Summary & References: freeze-thaw sucrose gradients

Bradley Turner bsturner at
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
330 Brookline Avenue            bturner at
Boston, MA 02215                bsturner at

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>
> %----------------------------------------------------------------------%
> On Tue, 5 Sep 2000, Dr. Duncan Clark wrote:
> > In article <OF953379A1.5E172E74-ON87256950.00798779 at>,
> > tfitzwater at 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

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

Date: Wed, 06 Sep 2000 14:36:38 -0400
From: Steven Hecht <hechts at>
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>
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
Subject: freeze thaw gradients medline refs

1: Centelles JJ, Franco R.
Heterogeneity of the gradients performed by the freeze-thaw 
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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