Summary & References: freeze-thaw sucrose gradients

Bradley Turner bsturner at mbcrr.harvard.edu
Thu Sep 7 19:51:44 EST 2000


Hello All,

Thank you to everyone for your help in tracking down information
and sharing protocols about a very interesting technique for 
(mass)producing sucrose gradients.  Below I've listed the various
methods that were sent to me along with some references from 
Medline that I found on the topic.  I hope others will also
find this helpful.

Thanks to Duncan Clark for starting me on this quest by 
ressurecting this seemingly lost technique.

Brad Turner


****************************************************************
                    Bradley Turner
                Beth Israel Deaconess
                    Medical Center

Harvard Medical School          617-667-1215 phone
Division of Gastroenterology    617-667-2767 fax
Room Dana 605                   bsturner at biosun.harvard.edu
330 Brookline Avenue            bturner at caregroup.harvard.edu
Boston, MA 02215                bsturner at mbcrr.harvard.edu
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On 7 Sep 2000, Thanit Pewnim wrote:

> Hello Dr. Clark,
> 
> We will greatly appeciate if you would give us a physical basis behind the
> freeze-thaw method that generates a sucrose gradient.
> 
> Thank you
> 
> 
> 
> %----------------------------------------------------------------------%
> 	Thanit Pewnim, Department of Chemistry, Silpakorn University
> 	Nakornpathom 73000, THAILAND >>>>> Phone +66 34 255797
> 	Fax +66 34 271356 or 255820, E-mail <thanit at su.ac.th>
> %----------------------------------------------------------------------%
> 
> On Tue, 5 Sep 2000, Dr. Duncan Clark wrote:
> 
> > In article <OF953379A1.5E172E74-ON87256950.00798779 at nexstar.com>,
> > tfitzwater at gilead.com writes
> > >While   many   sources   indicate   that  magnesium  stock  solutions  form
> > >concentration  gradients  during  multiple freeze/thaws,
> > 
> > That is also a great way of producing sucrose gradients. Start with
> > 12.5% and do three slow (in a fridge) freeze thaws and you will generate
> > a nice low to high gradient for centrifuging in swing out rotors etc.
> > 

-------------------------------------------------------------
>From Dr Coffin at Tufts Univ.


From: John Coffin
Sent: 9/7/00 12:36 PM
Subject: Re: FW: frozen Sucrose gradient reference request

It's very simple.  For a relatively low density (e.g. 5-20%) 
gradient, fill the tube (or a rack of tubes) with the intermediate 
concentration (e.g. 12.5%) sucrose in the appropriate buffer, freeze 
in a -20 freezer, thaw at +4, and repeat 2 more times.  Gradients can 
be stored at the last freeze for quite a long time and thawed as 
necessary.  For higher density gradients (e.g. 15-65%), you can start 
by layering an equal volume of the less dense (15%) on the more dense 
solution, then proceed as above.  This procedure works very well for 
4-5 ml gradients.  for larger ones, you may have to experiment a bit.
This method has saved us a lot of work over the years.  I hope it is
helpful.

--------------------------------------------------------------------
>From Dr S. Hecht at Grand Valley St. Univ.


Date: Wed, 06 Sep 2000 14:36:38 -0400
From: Steven Hecht <hechts at gvsu.edu>
Subject: Sucrose gradient

    What we did was the following: A gradient solution was made with 30
ml of saturated sucrose (density gradient grade), 21 ml of 10X TKM
buffer (10X is 0.5M Tris-HCl,pH 7.5; 0.8M KCl, 0.05M MgCl2), and 147 ml
water. The refractive index was checked and water used to adjust to 14
to 15% sucrose. 10.5 ml of the solution was put in clean SW41
polyallomer tubes and frozen at minus 20 C. The frozen tubes were
allowed to thaw at 4 C. The cycle was repeated one more time, and then
the tubes stored at -20 C. A sample was chosen from each set and checked
to see that a 5-23% gradient had formed. Prior to using, they were
thawed at 4 C overnight. I was putting 0.5 ml of bacterial lysate on the
gradient and spinning at 35K rpm, 5 C, for 90 minutes.


Steve Hecht
Grand Valley State Univ.

--------------------------------------------------------------------
>From A. McLennan at Liverpool Univ.


Date: Thu, 7 Sep 2000 12:01:11 +0100
From: "Dr. A.G. McLennan" <A.G.Mclennan at liverpool.ac.uk>
Subject: Re: Seeking reference about 'freeze-thaw' sucrose gradients

Dear Dr. Turner

I'm afraid I cannot locate the original reference to this procedures 
(FEBS letters, early 70's I think).  Basically, to prepare a 5-20% 
sucrose gradient, 12.5% sucrose was freeze-thawed in the centrifuge 
tube 3 or 4 times.  I seem to recall the ends of the gradient were a 
bit flat, but you could check yourself with a refractometer.  Very 
useful method if you want large numbers of gradients (we only ever 
used 5 ml so I don't know how well it works with larger ones).

Hope this helps

Sandy McLennan


------------------------------------------------------------------
The references:


Date: Thu, 07 Sep 2000 13:31:31 -0400
From: bsturner at mbcrr.harvard.edu
Subject: freeze thaw gradients medline refs

1: Centelles JJ, Franco R.
Heterogeneity of the gradients performed by the freeze-thaw 
method.
J Biochem Biophys Methods. 1989 May;18(3):177-82.
PMID: 2732420; UI: 89278508

2: Fourcroy P, Cuiller S, Largitte FC, Lambert C.
Polyribosome analysis on sucrose gradients produced 
by the freeze-thaw method.
J Biochem Biophys Methods. 1981 Mar;4(3-4):243-6.
PMID: 7240652; UI: 81216413

3: Haff LA.
Production of Ficoll, Percoll, and albumin gradients by 
the freeze-thaw method.
Prep Biochem. 1979;9(2):149-56.
PMID: 220602; UI: 79180090

4: Chanas AC, Johnson BK.
Sucrose density gradient formation by freezing and thawing.
Med Lab Sci. 1980 Jan;37(1):89-90. No abstract available.
PMID: 7382750; UI: 80209032

5: Cooper AJ, Perry S.
The applicability of freeze-thaw Percoll gradients to 
whole-cell isopycnic fractionations: preliminary results.
J Immunol Methods. 1980;37(3-4):353-61.
PMID: 6256448; UI: 81095183

6: Davis PB, Pearson CK.
Characterization of density gradients prepared by freezing 
and thawing a sucrose solution.
Anal Biochem. 1978 Nov;91(1):343-9.
PMID: 9762116; UI: 98434864

7: Luthe DS.
A simple technique for the preparation and storage 
of sucrose gradients.
Anal Biochem. 1983 Nov;135(1):230-2.
PMID: 6670744; UI: 84151754

8: Cooper AJ, Smallwood JA, Morgan RA.
The preparation of freeze-thaw density gradients with homogeneous 
solute concentrations.
J Immunol Methods. 1984 Jul 6;71(2):259-64.
PMID: 6547463; UI: 84241168

9: Michov BM.
A concentration gradient system.
Anal Biochem. 1978 Jun 1;86(2):432-42. No abstract available.
PMID: 655408; UI: 78185032

10: Hollender A.
A simple method for concentrating serum by freezing and thawing.
Scand J Clin Lab Invest. 1970 Jan;25(1):63-4. No abstract available.
PMID: 5444962; UI: 70186396

11: Hirano T, Yoneyama T, Matsuzaki H, Sekine T.
Simple method for preparing a concentration gradient of 
serum components by freezing and thawing.
Clin Chem. 1991 Jul;37(7):1225-9.
PMID: 1855294; UI: 91309202

12: Pertoft H.
Fractionation of cells and subcellular particles with percoll.
J Biochem Biophys Methods. 2000 Jul 10;44(1-2):1-30.
[MEDLINE record in process]
PMID: 10889273; UI: 20349496

13: Fullerton GD, Keener CR, Cameron IL.
Correction for solute/solvent interaction extends accurate 
freezing point depression theory to high concentration range.
J Biochem Biophys Methods. 1994 Dec;29(3-4):217-35.
PMID: 7699200; UI: 95213524

14: [No authors listed]
Concentration of solutes by freezing and centrifugation.
Prep Biochem. 1993 Feb-May;23(1-2):79-80. No abstract available.
PMID: 8367407; UI: 93376651

15: Murase N, Franks F.
Salt precipitation during the freeze-concentration of 
phosphate buffer solutions.
Biophys Chem. 1989 Nov;34(3):293-300.
PMID: 2611352; UI: 90123080

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