[RASMB] radial scans 6 sector cells

[John Philo]

Lake, regarding the UV damage, remember that pathway is independent of
whether your protein is stable enough in solution at RT to do equilibrium.
Every biopharmaceutical has to be tested for light stability, so we biotech
types tend to be more aware of these issues than academics. A very common
result of UV exposure is irreversible aggregation. Many SE protocols involve
quite a few scans, for example to try to decide whether equilibrium has been
reached, and often those scans have rather high repetitions at each radial
position. If you do have UV damage during an SE experiment, you are just
going to see that the profile keeps changing as you do more scans (you don't
really reach equilibrium). You could easily mistake this as lack of physical
stability, and may not realize it is the absorbance scans themselves that
are causing the problem. I hear that photodamage can vary a lot from one
protein to another, and I'm not saying that in practice this is a common
problem for SE or SV, but it may not be at all obvious if and when it is
actually a problem.
 
Here again is an issue where Beckman could easily help us by finally
providing software that scans the 6-channel cells properly and doesn't
needlessly expose all the samples to UV while it is fruitlessly scanning
through regions of charcoal-filled epon.
 
John

  _____  

From: Dr. Lake Paul [mailto:lpaul at purdue.edu] 
Sent: Thursday, December 09, 2010 2:47 PM
To: John Philo; 'Roy Hantgan'; rasmb at rasmb.bbri.org
Subject: Re: [RASMB] radial scans 6 sector cells


John,
I agree to a point. I usually like to see
my menicus region just in case I sprung a leak. It is always easier to
subtract data than to add it. As for UV damage, I think if you are going to
be doing a SE experiment your protein has to be stable as a rock for long
periods of time near room temperature, so a little UV damage is not going to
be fatal to the experiment. If UV does damage the protein then I would use
interference instead. 
Lake


----- Reply message -----
From: "John Philo" <jphilo at mailway.com>
Date: Thu, Dec 9, 2010 5:00 pm
Subject: [RASMB] radial scans 6 sector cells
To: "'Roy Hantgan'" <rhantgan at wfubmc.edu>, <rasmb at rasmb.bbri.org>


Lake, it seems to me for equilibrium there is no need to capture any data to
the left of the meniscus, or even the meniscus itself. You'll just have to
trim all that region away before you analyze anyway. So the default start at
5.8 cm should capture everything you will need. On the other end I find
there is no useful (fittable) data beyond ~7.12 cm. Also remember that UV
does damage proteins, and that all three samples (3 channels) get exposed
with each flash, so at some point extra flashes to collect data you aren't
going to use does become harmful.
 
Roy, you may find it useful to have both a velocity method and an
equilibrium one open. I usually do a quick velocity-mode scan to be sure
everything is loaded okay and to figure out the radial range I want, and
then set up the equilibrium method window based on the quick velocity-mode
scan results.
 
John
 

  _____  

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

 

 

 

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