[RASMB] Re: Working with glycerol solutions

Wed Feb 15 15:16:01 PST 2006

Hi Jo,

In order to test if the study of protein oligomeric states works well in high glycerol, I made a sedimentation velocity runs of BSA in PBS in the presence of 0 and 5% glycerol (volume 200 ul) and BSA in (PBS + 5% DMSO) in the presence of 0, 5, and 15% glycerol (volume 400 ul), all at 20 C.  I have not seen any influence of glycerol on the state of protein oligomerization. The c(s) fit worked well and resolved the dimers and trimers. I did not take density gradient of glycerol into account. The weight averages of the sedimentation coefficient distribution were decreased, the width of the c(s) distributions had become narrower and the apparent frictional ratio increased in the presence of glycerol. 

Regards,
Inna 

Inna Gorshkova, Ph.D.
DBEPS,  ORS, NIH
13 South Drive
Bldg.13, Room 3E49
Bethesda, MD 20892
tel. 301-402-2955
fax 301-4801242
e-mail gorshkoi at helix.nih.gov

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Subject: RASMB Digest, Vol 5, Issue 17

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Today's Topics:

   1. Working with glycerol solutions (pjgb at mrc-lmb.cam.ac.uk)
   2. Re: Working with glycerol solutions (Tom Laue)
   3. FW: [RASMB] Working with glycerol solutions (Cole, James)

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

Message: 1
Date: Wed, 15 Feb 2006 12:58:06 +0000
From: pjgb at mrc-lmb.cam.ac.uk
Subject: [RASMB] Working with glycerol solutions
To: RASMB-Noticeboard <rasmb at server1.bbri.org>
Message-ID: <2147483647.1140008286@[10.2.1.127]>
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Dear RASMBers,

Has anyone had experience of working with significant percentages of glycerol (10-25%), for either velocity or equilibrium runs?
Obviously the main problem is going to be the greatly increased viscosity, particularly at 5°C with 20°C less of a problem, but what other snags are there?  Also, does the theoretical reduced diffusion, at these high viscosities, work out in practice so that the boundaries are no broader after spinning for the extra time needed?

Jo

P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH, UK.
Tel. +44 (0)1223 402296

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

Message: 2
Date: Wed, 15 Feb 2006 09:12:14 -0500
From: Tom Laue <Tom.Laue at unh.edu>
Subject: Re: [RASMB] Working with glycerol solutions
To: pjgb at mrc-lmb.cam.ac.uk
Cc: RASMB-Noticeboard <rasmb at server1.bbri.org>
Message-ID: <43F336BE.8000804 at unh.edu>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

Hi Jo-
Both S and D are affected by the same amount, assuming that there is no glycerol-dependent changes in oligomerization of the protein.
The time to reach equilibrium is much longer, so it is useful to use shorter solution columns if possible (e.g. cutting the column height in half will reduce the equilibration time by a factor of four).
There will be a slight density and viscosity gradient in the solution due to the redistribution of the glycerol. I believe Sedfit can take this into account.
Best wishes,
Tom

pjgb at mrc-lmb.cam.ac.uk wrote:

> Dear RASMBers,
>
> Has anyone had experience of working with significant percentages of 
> glycerol (10-25%), for either velocity or equilibrium runs?
> Obviously the main problem is going to be the greatly increased 
> viscosity, particularly at 5°C with 20°C less of a problem, but what 
> other snags are there?  Also, does the theoretical reduced diffusion, 
> at these high viscosities, work out in practice so that the boundaries 
> are no broader after spinning for the extra time needed?
>
> Jo
>
> P.J.G. Butler,
> MRC Laboratory of Molecular Biology,
> Hills Road, Cambridge, CB2 2QH, UK.
> Tel. +44 (0)1223 402296
> _______________________________________________
> RASMB mailing list
> RASMB at rasmb.bbri.org
> http://rasmb.bbri.org/mailman/listinfo/rasmb
>

--
Department of Biochemistry and Molecular Biology University of New Hampshire Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
E-mail: Tom.Laue at unh.edu
www.bitc.unh.edu
www.camis.unh.edu

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

Message: 3
Date: Wed, 15 Feb 2006 09:43:21 -0500
From: "Cole, James" <james.cole at uconn.edu>
Subject: FW: [RASMB] Working with glycerol solutions
To: <rasmb at rasmb-email.bbri.org>
Message-ID:
	<DE444C0E33E9734689769D8FF3FD40236D84BF at EXCHANGEB.mgmt.ad.uconn.edu>
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Dear Jo
In addition to the stuff that Tom mentioned you should also be aware that preferential hydration in glycerol-containing solutions increases the partial specific volume. This effect is pretty small: by averaging some published data from four proteins  [Gekko and Timasheff (1981) Biochemistry 20, 4667] we calculated a linear change in v-bar of: delta v-bar/% Glycerol = 3.33 +/- 0.38 x 10^-4 (ml/g)/%. Also, glycerol can enhance oligomerization of proteins. For example, the Kd for dimerization of cmv protease drops by about 10-fold in the presence of 20% glcyerol [Cole (1996) Biochemistry 35, 15601].

Regards,
Jim

-----Original Message-----
From: rasmb-bounces at server1.bbri.org [mailto:rasmb-bounces at server1.bbri.org] On Behalf Of pjgb at mrc-lmb.cam.ac.uk
Sent: Wednesday, February 15, 2006 7:58 AM
To: RASMB-Noticeboard
Subject: [RASMB] Working with glycerol solutions

Dear RASMBers,

Has anyone had experience of working with significant percentages of glycerol (10-25%), for either velocity or equilibrium runs?
Obviously the main problem is going to be the greatly increased viscosity, particularly at 5°C with 20°C less of a problem, but what other snags are there?  Also, does the theoretical reduced diffusion, at these high viscosities, work out in practice so that the boundaries are no broader after spinning for the extra time needed?

Jo

P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH, UK.
Tel. +44 (0)1223 402296
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