[RASMB] Fwd: Re: AUC capabilities

[Arthur Rowe]

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
This message and any attachment are intended solely for the addressee and may contain confidential information. If you have received this message in error, please send it back to me, and immediately delete it.   Please do not 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
may still contain software viruses which could damage your computer system:
you are advised to perform your own checks. Email communications with the
University of Nottingham may be monitored as permitted by UK legislation.
-------------- next part --------------
A non-text attachment was scrubbed...
Name: List of buffers for far uv.doc
Type: application/applefile
Size: 424 bytes
Desc: not available
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20110127/fc96745e/attachment.bin>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: List of buffers for far uv.doc
Type: application/msword
Size: 39936 bytes
Desc: not available
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20110127/fc96745e/attachment.doc>
-------------- next part --------------


************************************************************************ 
*******
Arthur J Rowe
Professor of Biomolecular Technology / Director NCMH Business Centre
School of Biosciences
University of Nottingham
Sutton Bonington
Leics LE12 5RD

TEL:  0115 9516156

************************************************************************ 
*******

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
>>>
>
> _______________________________________________
> RASMB mailing list
> RASMB at rasmb.bbri.org
> http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb
>
>


This message and any attachment are intended solely for the addressee and may contain confidential information. If you have received this message in error, please send it back to me, and immediately delete it.   Please do not 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
may still contain software viruses which could damage your computer system:
you are advised to perform your own checks. Email communications with the
University of Nottingham may be monitored as permitted by UK legislation.