[RASMB] concentration range for non-ideal behavior

[David Hayes]

Hi John,
The opposite of non-ideality is reality.  All real molecules take up space, so all real molecules sediment with some excluded volume non-ideality.  Charge is another source of non-ideality:  the ideal model of a molecule, besides being a geometric point with no volume, also has no attractive or repulsive forces.The type of non-ideality that is not avoidable is excluded volume.  So it is not exactly mg/ml that determines whether or not the non-ideality is significant, but the excluded volume.  For most proteins it is reasonable to see little effect from concentration alone up to 1-2 mg/ml.  But, put a molecule in a salt free buffer where charge is not screened (Actin) or take a molecule that has a really big carbohydrate attached (Pegasys) and nonideality can be evident at 0.5 mg/ml and below.
In sedimentation velocity, the other non-ideality is the back flow.  When a real molecule moves down the cell, real buffer has to move up the cell to fill in the otherwise to be empty spot where the molecule was.  This hydrodynamic non-ideality is also always there, but usually negligible below 1-2 mg/ml.
Did you do a concentration series of your antibody?  If so, it is expected that around 5 mg/ml; you would have measurable concentration dependence of the sedimentation coefficient.  If your peak moves the same S at 0.5 and 5 mg/ml I would suspect self association is adding S and offsetting the concentration dependence of S.  Static light scattering is helpful to use as an orthogonal method when self association and non-ideality balance out in AUC-SV.
David

       From: John Sumida <jpsumida at u.washington.edu>
 To: rasmb at rasmb.org 
 Sent: Tuesday, January 20, 2015 9:00 PM
 Subject: [RASMB] concentration range for non-ideal behavior
   
<!--#yiv6573107090 _filtered #yiv6573107090 {font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;}#yiv6573107090 #yiv6573107090 p.yiv6573107090MsoNormal, #yiv6573107090 li.yiv6573107090MsoNormal, #yiv6573107090 div.yiv6573107090MsoNormal {margin:0in;margin-bottom:.0001pt;font-size:11.0pt;font-family:"Calibri", "sans-serif";}#yiv6573107090 a:link, #yiv6573107090 span.yiv6573107090MsoHyperlink {color:blue;text-decoration:underline;}#yiv6573107090 a:visited, #yiv6573107090 span.yiv6573107090MsoHyperlinkFollowed {color:purple;text-decoration:underline;}#yiv6573107090 span.yiv6573107090EmailStyle17 {font-family:"Courier New";color:windowtext;}#yiv6573107090 .yiv6573107090MsoChpDefault {font-family:"Calibri", "sans-serif";} _filtered #yiv6573107090 {margin:1.0in 1.0in 1.0in 1.0in;}#yiv6573107090 div.yiv6573107090WordSection1 {}-->Dear RASMB,  My recollection is that concentrations in excess of 1.0 mg/ml should be avoided due to the possibility of non-ideal interactions that would complicate the analysis of SV and SE measurements.  However in two recent cases (in one case a mAb was being studied, and in another a globular viral capsid protein), we exceeded this value by a factor of five and failed to observe any evidence of non-ideal behavior. In both cases, it was possible to fit both SV and SE data without assuming any non-ideal interactions when the experiment was performed in PBS or HEPES.  I would be curious to hear if it is the experience of the community that non-ideality is commonly to be expected at concentrations >1.0mg/ml or whether the concentration cutoff for non-ideality is less of an actual cutoff being more a function of the particular properties of the molecule being studied.  Best regardsJohn SumidaUniversity of Washington
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