[RASMB] MALLS

Ewa Folta-Stogniew stogniew at yahoo.com
Thu Feb 27 15:26:59 PST 2003


John,

I never saw the instrument or the data from it-
besides what is on their WebPage.

I belive that the sample shown in Fig.3 
http://www.viscotek.com/proteinfig3.php3

is more likely a polydisperse sample and their results
do not indicate this fact.

For a sample that is 6,000,000 Da, I would expect a
considerable angular dependence of scattered light and
I wonder how do they correct for this?

Ewa


--- john.e.harlan at abbott.com wrote:
> One system I have not seen mentioned here is the set
> up from Viscotek. 
> Their unit does not use multiple angles to get MW,
> but rather a single 
> angle, and a four capilary viscometer to measure
> intrinsic viscosity.  The 
> combination of intrinsic viscosity and single angle
> light scattering, 
> along with concentration from RI, allows for the
> calculation of MW.  There 
> is a bibliography list at their website
> (http://www.viscotek.com) that has 
> papers to expalin the theory.  The device they use
> for this purpose is a 
> triple detector (IV, LS, and RI) that combines all
> three measurements in a 
> very small footprint, with minimal flowpath
> differences between detectors.
> 
> I am wondering how well this type of measurement
> works relative to the the 
> more traditional multiple angle light scattering.  I
> have tried their 
> instrument on a few samples, it gave the expected MW
> (based on analytical 
> ultracentrifugation measurements). I remember the
> software being extremely 
> easy to use.  Within a very short time, I was able
> to analyze data and 
> arrive at an answer that made sense.  I would be
> interested in what people 
> much better versed in the theory of these
> measurements then I am think 
> about this alternative approach.
> 
> John Harlan
> 
> Abbott Laboratories
> 
> 
> 
> 
> Ewa Folta-Stogniew <stogniew at yahoo.com>
> Sent by: rasmb-admin at rasmb-email.bbri.org
> 02/26/2003 04:01 PM
> 
>  
>         To:     Joel Mackay
> <j.mackay at mmb.usyd.edu.au>
>         cc:     rasmb at rasmb-email.bbri.org
>         Subject:        Re: [RASMB] MALLS
> 
> 
>
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> 
> --- Joel Mackay <j.mackay at mmb.usyd.edu.au> wrote:
> 
> > Hi all,
> > We are currently considering the purchase of a
> > multiangle laser light 
> > scattering instrument for getting a snapshot of
> > protein molecular weight - 
> > we intend to hook up to one of our chromatography
> > systems. [Of course it 
> > isn't intended as a replacement for our venerable
> > XLA... - we just thought 
> > it would be handy for getting a feel for molecular
> > weight during the 
> > chromatography stage].
> > 
> 
> It sure is.
> 
> I have been using Wyatt's detector for a few years
> already and I really liked them (although I would
> buy
> Waters' RI detector since it has better dynamic
> range
> than the OPTILab from Wyatt).
> 
> I do not have any experience with Precision
> Detectors'
> instrument- I have DynaPro from former Protein
> Solution, which is primarily dynamic LS detector,
> but
> can collect static data as well. The weak part for
> DynaPro is the software, which cannot really process
> the static data efficiently.
> 
> You may check:
>
http://info.med.yale.edu/wmkeck/biophysics/Presentation_04_01_02_final.pdf
> 
> or
>
http://info.med.yale.edu/wmkeck/biophysics/Final_10_25_02a_Wyatt.pdf
> 
> to get a "feel" for what kind of information one can
> get using these detectors.
> 
> You may check:
>
http://info.med.yale.edu/wmkeck/biophysics/Lsmemoa.htm
> for more info about sample requirements et.c.
> 
> Yes, they cannot replace the centrifuge, but one can
> get  a  lot of information even for not such a clean
> sample. 
> 
> For protein work on complexes that are not greater
> than 500 kDa the miniDAWN with 3 angles should be
> absolutely sufficient. 
> 
> If you want to e-mail me directly, please use:
> Ewa.Folta-Stogniew at yale.edu address.
> 
> Answers to your Qs. below:
> 
> 
> > 2. What sort of masses can be measured with
> > confidence on an instrument 
> > like the mini-Dawn from Wyatt - we often deal with
> > proteins that are ~5-10 kDa.
> > 
> 
> you will just need more material to get a decent
> signal in LS.  I worked with 5kDa protein/peptides
> easily.
> 
> > 3. Wyatt seems to be the major player in the
> field,
> > but i have been getting 
> > info from Precision Detectors as well. Does anyone
> > have any comments 
> > (printable!) to make about either manufacturer's
> > instruments etc?
> > 
> I had not yet met anybody who uses Precision
> Detectors, and had never worked on one.
> 
> I like Wyatt's detectors a lot; DynaPro is OK for
> data
> collection but is lacking the software side to make
> the data analysis user-friendly.  It has however a
> nice way of using UV detector as a mass detector,
> which Wyatt's ASTRA software is lacking (one can use
> UV, but it is cumbersome).
> 
> > 4. Do people tend to use a refractometer to
> measure
> > refractive indices, or 
> > use calculated values?
> > 
> 
> In a given buffer all unmodified proteins will have
> the same dn/dc, so one can run standards and
> starting
> with the commonly used 0.185 ml/g "guessed" the
> dn/dc
> for polypeptides.  There is only one number that can
> fit all the data correctly and give appropriate MWs.
> 
> (the RI detectors needs to be calibrated and this
> needs to be checked).
> 
> The dn/dc will change slightly when going to high
> salts, but this can be easily corrected without
> measuring it.
> 
> For modified proteins, like for example
> glycoproteins,PEG_ylated proteins, or
> protein-detergent complexes, one needs to utilize
> signals from all three det., UV,RI and LS and the MW
> for the polypeptide portion of the complex can
> easily
> be determined (see the *.pdf files for references
> for
> "three det. approach").
> 
> Ewa
> 
> > Any comments most appreciated!
> > 
> > cheers
> > joel
> > 
> > 
> >
>
*****************************************************************
> > Dr Joel Mackay
> > Senior Lecturer
> > School of Molecular and Microbial Biosciences
> > Building G08
> > University of Sydney,
> > Sydney, NSW 2006
> > Australia
> > ph +61-2-9351-3906
> > fax +61-2-9351-4726
> > WWW: http://www.mmb.usyd.edu.au/mackay/
> > Sydney Protein Group Website:
> > http://www.mmb.usyd.edu.au/spg/
> >
>
****************************************************************
> > 
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> 
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