Technical Note: TNCFGLYNXAMSULF 1012
Large Volume Precipitation of Proteins with
Ammonium Sulfate Using Thermo Scientific
Fiberlite Carbon Fiber Rotors
Key Words
Ammonium Sulfate Precipitation, Superspeed Centrifuges, Carbon Fiber Rotors
Introduction
Ammonium sulfate precipitation is a method of protein
purification by altering the solubility of protein.
Ammonium sulfate is commonly used due to its high
solubility that allows salt solutions with high ionic strength.
At sufficiently high ionic strength, the protein will
be almost completely precipitated from the solution.
Differential precipitation of proteins by ammonium sulfate
is one of the most widely used preliminary purification
procedures. It is based on proteins having differing
solubility in ammonium sulfate solutions and can result
in a two- to five-fold increase in specific activity.
Provided that appropriately buffered ammonium
sulfate solutions are used to protect the desired activity,
recoveries approaching 100% can be expected. A typical
protocol consists of adding ammonium sulfate to give
specific percentage saturation, followed by a period of
time for proteins to precipitate and a centrifugation
step to collect the precipitate. The adapted protocol1
described below was carefully followed to precipitate
protein removed by centrifugation using the Thermo
Scientific Sorvall LYNX 6000 superspeed centrifuge and
the large volume Thermo Scientific Fiberlite F12-6x500
LEX carbon fiber rotor. The total capacity of the
Fiberlite® F12-6x500 LEX rotor is 3 liters and is rated
for RCF up to 24,471 x g.
The ammonium sulfate concentration was increased
to a value that will precipitate most of the protein of
interest, while leaving the maximum amount of protein
contaminants still in solution. The precipitated protein of
interest was recovered by centrifugation and dissolved in
fresh buffer for the next stage of purification.
PROTOCOL: Ammonium Sulfate Precipitation
1. Measure the volume of protein solution to obtain
1500 mL. If necessary add 1 M HEPES, pH 7.4, or 1 M
Tris-HCl, pH 7.5, to a final concentration of 50 mM.
Figure 1: Thermo Scientific Fiberlite F12-6x500 LEX
carbon fiber rotor.
2.While gently stirring, slowly add ammonium sulfate to
45% saturation (277 g ammonium sulfate/liter solution).
Stir gently for a minumum of 4 hr and a maximum of
16 hr at 4 ºC.
Note: Addition of ammonium sulfate acidifies the solution,
therefore protein stability may be improved by buffering
with at least 50 mM HEPES or Tris buffer. It is important
that no foaming of the solution occurs during ammonium
sulfate addition as this will cause denaturation of proteins.
If foaming occurs, slow the addition of ammonium sulfate.
Make sure that the ammonium sulfate dissolves nearly as
quickly as it is added.
3. Transfer the ammonium sulfate precipitated solution
to 500 mL Thermo Scientific Nalgene polycarbonate
centrifuge bottles with screw caps (PN 3140-0500).
Perform centrifugation using the Fiberlite
Conclusion
SPANISH
LYNX 6000
This protocol is useful to quickly remove large amounts
superspeed
centrifuge
with
the
following
parameters:
of contaminant proteins, as done in the first step in many
© 2012 Thermo Fisher Scientific Inc. Reservados todos los derechos. Las otras marcas comerciales son propiedad de Thermo Fisher Scientific Inc. y sus
25,000
x gcondiciones
for 20 ymin
4 sujetos
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purification
Itlosis also often employed during the
filiales.
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países. Consulte
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later stages of purification to concentrate protein from a
4. Decant the supernatant, being sure not to disturb the
2
dilute solution
following
Alemania +49 6184 90 6000
Francia +33 2 2803 2180
Rusia +7 812
703 42 15 procedures such as gel filtration.
relatively loose protein-ammonium
sulfate pellet.
Australia +61 39757 4300
Holanda +31 76 579 55 55
Suiza +41 44 454 12 22
Austria +43 1 801 40 0
India +91 22 6716 2200
UK/Irlanda
+44 rapid
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In addition
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At 53
this
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the
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F12-6x500 LEX rotor and the Sovall®
thermoscientific.com
5. Re-suspend the pellet in PBS, pH 7.2. The solution will
remain turbid, but discreet flocculent is not visible.
References
1. Elder, G.H., Tovey, J. A. and D. A. Sheppard, (1983).
Purification of uroporphyrinogen decarboxylase from
ITALIANthe clarified
thermoscientific.com
6.Remove
residual precipitate and collect
human erythrocytes. Immunochemical evidence for a
proteins
by performing
centrifugation
thedifollowing
protein
decarboxylase activity in human
© 2012
Thermo Fisher
Scientific Inc. Tutti i diritti
riservati. Tutti gli altriwith
marchi sono
proprietà di Thermo Fisher single
Scientific Inc.
e delle suewith
filiali. Specifiche,
condizioni
e tariffe possono
subire variazioni.
Non tutti
consultare il rivenditore
locale
parameters:
25,000
x g for
10i prodotti
min sono
at 4disponibili
ºC. in tutti i paesi. Per maggiori dettagli
erythrocytes
and
liver. Biochem. J. 215: 45-55.
Australia +61 39757
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22 6716
7.Remove
the clarified
proteins to fresh
tubes
and2200
store at
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+32for
53 up
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Nuova Zelanda +64 9 980 6700
8.
if column purification
is going
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+86Alternatively,
10 8419 3588
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del Nord/CIS
Francia +33 2 2803 2180
+358 9 329 10200
performed
immediately,
dialyze
protein
against
column
Germania +49 6184 90 6000
Russia +7 812 703 42 15
binding
Pass the solution through
a 0.45+34
μm93filter
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+81buffer.
45 453 9220
Spagna/Portogallo
223 09 18
and apply to the appropriate column.
thermoscientific.com
+41 44 454
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2. Koch,Svizzera
U., Choksi,
S.,12Marcucci,
L. and R. Korngold,
UK/Irlanda +44 870 609 9203
(1998).
A Synthetic
Peptide Analog
USA/Canada
+1 866 CD4-CDR3
984 3766
Enhances Skin Allograft Survival Across a MHC Class II
Altre Nazioni Asiatiche +852 2885 4613
Barrier.
Journal
of Immunology,
161: 421-429.
Nazioni
non in elenco
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