[RASMB] AUC interference concentration limits

[David Hayes]

Hi all,Just a quick note to add to Walter's calculations.  You can use a 3 mm thin centerpiece and then multiply concentration by 4.  I have done 50 mg/ml solutions in a 3 mm cell.  The last one I did recently had to be run at 20,000 RPM.  Previously, I have done 50 mg/ml at 40,000 RPM.  The steepness of the boundary at high concentration depends on the non-ideality more than the size of the molecule and its diffusion coefficient.  Without meniscus matching, you do get a noisy baseline, but compared to 50 mg/ml signal, it is insignificant.
But I did hear once on the phone about high concentration experiments:  "anyone can collect that data, no-one understands it."  Here "understands" means fitting to a valid hydrodynamic equation.  If you have 50 mg/ml in a typical no salt high trehalose buffer, I doubt it will fit to a linear ks term.  I recommend doing many concentrations:  you can mistake a dimer with lots of non-ideality for a monomer with less non-ideality.
Kind Regards,
David Hayes
      From: Walter Stafford <wstafford3 at walterstafford.com>
 To: John Sumida <jpsumida at uw.edu> 
Cc: rasmb at list.rasmb.org 
 Sent: Tuesday, October 20, 2015 6:31 PM
 Subject: Re: [RASMB] AUC interference concentration limits
   
As said by others. the limiting thing here is not the concentration, but the concentration gradient. There really is no practical limit to the max concentration. The limit is the ability to resolve the fringes in the radial direction. The steepness for the fringes and the radial resolution of the camera determines that. The vertical distance between fringes is constant and is something like 22 pixels/per fringe, and in the radial dimension the distance between fringes is about 0.007 cm per pixel. So 1 fringe displacement over one pixel in the radial direction correspond to an upper limit of 142 fringes per cm. The way to avoid the problem you are seeing, if it's feasible, is to run at a slower speed and wait for the boundary to spread enough so that the fringes can be resolved. The actual practical limit is probably about half that, or about 70 fringes per cm, or a gradient of about 21 mg/(mL-cm).

Walter Stafford
wstafford3 at walterstafford.com
"Twenty years from now, you will be more disappointed by the things you didn't do than those you did. So throw off the bowlines. Sail away from safe harbor.Catch the wind in your sails. Explore. Dream. Discover."  — Mark Twain, Great American Writer


On Oct 20, 2015, at 15:32, John Sumida <jpsumida at uw.edu> wrote:


#yiv0166349145 #yiv0166349145 -- _filtered #yiv0166349145 {panose-1:2 4 5 3 5 4 6 3 2 4;} _filtered #yiv0166349145 {font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;}#yiv0166349145 #yiv0166349145 p.yiv0166349145MsoNormal, #yiv0166349145 li.yiv0166349145MsoNormal, #yiv0166349145 div.yiv0166349145MsoNormal {margin:0in;margin-bottom:.0001pt;font-size:11.0pt;}#yiv0166349145 a:link, #yiv0166349145 span.yiv0166349145MsoHyperlink {color:#0563C1;text-decoration:underline;}#yiv0166349145 a:visited, #yiv0166349145 span.yiv0166349145MsoHyperlinkFollowed {color:#954F72;text-decoration:underline;}#yiv0166349145 span.yiv0166349145EmailStyle17 {color:windowtext;}#yiv0166349145 .yiv0166349145MsoChpDefault {} _filtered #yiv0166349145 {margin:1.0in 1.0in 1.0in 1.0in;}#yiv0166349145 div.yiv0166349145WordSection1 {}#yiv0166349145 Dear RASMB,  I have been reviewing the thread posted on RASMB between September 1 2008 and September 15 2008 under the subject heading “upper concentration limit AUC”.     We attempted to perform an SV measurement of a sample using interference detection at a sample concentration of 17 mgs/ml.    At this concentration however I observe a gap in the boundary data that resulted from the spin (see bmp image attached); this behavior was not observed at lower concentrations up to 2-5 mgs/ml.    The gap in the data appears to be correlated with the absence of a fringe pattern in the ccd image.  Adjustment of the duration setting does not resolve the issue with the data, but it does improves the ccd image so that I can make out the fringes in the region where I’m observing the gap in the data.  Counting the fringes/mm in this region, I estimate the fringe gradient to be approximately 50 fringes/mm. This is less than the 76 fringes/mm noted in the RASMB thread but my count may be off.  On a separate note, since I suspect that the artifact I’m seeing is associated with the observed steep fringe gradient I had wondered if our instrument was focused at the 2/3rds plane or not.  I have been informed, by the vendor, that the process of focusing the interference optics at the 2/3rds optical plane is not something that is possible in the “real world” (their words, not mine), and that this is that this is only something that is only “theoretically” possible.  This appears to be in direct contradiction to the descriptions provided on pages 311 and 312 of Yphantis et. al. Biochemistry 1964 vol.3  (see attached) where concentrations of 12.5 and higher were measured (if my reading is correct) where measurements were performed by using the 2/3rds plane focusing.  Questions.Is the displacement in the fringe data along the radial axis consistent with a concentration gradient that is too steep?  What is the best way to measure the fringe/mm value?Are concentrations up to 30mgs/ml possible in a 1.2 cm cell using interference optics, and if so how should the optics be focused?  Thank you in advance for your time, comments and your advice.  Best regards,John Sumida University of WashingtonMolecular Engineering & Sciences  




_______________________________________________
RASMB mailing list
RASMB at list.rasmb.org
http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org



_______________________________________________
RASMB mailing list
RASMB at list.rasmb.org
http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org


  
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20151021/94b9269b/attachment.htm>