[RASMB] (no subject)

[Leech, AP]

John Burgner wrote:


> Dear All,
> 
> Does anyone with recent experience with "state of the art" CD spectrometers
> have strong feelings on which  instruments would best fulfill the
> description given below. 
> Based on an intial investigation, it lookes like both  the Aviv 215 and the
> Jassco J-810 fit the bill. Both Olis and Applied Photophysics seem to be
> close 

We have gone through some of these questions here at York. I will just add
some personal opinions based on our experience, for what it's worth. We
ended up with a Jasco J810 and BioLogic stop-flow system, but that is not
to say you should do the same. (We already had a Photophysics stopped-flow.)


> Based on a survey of proven and potential users, high-quality sample
> temperature control with automatic temperature ramping is an absolute
> requirement for all.

How many cells and what sort do you need to measure? Peltier systems are
good for rapid temperature changes, but may be limited to one cell, and that
rectangular. If you want to do multiple cells with a sample changer you
may need a water-thermostatted system (bath). Also ask how the temperature
is measured - in the block or (optionally) by cuvette probe. We had some
problems with the Peltier system "hunting" (oscillating about the required
temperature) using 10 mm cuvettes.

> A rapid stopped flow system (less than or equal to 1
> ms dead time) is also essential for several users and will provide the fast
> kinetic circular dichroism capabilities that are currently absent from
> campus.

Look carefully at the mixing system and flow cell parts. For high denaturant
concentrations you may need a special mixer. The Biologic system uses motor
driven syringes, more flexible for changing mixing ratios, whereas the
Photophysics uses a pneumatic system with ratios changeable only by changing
syringe. On the other hand I feel that the BioLogic flow cell cuvettes are
fragile and expensive (we have already broken one) and the Photophysics
system may be more robust for frequent use.

> An automatic microtitrator will eliminate tedious and error-prone
> manipulations and provide for time-saving automated data collection.  This
> last capability was requested by several users, particularly for ligand
> binding studies and the determination of protein and nucleic acid stability
> as a function of denaturant concentrations.  Automatic titration will also
> require a device for the effective mixing of sample and titrant in the
> cuvette.

You can do titration with the BioLogic stop flow unit. Volumes required in
most of the titrator accessories are rather large (you need a 10 mm x 10 mm
cell for effective stirring).

> 
> An additional optics and data processing package to allow the detection of
> fluorescence polarization and anisotropy in the same instrument would be a
> very valuable addition.  This polarization and anisotropy capability is not
> replicated anywhere else on campus.

The arrangements for FP may vary between prism and grating spectrometers. I
do not know if this has performance implications.


> Since these packages also allow
> collection of total fluorescence emission, this addition would enhance the
> current fluorescence stopped flow capabilities on campus.  The fluorescence
> package will allow simultaneous collection of circular dichroism and
> fluorescence signals on the same sample, providing immediate confirmatory
> data and reducing sample preparation requirements.  When combined with the
> proposed automatic titrator, this package would enable stability and ligand
> binding studies by complementary methods on samples in precisely the same
> state (e.g. same ligand and/or denaturant concentration).  

Beware of the problem that for decent data from the two techniques, you may
need to operate at different concentrations (or path lengths etc), so it may
not be the time-saver you thought. Don't forget you will need filters etc.

Also don't forget to budget for the nitrogen requirements of the machinery
- a small constant flow at all times and more when the lamp is on, possibly
quite high flows at short wavelengths, and of good purity.

Regarding the different companies (and Ewa's comments), I have had excellent
service from both Jasco and Applied Photophysics, both of whom have service 
operations here in the UK, and from BioLogic who are based in France. As to
Photophysics and the Acorn computers, I understand their new "Chirascan" system
uses Windows, but I have not seen the machine myself. I cannot comment on Olis
or Aviv as we do not have any of their equipment.

There is no real substitute for seeing the equipment and if possible trying
it out, but this is not always easy to arrange with such specialised kit!

Andrew

-- 
Dr Andrew Leech                   *  Laboratory Manager
Technology Facility               *  Molecular Interactions Laboratory
Department of Biology (Area 15)   *  Tel   : +44 (0)1904 328723
University of York                *  Fax   : +44 (0)1904 328804
PO Box 373,  York  YO10 5YW       *  Email : apl3 at york.ac.uk