Hi Wayne and Andrew.
What Andrew said is absolutely correct!
Most often protein quantity is limited so we try to use as least as possible
and that implies that in a titration experiment we use the same sample of
protein in every step and just add the ligand to it.
Therefore, according to Wayne's explanation, the total concentration of A
(a) in a sample decreases because the total volume is increasing. Thus, at
each point of the titration we have a known (but different from the
previous) concentration of protein and ligand.
Regarding the question about dV, at least in my case its only small ammounts
(few uL in 500 uL)
Thank you for your help,
Aldino
-----Mensagem Original-----
From: Wayne Boucher
Sent: Wednesday, February 08, 2012 5:24 PM
To: [log in to unmask]
Subject: Re: curve fitting
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-analysis/
>>> 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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