that one extra parameter as input, so dV/bV, where V is the fixed
volume started with and dV is the volume added for a specific
concentration b of B (so you have an extra 2dV for 2b, etc.). This might
be best specified as far as the user is concerned as three pieces of data:
V and then also dV for some known b).
Wayne
On Wed, 8 Feb 2012, Fowler, Andrew wrote:
> Hi Wayne,
>
> I think this is effectively what I was getting at - and yes, dV/V should
> be proportional to the amount of B that is added.
>
> As for whether dV is large enough, I suppose that depends on what your
> criteria are. If the ligand is poorly soluble and/or Kd is small enough
> that you need to add 20 equivalents or more of ligand to get near
> saturation, then that's quite possible. I've certainly done titrations
> where the total volume has changed by 15-20%. On the other hand, sometimes
> the total change can be no more than 2-3% and that's probably within
> fitting error.
>
> Now, a in your original question can be kept fixed by dissolving the
> ligand stock in the same starting protein, but (as I just saw in Aldino's
> most recent message) sometimes that's just not possible.
>
> Andrew
>
> --
> C. Andrew Fowler, Ph.D. | University of Iowa
> Associate Director | B291 Carver Biomedical Research
> Building
> Medical NMR Facility | Iowa City, IA 52242
> 319-384-2937 (office) | 319-335-7273 (fax)
> [log in to unmask]
>
>
>
>
> On 2/8/12 11:24 AM, "Wayne Boucher" <[log in to unmask]> wrote:
>
>> Ah, Tim/Magnus are suggesting that if V is the original volume then you
>> are adding dV extra volume and so the concentration of A (including that
>> in AB) has changed from a to aV/(V+dV). If dV is large enough then that
>> could be a problem. Is dV large enough? And if it is a problem then we
>> would have to have some way for dV/V to be input (not for each point,
>> because presumably this is proportional to how much B is added, so if
>> you add twice as much extra volume that is because you are adding in
>> twice as much B).
>>
>> Wayne
>>
>> On Wed, 8 Feb 2012, Wayne Boucher wrote:
>>
>>> Hello,
>>>
>>> Just to make sure what we are talking about (!), if we have the
>>> reaction A +
>>> B <--> AB where A is the protein (say) and B the ligand, then the total
>>> concentration of A is a = [A]+[AB] and the total concentration of B is
>>> b =
>>> [B]+[AB]. So are you just saying that [A] decreases when you add B,
>>> because
>>> that I agree with. The Analysis code assumes not that [A] is fixed but
>>> that
>>> a is fixed (and perhaps I'm using the wrong terminology to describe
>>> that
>>> situation). So, alternatively, are you saying that a itself might
>>> change??
>>>
>>> Wayne
>>>
>>> On Wed, 8 Feb 2012, Fowler, Andrew wrote:
>>>
>>>> I may be wrong, but I think what Aldino means by changing both the
>>>> ligand
>>>> and protein concentration in a titration is that one starts with free
>>>> protein at a given concentration and volume and adds increasing
>>>> amounts of
>>>> ligand to that. So the protein concentrations decreases while the
>>>> ligand
>>>> concentration increases, but both are known at each point so you really
>>>> don't have to fit two independent variables. It's not be the ideal
>>>> case,
>>>> but for various reasons many (most?) titrations get done this way.
>>>>
>>>> Analyzing this sort of titration data would be a very nice addition to
>>>> Analysis - I know I would find it useful. The equation to fit the data
>>>> is
>>>> a bit messier but input would be both ligand and protein concentration
>>>> for
>>>> each point (or if you wanted to be really nice, starting concentration
>>>> and
>>>> volume of protein, ligand stock concentration, and the volume of ligand
>>>> added at each step with the program calculating from that).
>>>>
>>>> Cheers,
>>>> Andrew
>>>>
>>>> --
>>>> C. Andrew Fowler, Ph.D. | University of Iowa
>>>> Associate Director | B291 Carver Biomedical Research
>>>> Building
>>>> Medical NMR Facility | Iowa City, IA 52242
>>>> 319-384-2937 (office) | 319-335-7273 (fax)
>>>> [log in to unmask]
>>>>
>>>>
>>>>
>>>>
>>>> On 2/8/12 10:22 AM, "Wayne Boucher" <[log in to unmask]> wrote:
>>>>
>>>>> Hello,
>>>>>
>>>>> The two titration type functions that are in Analysis are for
>>>>> protein-ligand in fast exchange where you vary the ligand
>>>>> concentration
>>>>> but keep the protein concentration fixed, and a monomer-dimer in fast
>>>>> exchange, where you vary the total concentration. The equations are
>>>>> discussed in a PDF file obtainable at:
>>>>>
>>>>>
>>>>> https://sites.google.com/site/ccpnwiki/Home/documentation/ccpnmr-analys
>>>>> is/
>>>>> FAQs/data-analysis
>>>>>
>>>>> If you want to vary two concentrations then that complicates any
>>>>> attempt
>>>>> at fitting (it looks like to me but someone out there probably knows
>>>>> better).
>>>>>
>>>>> The current Analysis does the fitting in C functions. Unfortunately
>>>>> that
>>>>> turns out to be some of the worst C code in Analysis because stuff
>>>>> kept
>>>>> getting bolted onto it, so I wouldn't recommend anyone try to adjust
>>>>> that
>>>>> (although if you want to I can tell you what needs doing).
>>>>>
>>>>> I've written a Pythonic version of the fitting but it has not been
>>>>> hooked
>>>>> into the user interface. I will send you information about that in a
>>>>> separate post, because it's likely to be the easiest way for you to
>>>>> add
>>>>> your own functions, it's just that the visualisation is not there, so
>>>>> would have to be done in (say) Excel. (You can also let us know what
>>>>> you
>>>>> want added and I can add it into the C functions.)
>>>>>
>>>>> Wayne
>>>>>
>>>>> On Wed, 8 Feb 2012, Aldino wrote:
>>>>>
>>>>>> Hi all,
>>>>>> I have some questions regarding curve fitting on analysis:
>>>>>>
>>>>>> 1) is there any function on analysis to fit a concentration titration
>>>>>> set in
>>>>>> which you change both the ligand and the receptor concentrations?
>>>>>>
>>>>>> 2) is the Michaelis-Menten funtion defined?
>>>>>>
>>>>>> 3) where can I find information on the several funtions in avalysis?
>>>>>>
>>>>>> 4) can I write my own funtions?
>>>>>>
>>>>>>
>>>>>> Cheers,
>>>>>> Aldino Viegas
>>>>>>
>>>>>>
>>>>>> ______________________________________________
>>>>>> Aldino Viegas, PhD Student
>>>>>> Dep. Química, REQUIMTE
>>>>>> Faculdade de Ciências e Tecnologia
>>>>>> Universidade Nova de Lisboa
>>>>>> 2829-516 Caparica, Portugal
>>>>>>
>>>>>> Tlf. +351 212948300
>>>>>> Ext. 10900 Lab. 106-A
>>>>>> [log in to unmask]
>>>>>>
>>>>
>>>>
>>>>
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