[RASMB] MALLS

[Ewa Folta-Stogniew]

--- 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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