[RASMB] Fwd: Re: AUC capabilities

[Ewa Folta-Stogniew]

Hi Arthur,

I have no AUC data so far.  I am just asking 
experts to whether AUC experiment may give us 
confirmation of the Kd I had already measured by 
light scattering.  I and the researchers on this 
project are no AUC users, so we would need to 
send the sample somewhere (or ask Yale AUC users 
for help), or use Allen's approach (if it is feasible with 25 ug of protein).

So far, I am not encouraged as the protein is 
produced in ~20 ug per prep (a very successful 
prep), and I have not yet gotten estimates how 
much protein will be needed for any of the proposed AUC approaches.

For your approach, what will be the amount of the protein needed?

Ewa







At 11:23 AM 1/27/2011, Arthur Rowe wrote:
>Hi Ewa (and everyone)
>
>I'm afraid I cannot accept the basis on which 
>some of the arguments made by Borries and Allen 
>are advanced. It is just not true that you need 
>to do SE experiments over a range of 
>concentrations which spans Kd. I and my 
>co-workers have over recent years shown in 
>detail, by simulation and by experiment, that a 
>single run suffices, provided that you analyse 
>your data correctly, and that the SE run can be 
>at a solute concentration removed by 1+order of 
>magnitude from Kd without too seriously 
>affecting the precision (see Ang & Rowe, 2010). True, my own
>interest is in ultra-weak interactions, where we 
>are at the other end of the concentration 
>spectrum, But there we are obliged to contend 
>with thermodynamic interaction (in addition to 
>the mass interaction) terms. Basically, for this 
>reason, weak dissociation is easier to cope with 
>than weak association, as it is Kd as a measure 
>of interaction which is all that we need to bother about.
>
>Your protein, you write, has an extinction 
>coefficient of 0.10 at 220 nm. So an SE run will 
>provide values spanning the range of 0.01 (near 
>meniscus) to around 0.8 (near base). That's at 
>sigma = 2, which avoids getting outside Beer's 
>Law.If you can establish a true baseline (e.g. 
>overspeeding, or taking an A320) then this is 
>plenty of signal to work with. In any case, if 
>you have taken a scan immediately on reaching 
>speed, then you have the means to correct for TI 
>noise (e.g. bumpy bits!) in the baseline. And 
>you can validly float the baseline offset, if 
>you are fitting with proper separation of 
>variables, via the inverse (INVEQ) fitting 
>algorithm. The software for the latter (which I 
>can provide) runs under pro Fit™ on a Mac, or 
>under Grafit on Windows (the latter a bit primitive, but it gives an answer).
>
>Or you could just mail me the data. It would 
>take me about 5 minutes - if a do it very slowly 
>- to get a Kd out. OK - allow another 5 minutes 
>to finish off the boot-strapping to get the 
>error distribution (I reckon that so-called 
>'confidence limits' are only 1 stage better than 
>believing what Marquardt-Levenberg tells you 
>about error bounds). And to anticipate an 
>obvious question, yes, I have on occasion looked 
>at weak dissociations, albeit not under 
>conditions where I am free to communicate the results.
>
>I attach a list of buffers compatible with 
>working in the far u/v - based upon measurements made locally.
>
>Kind regards
>
>Arthur
>
>Ang, S. & Rowe, A.J. (2010) "Evaluation of the 
>Information Content of Sedimentation Equilibrium 
>Data in Self-Interacting Systems" Macromolecular Bioscience 10 798-807
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>************************************************************************ 
>*******
>Arthur J Rowe
>Professor of Biomolecular Technology / Director NCMH Business Centre
>School of Biosciences
>University of Nottingham
>Sutton Bonington
>Leics LE12 5RD
>
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>On Jan 27, 2011, at 14:29, Ewa Folta-Stogniew wrote:
>
>>
>>>Date: Thu, 27 Jan 2011 09:19:28 -0500
>>>To: Allen Minton <minton at helix.nih.gov>
>>>From: Ewa Folta-Stogniew <ef55 at email.med.yale.edu>
>>>Subject: Re: [RASMB] AUC capabilities
>>>
>>>Allen,
>>>
>>>thanks for reminding me... I only thought about this approach for
>>>high concentrations..
>>>
>>>Is everything I may need for this approach (assuming minimal sample
>>>volume) commercially available?
>>>
>>>Ewa
>>>
>>>At 08:46 AM 1/27/2011, Allen Minton wrote:
>>>>At 09:41 PM 1/26/2011, you wrote:
>>>>>Hello,
>>>>>
>>>>>is it possible to determine dimerization constant in the order of
>>>>>25 nM using AUC?  What will be the minimal amount of protein
>>>>>needed?
>>>>>
>>>>>Ewa
>>>>
>>>>The answer is yes, but you will have to employ the non-traditional
>>>>approach we use in our lab, which involves centrifugation to sed.
>>>>eq., followed by fractionation of the cell contents (see attached
>>>>reprints).  Then you can use any technique available to measure the
>>>>relative concentration of the protein in each fraction and thereby
>>>>reconstruct the equilibrium gradient.  There are many analytical
>>>>methods for measuring protein concentration at much lower
>>>>concentrations accessible by either the absorbance or RI scanner in
>>>>your analytical ultracentrifuge, such as the widely used BCA
>>>>colorometric assay.  If you are willing to radiolabel your protein
>>>>or to employ an immunoadsorbent assay you can go one or two orders
>>>>of magnitude even lower in concentration.
>>>>
>>>>I realize that this would require you to learn a whole new way of
>>>>doing SE measurements, but it would provide you with a lot of new
>>>>capabilities.  For example, we are now using this method to study
>>>>associations of dilute proteins in solutions containing high
>>>>concentrations of other macromolecules.  Can't do that with your
>>>>Beckman!
>>>>
>>>>Allen
>>
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>
>
>This message and any attachment are intended 
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>confidential information. If you have received 
>this message in error, please send it back to 
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>use, copy or disclose the information contained 
>in this message or in any attachment.  Any views 
>or opinions expressed by the author of this 
>email do not necessarily reflect the views of the University of Nottingham.
>
>This message has been checked for viruses but the contents of an attachment
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