[RASMB] Fwd: Re: AUC capabilities

[Ewa Folta-Stogniew]

>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
>>