[RASMB] radial scans 6 sector cells

Fri Dec 10 08:52:56 PST 2010

Steve & Walter, 

Well I don't like to be belabor a point, but since I'm getting beat up a bit
here... 

Yes, the conservation of mass calculations do need to know the meniscus and
bottom positions, but that doesn't mean those positions have to be present
in the final equilibrium scan you are actually fitting. (Clearly they don't
since I have been using this data acquisition approach for many years,
including when using my 'soft' conservation of mass constraints). From an
experimental point of view it is also not very clear how to accurately
define those locations from scans that include them (e.g. the infamous
"where is the meniscus, really?" controversy).

Further, regarding the bottom position, what does the 'true' position
actually mean? The bottom of the standard Beckman 6-channel cells is flat,
not cut on a radius, so (without FC43) the length of the solution column
varies as you move across the width of the cell. Even when you use FC43
there is a radial region at the bottom where the absorbance optics are
detecting part sample solution and part FC43, so you really can't possibly
get good data for the entire solution column (this issue should pretty much
go away when you use interference slits). Often mass conservation approaches
are not actually considering the rectangular rather than sector shape of the
channel at all.

These issues of not knowing precisely where the column ends are located,
non-sector geometry, and not being able to get good data over the full
column height, are exactly why true accurate mass conservation usually
cannot be achieved and precisely why I implemented the 'soft' mass
conservation constraints about 16 years ago [Philo, J. S. (2000). Improving
sedimentation equilibrium analysis of mixed associations using numerical
constraints to impose mass or signal conservation. Methods Enzymol. 321,
100-120].

John
  _____  

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Walter Stafford
Sent: Thursday, December 09, 2010 8:21 PM
To: Stephen Harding; rasmb at rasmb.bbri.org
Subject: Re: [RASMB] radial scans 6 sector cells

So I agree with Steve:

Those data may not be recordable, especially near the bottom at higher
speeds, but one must know those positions accurately to do the curve fitting
properly (for two more components) because the best fit must match the
observable part of the data (i.e. that range over which the data are being
compared to the model for computation of the rms residuals),  and the proper
best fit (i.e. the model) is computed assuming those positions are known.
For a self-associating system (which is a single component, albeit
multi-species)  it is not so important since the only error in that case
would be the loading concentration - which we're not especially interested
in. But for a hetero-associating system, say A+B=AB,  the relative loading
concentrations of the two components do matter in the fitting and in getting
accurate values of the equilibrium constants. 

Since I do most of my runs using interference optics and always do a blank
run both before and after the main run in that case, at the speed of the
run, I can use those scans to get the positions of the bases of the
channels. And SEDANAL will do an interpolated blank subtraction.

Walter

Stephen Harding wrote: 

I also disagree with John and agree almost 100% with Walter, i.e. meniscus
and base positions have to be identifiable (and not just for SEDANAL) but I
don't think its possible to record (reliable) concentration data at the
solution boundaries, particularly for heterogeneous systems.

Steve

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Walter Stafford
Sent: 09 December 2010 23:14
To: jphilo at mailway.com
Cc: rasmb at rasmb.bbri.org
Subject: Re: [RASMB] radial scans 6 sector cells

I disagree with John. 

For example, my software (SEDANAL) needs to have both the menicus and the
base positions identifiable in order to do consrevation of mass calculations
for multi-compnent systems. For an equilibrium run - since time is not of
the essentce here - is to collect enough data to include the solution
boundaries.

actually, I usually collect all three channels of data in one scan and
process it afterward - once for each channel. Less muss and less fuss.

===============================================
Walter F. Stafford, Ph.D.
Senior Scientist
Boston Biomedical Research Institute
64 Grove Street
Watertown, MA 02472
617-658-7808
skype: w.stafford3
----------------------
"Be kind, for everyone you meet is fighting a hard battle."  -Plato
------------------------------------ 

  _____  

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Lake Paul
Sent: Thursday, December 09, 2010 1:26 PM
To: 'Roy Hantgan'; rasmb at rasmb.bbri.org
Subject: Re: [RASMB] radial scans 6 sector cells

Roy,

Funny you should mention this, I did my first 6-sector experiment yesterday.
The 5.7 default might not cover the inside sector, so you might have to
adjust it to 5.65. I did this by trial and error and by getting help from
Dr. John Burgner. The 5.65 worked well I get alittle of the top of the cell
and both menisci. Hope this helps.

Lake

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Roy Hantgan
Sent: Thursday, December 09, 2010 3:42 PM
To: rasmb at rasmb.bbri.org
Subject: [RASMB] radial scans 6 sector cells

Dear colleagues:

Can you please remind me how you set the radial distance for sed eq runs in
6-sector cells?
I've only run sed eq in double sector cells. 

Thanks for your advice,

Roy 

Roy  R. Hantgan, Ph.D.

Associate Professor of Biochemistry / Associate in Molecular Medicine

Director, Macromolecular Interactions Core Laboratory (MICL)

Wake Forest University School of Medicine

3058 Hanes Bldg / TEL 336-716-4675

_______________________________________________
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. 

-- 

===============================================

Walter F. Stafford, Ph.D.

Senior Scientist

Boston Biomedical Research Institute

64 Grove Street

Watertown, MA 02472

617-658-7808

skype: w.stafford3

----------------------

"Be kind, for everyone you meet is fighting a hard battle."  -Plato

------------------------------------ 
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20101210/407c8b25/attachment.htm>